The development of effective and personalized treatment options for patients with rare cancers like chordoma is hampered by numerous challenges. Biomarker-guided repurposing of therapies approved in other indications remains the fastest path to redefining the treatment paradigm, but chordoma’s low mutation burden limits the impact of genomics in target discovery and precision oncology efforts. As our knowledge of oncogenic mechanisms across various malignancies has matured, it’s become increasingly clear that numerous properties of tumors transcend their genomes – leading to new and uncharted frontiers of therapeutic opportunity. In this review, we discuss how the implementation of cutting-edge tools and approaches is opening new windows into chordoma’s vulnerabilities. We also note how a convergence of emerging observations in chordoma and other cancers is leading to the identification and evaluation of new therapeutic hypotheses for this rare cancer. Chordoma is an ultra-rare bone cancer that arises in the skull base or spine of pediatrics and adults, and originates from vestigial remnants of the embryonic notochord. Normally a low-grade but locally invasive disease, current standard of care for chordoma involves maximum surgical resection and/or radiotherapy . DespiteOver the same time period, the continued growth of genome-guided precision oncology prompted an explosion of drug repurposing efforts for molecularly-defined tumor types – a trend that also extended into the realm of rare cancers. For example, following its approval in chronic myelogenous leukemia, imatinib was successfully repurposed for KIT-mutant gastrointestinal stromal tumors , and dabper se , indicating an opportunity to explore repurposing of inhibitors targeting PI3K or its downstream effector mTOR. One of the most frequently altered genes in chordoma is PTEN . The majority of these models have been characterized by whole-exome and whole-transcriptome sequencing and will undergo additional multi-omics characterization in the future, with a view to facilitating hypothesis testing through the establishment of models representing the full diversity of chordoma. Moreover, these models are available to the research community, as are in-kind drug testing services offered through the Chordoma Foundation’s Drug Screening Program. As emerging drug repurposing concepts are evaluated in the Drug Screening Program, resulting data are publicly shared, whenever possible , the gene encoding brachyury, appears to be the most selectively essential gene in chordoma. Since brachyury (like most transcription factors) is a challenging drug target, the authors performed a drug repurposing screen and found that inhibitors of CDK9 or CDK7/12/13 (TBXT transcription and suppress chordoma cell proliferation. These results have motivated further in vivo testing of transcriptional CDK inhibitors, including KB-0742 (Patient avatars like PDXs, PDOs and cell lines also serve as key platforms for target discovery because they allow functional studies capable of revealing or validating non-oncogene dependencies in chordoma. Genome-scale loss-of-function screens in various cancer cell lines have enabled the creation of “dependency maps” , 117, anK7/12/13  downregu KB-0742 , in the  KB-0742 .Ongoing systematic screening of genetic and chemical vulnerabilities in chordoma is facilitating the development of new therapeutic hypotheses. For example, CDK6 – but not CDK4 – appears to be a genetic essentiality in some chordoma cell lines . OutsideChordoma’s apparent CDK6 dependence and potential ferroptosis susceptibility raises intriguing and unexpected parallels with clear-cell carcinomas , 125. HiAnother key implementation of systematic functional screens involves the discovery of synthetic lethal and combination therapy strategies. Loss-of-function screens in large cell line panels have led to identification of new synthetic lethal interactions , 135, inPreclinical and clinical research has yet to identify a therapy capable of producing frequent responses in chordoma , motivatAs one of the most well-validated therapeutic targets in chordoma, inhibitors of wild-type EGFR are arguably the best ‘anchors’ to initially explore in unbiased screens or rational combination studies. Indeed, combination therapy investigations with afatinib  or cetuxAchieving deep and durable responses in chordoma will likely require identification of therapeutic concepts capable of invigorating antitumor immunity. Despite a low tumor mutational burden, a significant proportion of chordomas appear to be characterized by complex genomic rearrangements , 37, whiTumorigenesis appears to require three main ingredients: an oncogenic signaling input, deregulation of the signal through tumor suppressor loss , 151, anThe development of functional inhibitors is a challenging endeavor, given that brachyury lacks the deep binding pockets commonly associated with enzymatic activity. Yet, transcription factors like brachyury are often involved in multiprotein complexes, pointing to the development of compounds that modulate protein-protein interactions as an attractive strategy. For example, brachyury associates with the histone acetyltransferase p300 using an interface involving amino acid residue Y88 . The proAlthough new drug discovery in an ultra-rare indication presents numerous challenges, the concept of “reverse” drug repurposing – that is, repurposing drugs initially developed in a rare cancer to more common indications – represents a promising path. Further highlighting the intriguing parallels between chordoma and kidney cancer, brachyury expression is associated with poor survival in ccRCC and papillary RCC Figure 4Even when macroscopic complete resection is achieved using cutting-edge surgical approaches, the majority of chordoma patients experience disease recurrence and are unlikely to be cured , 2. At sIn this review, we highlight several therapeutic hypotheses inspired by developing knowledge of chordoma biology and its parallels with other cancer types. In particular, we focus on emerging therapeutic opportunities based on emerging knowledge linking drug sensitivity to specific biomarkers. Nevertheless, through the lens of genomic sequencing, most chordomas still lack actionable alterations – underscoring the need to implement more sophisticated multi-omics approaches. Indeed, genomics is only one piece of the puzzle; tumor growth is controlled by multiple integrated systems, with each contributing uniquely to chordoma’s biology. Therapeutic opportunities exist within each of these systems, and efforts focused on elucidating and integrating them will provide a fuller view of chordoma’s biology. A key goal of multi-omics profiling efforts will be the identification of molecular subtypes, stratified by risk and therapeutic vulnerabilities, as has been demonstrated in other cancers –170.In parallel, unbiased functional assays, utilizing genome-wide CRISPR or high-throughput drug screening, may reveal non-oncogene dependencies or combination therapy strategies that would otherwise be difficult to detect through multi-omics profiling approaches. The identification of additive or synergistic therapeutic combinations is of particular interest, given the low historical response rates in chordoma . In addiFinally, we highlight how technological advances are opening the door to targeting the transcription factor brachyury, the main Achilles heel of chordoma. The identification of binding pockets on brachyury can serve as target sites for PROTAC warheads or molecular glues, but they may also be functionally important. One such potential site is pocket A’ or F (All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was funded by the generous donors to Chordoma Foundation.We acknowledge and thank our patient community for giving us constant motivation to help identify and develop better treatments for chordoma. We are also grateful for our generous donors who provide the necessary funding – and to our colleagues at Chordoma Foundation and the research community for their efforts – to work towards this goal. Finally, we thank Champions Oncology for their assistance with figure generation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nonalcoholic fatty liver disease (NAFLD) has emerged as the most frequent chronic liver disease globally. NAFLD is strongly associated with metabolic syndrome and it has been recently suggested that to rename NAFLD as metabolic dysfunction-associated fatty liver disease (MAFLD). NAFLD has been studied in different endocrine axes and accumulating body of clinical and experimental studies have suggested that NAFLD is associated with polycystic ovarian syndrome (PCOS), hypopituitarism, growth hormone deficiency (GHD), hypogonadism and other endocrine disorders. In fact, endocrine dysfunction may be considered as the major contributor for the development, progression, and severity of NAFLD. In the present comprehensive review, we discussed the epidemiological and clinical evidence on the epidemiology, pathophysiology, and management of NAFLD in endocrine disorders, with an emphasis on the effects of sex-specific hormones/conditions as well as molecular basis of NAFLD development in these endocrine diseases. The liver, a dynamic endocrine organ, displays a crosstalk with extra-hepatic organs in order to mediate numerous metabolic, endocrine and hormonal signaling pathways . Non-alcNAFLD-associated endocrinopathies, may be clinically displayed as other pathological conditions such as sex hormone imbalance. PCOS-a multifaceted disorder with a spectrum of clinical presentations- is known as the most prevalent endocrine disease in women of childbearing age . PCOS isvia assessing serum liver enzymes, ultrasonography, transient elastography, magnetic resonance spectroscopy (MRS) and liver biopsy. Brown et al. via their obesity . A vicio obesity . EvidencAbdominal obesity is another common finding in both NAFLD and PCOS patients and at the same time is associated with other related mutual risk factors . The terde novo lipogenesis  suppressing adenosine monophosphate-activated protein kinase (AMPK) activation in adipocytes that results in higher lipogenesis and increased visceral adiposity and (b) exerting alterations in insulin sensitivity or both mechanisms together  aimed to identify the molecular and genetic pathways involved in pathological conditions . The firNewly, a body of evidence indicates the role of mitochondrial dysfunction in NAFLD-PCOS connection. Patients with PCOS have shown mutations in mitochondrial genes and unusual mitochondrial DNA copy numbers . Moreove“Gut-liver axis” has a predominant role in the liver health. The intestinal factors such as dysbiosis and intestinal barrier dysfunction along with hepatic factors such as inflammatory/fibrotic mediators and hyperleptinemia lead to NAFLD progression through disturbed lipid and carbohydrate metabolism . On the To date, over-activation of the endocannabinoid system (ECS) is also involved in both diseases. ECS- a complex cellular signaling network- modulates a wide range of human functions and regulates inflammation, auto-immune and energy homeostasis . ECS is A commonly observed condition in women with PCOS and NAFLD is obstructive sleep apnea (OSA). Subjects with OSA have a 2.6- fold increased risk of NAFLD and fibrosis progression, while this number increases to 7.6-fold in patients with PCOS and OSA . OSA is via disturbed hepatic lipid metabolism during diseases  conducting more detailed compensatory meta-analysis studies seems desirable. The mechanisms underlying mitochondrial dysfunction during either disease or both together must be noticed. Indeed, improvement of mitochondrial function and endocannabinoid system can be studied as new therapeutic targets. Finally, the most effective, accurate, and evidence-based approaches for the treatment of NAFLD in PCOS women remain unknown, multi-disciplinary research seems to shed light on this issue. NAFLD and PCOS are known as the hepatic and ovarian manifestations of Mets, respectively. In conclusion, the review of literature suggests the significant higher risk of PCOS in NAFLD. The underlying responsible mechanistic pathways are not completely explored. Further research is needed to evaluate the current issue from the “hepato-ovarian axis” to epidemiological, clinical and pharmacological aspects.The total or partial loss of pituitary gland function caused by pituitary or hypothalamic disorders is defined as hypopituitarism . PituitaGH and insulin-like growth factor-1 (IGF-1) are mainly mediated by “hypothalamic-pituitary somatotropic axis” and exert several physiological effects, ranging from promoting cell division/proliferation and growth development to macro-nutrients metabolism in adipocytes, liver and skeletal muscle through direct or indirect-complex mechanisms , 116. GHA number of epidemiological studies have demonstrated that GHD patients exhibit higher risk of NAFLD progression, and a negative correlation between GH levels and the severity of NAFLD has been documented. Meanwhile, some studies do not report same results due to the heterogeneity in the characteristics of study participants and sample sizes. The epidemiological studies that have assessed GH and related risk factors in NAFLD patients have been summarized in Recently, Xu et al.  conducteGHD is followed by a wide range of metabolic consequences that are linked to the onset of NAFLD, including visceral obesity, IR and dyslipidemia . Beside via the stimulation of hepatic gluconeogenesis and glycogenolysis. However, GHD is along with IR and impaired hepatic glucose metabolism due to the changes in lipid flux  and prevents cholesterol accumulation -arginine test . AlthougIn summary, NAFLD screening is needed in all patients with GHD. Despite the controversy among recent finding, treatment with GH or IGF-1 might be a useful strategy in NAFLD patients with GHD. However, subsequent well-designed studies can evaluate the efficacy of aforementioned therapeutic strategies.As shown before, metabolic disturbances are parallel to hormonal abnormalities, triggering each other. According to the “sex hormone-liver axis”, male hypogonadism and female hyperandrogenism are recently known as the independent risk factors for the development of NAFLD . The dimTo the best of our knowledge, a great number of studies have investigated the bi-directional relationship between hypogonadism and NAFLD –162. WomSerum TT levels are associated with central fat accumulation, IR and Mets . Seo et In this regard, the same association has been observed in children and adolescent, as Mueller et al.  in a croIn addition, some studies have investigated the relationship between lower dehydroepiandrosterone (DHEA) levels and liver-related complications in NASH patients and have suggested DHEA as a marker for assessing the risk of NASH with advanced fibrosis .The exact underlying mechanism in the incidence of “disturbed sex hormone- liver axis” state remains unclear, while deregulated pancreatic β-cell, adipocyte and hepatocyte function and changes in energy homeostasis could be considered as indirect changes during hormonal imbalances . Sex horDecreases in sex steroids are observed in women during menopause . Men andHypogonadism in females is commonly resulted from Turner syndrome-defined by premature ovarian failure and subsequent hypoestrogenism- that eventually leads to elevated hepatic enzymes, NAFLD and liver injury . In addiLow testosterone levels are observed in male hypogonadism. Low testosterone levels are correlated with IR in a bi-directional manner followed by abdominal/visceral fat deposition . In turnSex steroid deficiency is mainly involved in the pathogenesis of NAFLD as estrogen and testosterone play role in liver steatosis. Moreover, a number of other pathologic changes also occur during hypogonadal state.via inhibiting PPAR-α signaling pathway (Estrogen regulates energy and lipid homeostasis through its classic nuclear estrogen receptors (ERs) in the liver, exerts non-nuclear activities and inhibits liver fibrosis , 143. Es pathway . Indeed, pathway . Lower e pathway . Moreove pathway .The major circulating androgens-testosterone and dihydrotestosterone (DHT)-affect hepatic metabolism. Testosterone modulates gene expression by translocating to the nucleus after binding to the hepatic ARs in both male and females . The higCollectively, observational studies suggest that testosterone levels are associated with NAFLD in a sex- dependent manner, as higher serum TT levels lead to steatosis in women, while may be even protective in men. More precisely, TT levels in men and post-menopause women are negatively correlated with the incidence of NAFLD, whereas the correlation seems positive in PCOS women , 175. InDHEA deficiency is commonly observed in pan-hypopituitarism condition and amplifies the rapid progression of NASH to cirrhosis. DHEA is a steroid hormone with high abundance levels and modulates homeostasis, mediates ROS scavengers and OS, reduces IR, and increases the transcription levels of PPAR genes , 196. SeHypogonadism triggers a wide range of impairments in cardiovascular risk factors. The pattern of fat distribution as well as regional adiposity is dependent on sex hormones and hypogonadism which causes obesity, dyslipidemia, hypertension and dysglycemia and resulting in the higher progression of NAFLD . Based oBased on the evidence from animal studies, intestinal dysbiosis links hypogonadism to the development of NAFLD. Changes in Lactobacillus numbers as well as Firmicutes/Bacteroidetes ratio could be resulted from androgen loss in castrated male mice .Patients with hypogonadism-induced NAFLD should regularly be visited by both endocrinologist and hepatologist and undergo liver related assessments after excluding other steatogenic causes to prevent liver damage . The genFurther studies evaluating the potential impact of HRT in patients with hypogonadism and NAFLD are needed. As mentioned before, hypogonadism is linked to NAFLD in a bi-directional manner and these patients should be screened routinely.via modulating glucose and lipid metabolisms as well as stimulating adipogenesis .This study was supported by the Research Vice-Chancellor and Endocrine Research Center of Tabriz University of Medical Sciences (grant No 71003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Common Features highlights areas of disease pathology that share similar mechanisms of neurodegeneration across multiple diseases. This meeting report (Part 1 of 2) summarizes two of the hot topics discussed in the AD/PD 2021 Common Features, including cellular senescence and resilience/resistance (visit the meeting website for a complete agenda https://adpd2021.kenes.com/common-features-of-neurodegenerative-disease-exploring-the-brain-eye-connection-and-beyond). As funders of brain and eye diseases, BrightFocus supports research in each of these critical areas of disease pathology and anticipates that scientific discussions on these common elements will catalyze cross-disease collaborations and breakthroughs (information on how to apply for funding can be found at science.brightfocus.org/apply-grant). Since the topics and speakers change at every meeting, we look forward to your participation in the fourth installment of Common Features taking place at the AD/PD 2023 conference on March 28, 2023.BrightFocus Foundation sponsored the third iteration of the Alzheimer’s disease (AD) and related dementias, age-related macular degeneration (AMD), and glaucoma all are neurodegenerative diseases, with neuronal damages and losses in the eye or brain leading to functional impairment. The result can be side or central vision loss in glaucoma or AMD, or the onset of memory loss and dementia characteristic of AD.BrightFocus Foundation is currently funding $75 million in 287 active science projects around the globe through its three programs: Alzheimer’s Disease Research, Macular Degeneration Research, and National Glaucoma Research. BrightFocus is leading the way to explore the common neurodegenerative features to bridge what was once thought of as disparate fields of science, leveraging the learnings of one disease to better inform and help accelerate research progress in another.The 2021 program examined burning issues that unite diseases of mind and sight, and the lingering barriers to progress towards cures. Age is the single biggest risk factor that unites all three diseases of AD, AMD, and glaucoma. Accumulation of wear-and-tear on the nerves and supporting cells of the eyes and brain during aging may lead to common biological and biochemical features shared by these diseases. Lessons learned from tackling one disease could accelerate a solution to combat another. As such, this pre-symposium is a catalyst for future collaborations and scientific advances.Cellular senescence takes place when cells transition into a permanent, irreversible state of cell cycle arrest that can reduce the function and regenerative potential of terminally-differentiated tissues while increasing a senescent-associated pro-inflammatory phenotype (SASP). Cellular senescence may be particularly important for diseases where age is the number one risk factor. Current research is focused on identifying specific genetic and proteomic signatures of senescent cells and developing novel approaches to remove senescent cells, decreasing their pro-inflammatory phenotype, or reverting them back to a proliferating state.Darren Baker, PhD, Mayo Clinic, Rochester, focuses his research on novel treatment models to tackle senescence in AD. Senescence results in stem cell exhaustion, degradation of extracellular matrix molecules, and SASPs, where cells release a plethora of inflammatory molecules like cytokines, chemokines, proteinases, and growth factors. Senescent cells that express the cell cycle inhibitory protein, p16, can be targeted in animal models using gene therapy and senolytic drugs to activate the caspase pathway and induce apoptosis. Dr. Baker’s work has demonstrated that the primary senescent cells accumulating in the brain of PS19 tauopathy mice are astrocytes and microglia. These senescent cells promote insoluble tau aggregates and drive neurodegeneration. Clearance of senescent astrocytes and microglia using senolytic drugs attenuates tau phosphorylation and restores recognition memory [Senescence can occur as a response of normal cells to persistent damage that becomes resistant to apoptosis. The cells then undergo various cellular and molecular changes, including SASPs that affect the cellular environment. Dorota Skowronska-Krawczyk, PhD, University of California, Irvine, observed senescent cells in the retinal ganglion cells (RGCs) of glaucomatous eyes from human donor samples  and furtThe role of senescence in vascular diseases of the eye is being investigated by Przemyslaw (Mike) Sapieha, PhD, from the University of Montreal. Dr. Sapieha’s work on the oxygen-induced retinopathy model identified clusters of immune cells using single-cell RNA sequencing of the retina . They dehttps://genomics.senescence.info/about.html). This includes genes commonly altered during aging as well as a longevity map that serves as a repository for human genetic variants associated with longevity. Interestingly, using whole transcriptome sequencing, Dr. Magalhaes and colleagues identified many differentially expressed dark matter transcripts (transcripts that do not map to known exons) during aging [To provide a resource for geroscience researchers, Joao Pedro de Magalhaes, PhD, University of Liverpool, has developed an open database that identifies genetic signatures of cellular senescence in aging and longevity across different tissues and model organisms (Human Aging Genomics Resources ng aging . This wohttps://sennetconsortium.org/) program to generate a senescent cell atlas that will map the characteristics of different senescent cell types across different health states.An important question facing the field is how to identify senescent cells and distinguish them from post-mitotic cells. In the quest for a senescent cell biomarker, it may be possible to leverage the distinct metabolic profile that arises in senescent cells to not only identify senescent cells, but to better understand the intracellular mechanisms that underlie senescence. However, it is unclear whether the distinctive phenotypes of senescent cells in model systems recapitulate what occurs in human disease states. Having methods to identify and separate inflammatory signals from quiescent, reactive, and senescent cells will help to characterize cells across tissues and over time. To define a senescent cell, developing reliable biomarkers is critical, and efforts are underway through the NIH Cellular Senescence Network  and the APOE-Jacksonville (Jac) mutation (V236E), discovered by Dr. Guojun Bu , 9.How do we know if someone will be resilient to AD or other related dementias? Dr. Rik Ossenkoppele, Amsterdam UMC, Netherlands, utilizes a statistical residual model of resilience to obtain individualized resilience scores that can be used as a predictive tool for cognitive trajectories and/or to correlate proxies of resilience  and tau PET imaging signatures. Cognitive reserve is the adaptability of cognitive processes  while brain reserve is a neurobiological correlate that reflects the integrity of neural circuits. Cognitive and brain resilience may be associated with different mechanisms as a recent study that looked at tau pathology in clinically impaired individuals on the AD continuum, demonstrated that female sex and early age conferred greater brain resilience while higher education and global cortical thickness correlated with greater cognitive resilience .In the eye, selective RGC types have been shown to have resilience to damage or injury. Steven Barnes, PhD, Doheny Eye Institute, discussed the functional and morphological diversity of alpha RGC subtypes and their ability to modulate electrophysiological responses in the presence of reactive oxygen species (ROS). Dr. Barnes showed that during increased endogenous ROS, the ON-sustained alpha RGCs spike less and the transient RGCs had the opposite response showing increased spiking. The OFF-sustained spike less during increased ROS, and OFF-transient also show a small effect. Thus, sustained RGCs are set to handle long phases of slow spiking. Furthermore, ROS modulates the sodium (Na) channel gating to have a lower threshold in transient cells, permitting more spikes, but further increase in ROS production causes cessation of spiking due to Na channel inactivation. Different alpha RGCs have differentially tuned Na current activation properties that contribute to varied spiking responses in response to ROS levels.https://reserveandresilience.com/framework/). The ability to systematically identify people as resistant or resilient to disease across cohorts will allow for more segregated GWAS that can better identify genes critical for resistance and resilience. This will lead to better clinical trial cohort grouping, statistical analysis, and treatment outcomes, increased probability for the detection of sex differences in resistance and resilience and optimizing treatment regimens for a precision medicine approach.While mechanisms of resistance, resilience, and reserve have been studied for some decades, the lack of translation to a clinical setting has disadvantaged the field by neglecting to collect and provide valuable information for both basic and clinical studies. Resilience is driven by many factors including genetics, lifestyle, and environment, and it is important to understand risk factors and mechanisms of protection that can teach us how to treat disease. Recently, the Collaboratory on Research Definitions for Reserve and Resilience in Cognitive Aging and Dementia developed consensus definitions and research guidelines that should be used as standardized nomenclature for research on reserve and resilience (To Be Continued in Part 2 (Vascular Components and Conclusions).


Hydrochoerus hydrochaeris) with ahistory of chronic weight loss was euthanized following discovery by palpation of a largeintra-abdominal mass. Postmortem examination revealed a large, firm, tan mass expandingthe uterine body and extensively adhered to the jejunum and abdominal wall. Numerouspinpoint to 3-cm diameter, tan-to-red, raised masses were present throughout the parietalperitoneum, liver, lungs, and intestinal serosa. Histologic examination of the uterinemass revealed well-differentiated smooth muscle intermixed with abundant collagen,interspersed with a highly anaplastic spindle cell population extending to the serosa; themasses in the lung, liver, and peritoneum were histologically very similar to theanaplastic uterine spindle cells. Immunohistochemical staining of the uterus and lungconfirmed smooth muscle origin of the anaplastic cells. To our knowledge, leiomyosarcomahas not been reported previously in a capybara, and the widespread metastases in this caserepresent an unusually aggressive presentation of this rare malignancy. The animal alsohad an incidental dermal histiocytoma, a tumor that has also not been reported previouslyin this species, to our knowledge.A 10.5-y-old intact female capybara ( Hydrochoerus hydrochaeris) are the largest extant species ofrodent. They are indigenous to wetlands and forests of the Amazon basin and are commonly heldin zoologic collections.9 Numerousparasitic and bacterial diseases have been described in free-ranging capybaras, including brucellosis, paramphistomosis, and trypanosomosis. In addition, wild capybaras have been incriminated in transmission of disease agentssuch as Leishmania spp., Sarcocystis spp.,Neospora spp., Encephalitozoon spp., and orthopoxvirus todomestic animals and/or humans.19 Incontrast, there are few reports of disease in captive capybara. Fatal placental subinvolution was reported in an adult female, and neoplasia has only been reported twice: an intramuscular fibrosarcoma in an aged male and a cutaneous squamous cell carcinoma with regional lymph node metastasis in an adult male. We describe here leiomyosarcoma in an aged capybara.Capybaras . In addition, a section ofthe flank mass was stained with toluidine blue at the University of Surrey, and for ionizedcalcium-binding adapter molecule 1 (IBA1) by UG-VDS . All sta2; Microscopic examination revealed that the uterine mass consisted mainly of a well-demarcated,expansile mass of well-differentiated smooth muscle cells interspersed with broad bundles ofdense collagen with low numbers of slender fibrocytes , 2B. How2). All neoplastic round cells displayedmoderate-to-strong reactivity to IBA1; half of the population had membranous reactivity andthe remaining half had cytoplasmic reactivity, confirming histiocytic origin. The low degreeof cellular atypia was considered most consistent with histiocytoma.The dermal plaque on the right flank consisted of a well-demarcated, unencapsulated aggregateof large neoplastic round cells forming variably sized clusters separated by thick, denselyarranged collagen bundles, with numerous eosinophils intermixed. The overlying epidermis wasmarkedly thickened and formed prominent rete pegs. The neoplastic cells had variably distinctcytoplasmic margins and abundant faintly eosinophilic cytoplasm, rarely containing scant,basophilic granules that were not metachromatic with toluidine blue stain. Nuclei wereround-to-oval with dispersed chromatin and prominent, single nucleoli. Rare nuclei displayedindentation; anisokaryosis was mild with occasional binucleate cells throughout the mass; andmitotic count was 28 per 10 hpf  uterine origin with serosal and hematogenous metastasis, and 2) anunidentified primary site with hematogenous and serosal metastasis, including to the uterus.Uterine origin was considered most likely for several reasons. First, although the bulk of thelarge uterine mass had a relatively benign appearance consistent with fibroleiomyoma, therewas evidence of progressively increasing anaplasia approaching the serosal surface. This couldbe consistent with a slow-growing fibroleiomyoma in which the neoplastic cells graduallyaccumulated additional mutations, allowing for the eventual appearance of a highly malignantsubpopulation of neoplastic cells capable of breaching the serosa and metastasizing locallyand systemically. The pattern of gradual progression from well-differentiated to anaplasticcells is also not consistent with deep invasion of a serosal metastatic focus, which would beexpected to form invasive cords of anaplastic cells extending into and surrounded by thedeeper, well-differentiated tissue, as was observed in the hepatic and pulmonary metastases.Such cords were not observed; moreover, the densely collagenous structure of the uterineneoplasm would have acted as a barrier to deep invasion of a serosal metastasis from anotherprimary site.11 and suids maintained in zoologic collections, goats, dogs, and domestic cats. To date, there have been no reports of uterine tumors in capybara. Guinea pigs, theclosest domestic relative of the capybara, frequently develop uterine tumors; however, mostare leiomyomas. Leiomyosarcoma was identified concurrent with choriocarcinoma in one animal ina case series of 23 guinea pigs with uterine lesions, of which 13 of 37 lesions were neoplasms. Prevalence of uterine leiomyosarcoma in guinea pigs ranges from reports of 5 of 62uterine neoplasms to 9 of 83 uterine proliferative lesions. Two uterine leiomyosarcomas were also reported in a case series of 18 reproductiveneoplasms in guinea pigs of both sexes, although reproductive neoplasia comprised only 18 of341 neoplasia cases in that study. Thus, the incidence of uterine leiomyosarcoma in the closest relative of the capybarais also low. In all species, reports of uterine leiomyosarcoma without metastases greatlyoutnumber cases with confirmed metastases; thus, our case is also unusual because of thewidespread metastases. Reports of leiomyosarcoma have largely described differentiationsuggestive of smooth muscle origin based solely on routine H&E staining, without requiringimmunohistochemistry unless a second neoplastic population is present.20 Thus, the neoplasm described here was alsounusual in its high degree of anaplasia.Uterine smooth muscle tumors are uncommon in animals, although benign and/or malignant tumorshave been reported in a range of species including nondomestic felids7 There was no history ofcontraceptive administration to the female capybara in this collection. Age is well-recognizedas a risk factor for uterine smooth muscle neoplasms in various species.20 Given that the typical lifespan of acapybara is 8–10 y, the advanced age of this animal was likely the primary predisposing factor.The risk factors for development of malignant uterine neoplasms are incompletely understood.Notably, although captive wildlife are often administered contraceptive treatment, which hasbeen associated with non-neoplastic genital tract lesions and some reproductive neoplasms, noassociation has been found between contraceptive exposure and uterine smooth muscle tumordevelopment. The bullae in our case more closely resembled the idiopathic pulmonary bullae seenrelatively commonly in dogs, especially deep-chested breeds. Such bullae can occasionally rupture and cause spontaneous pneumothorax, but thiscomplication had not occurred in our case. Histiocytoma has also not been reported in acapybara to our knowledge, and no cases were reported in a series of 341 neoplasms in agingguinea pigs; hence, histiocytoma appears to be an uncommon lesion in this family of rodents.The large pulmonary bullae and the flank histiocytoma in this capybara are also of interest,although unrelated to the uterine neoplasm and weight loss. Pulmonary bullae have not beenreported in capybaras and do not appear to be a common lesion in aging guinea pigs but may beseen in guinea pigs used as a model for smoke exposure–related emphysematous changes.


Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity. Immune signaling processes occur over broad spatial and temporal scales. The spatial reorganization of immune signaling ranges from large micron-sized patterns, termed supramolecular activation clusters (SMACs) to signaling micro-clusters and nanoclusters below the resolution limit of conventional light microscopy ,2. The tThe triggering of the T-cell receptor (TCR) occurs within a few seconds of cell–cell contact , which iThe interface between a glass coverslip and immune cells presents an attractive platform for imaging signaling processes occurring at the plasma membrane. Total internal reflection fluorescence microscopy (TIRFM) can be applied to create an evanescent wave that penetrates 100–200 nm into the cell , enablinThese methods all attempt to model a cell–cell interface, typically by coating the coverslip with a supported lipid bilayer (SLB) that incorporates membrane proteins. However, this presents challenges as the physiological relevance of SLBs can be questioned due to unknown role of the glycocalyx , unexpecFluorescence images of an immunological synapse (IS) were first acquired using confocal microscopy in the late 90s ,19. CommWidefield fluorescence techniques utilize cameras to overcome scanning-induced speed limitations of confocal microscopy, but with reduced axial sectioning abilities. Structured illumination microscopy (SIM) is a widefield super-resolution microscopy technique that uses interference patterns to acquire multiple patterned fluorescence images from varying directions. These sequences then enable reconstruction of super-resolution images. This practically doubles resolution in all dimensions to achieve 125 nm and 350 nm resolutions in lateral and axial dimensions, respectively. In terms of immune signaling, most dynamic imaging studies to date have been performed in 2D . CombinaConfocal, SIM and epifluorescence microscopy are all prone to photobleaching as most of the sample is continuously exposed to laser excitation. To achieve gentler imaging, light-sheet microscopy  techniques have been developed, which selectively excite fluorophores within the focal plane of the sample. This combines the advantages of TIRFM and off-surface methods to enable 3D imaging with axial sectioning and high contrast due to reduced out-of-focus excitation. However, the difficulty in applying light-sheet microscopy to bioimaging has always involved the complexity of introducing the light sheet into the sample. Given the need for high numerical aperture objective lenses to visualize signaling processes, a variety of complex solutions have been developed including tilted objective lenses ,26, specA major improvement came with the development of lattice light-sheet microscopy (LLSM) that offered optical sectioning  over a large field-of-view (50 µm) ,32. ThisDynamic diffraction-limited 3D imaging of immune cells has mostly been solved with above introduced techniques, although there are needs in terms of imaging deeper into tissue and simplifying setups for increased accessability. While a great deal can be learned, understanding of immune signaling processes on the molecular scale requires nanoscale resolution, thus the field is in need to combine its efforts with super-resolution imaging techniques while maintaining the high temporal resolution offered by the dynamic 3D imaging techniques.Super-resolution imaging modalities paved the road to investigate immune signaling down to individual signal events initiated or mediated by single receptors, kinases, phosphatases, adaptor molecules or other contributing biomolecules. Nowadays commercial availability of super-resolution microscopes facilitates their active utilization to study cellular processes, such as immune signaling, on the nanometer scale. Common to all super-resolution modalities discussed in this section is the suppression of fluorescence emission from labels coupled to the biomolecule of interest to achieve spatial resolutions far below the classical resolution limit of light microscopy (∼250 nm).In stochastic and camera-based approaches like (direct) stochastic optical reconstruction microscopy ((d) STORM) ,39, flu.Recording thousands of frames for super-resolution imaging takes minutes to hours, rendering the observation of biomolecular dynamics challenging . TherefoIn contrast with these ‘single molecule localization microscopy' (SMLM) techniques, stimulated emission depletion (STED)  or grounOf note, long before the advent of super-resolution imaging, the high precision in localizing individual signals was utilized in single-particle or single-molecule tracking  to extract information about locations, kinetics, dynamics and interactions of individual molecule. The basic concept is to directly observe the motion of individual signals in live cell experiments and to extract the aforementioned information from trajectories generated from localized particles or molecules . High-spSuper-resolution techniques immensely contribute to our understanding of immune signaling by identifying spatiotemporal positions of individual key molecules on the nanometer-scale. However, the precise mode of action of these molecules within the signaling process needs to be addressed separately. One parameter gaining increasing attention in the field of immunology is the exertion of or response to mechanical forces .The structural plasticity of immune cells facilitated by cytoskeletal remodeling has been shown to exert a substantial amount of force on neighboring cells, on the extracellular matrix, and to the components of the IS. It changes the conformation of individual molecules, initiates cluster formation, and alters bond kinetics and biophysical properties such as membrane stiffness. Remodeling of the cytoskeleton, similar to cell spreading and motility, results in cycles of pushing and pulling ,73, whicWhile we reviewed techniques to probe mechanical forces elsewhere , the fieBesides the structural dynamics of IS formation , to trigTo detect forces on the single TCR–pMHC bond level, DNA-based tension sensors were instated, suggesting forces as high as 12 pN for ligand-bound TCRs  on glassCellular biophysical properties such as cellular deformability, stiffness, membrane fluidity, viscosity and tension differ for different cells, cellular states and diseases ,96. SuchImmune cells undergo extensive biophysical remodeling during their lifetime which is necessary for their function. A recent example by Lühr et al.  using enBiophysical properties of the IS attracted particular attention in the last decade. There is significant evidence suggesting a major role of biophysical properties of IS as regulating factors for signaling ,112. Owein vivo immune response was imaged in lymph nodes by light sheet dSTORM and SIM [While advanced imaging techniques reveal new important and complex details about the immune response, they come with technical, biological, and methodological limitations. In many applications mentioned before, the complexity of the biological systems was kept as low as possible to allow for high-contrast imaging. For example, imaging was performed on cells interacting with 2D surfaces to enable TIRFM, or simple cell–cell interactions in solution were observed using (lattice) light-sheet microscopy. Looking directly at immunological processes occurring in tissue, e.g. lymph nodes, demands new imaging strategies or modifications of existing techniques. Recently, the multiscale reorganization of TCRs during the  and SIM , demonst and SIM  or Lck [ and SIM . Softwar and SIM  or elabo and SIM , tempora and SIM  or using and SIM ) might oLimits are also set for the resolution of mechanical forces, where probes and methods sensing a high dynamic force range are still missing. Also, the ultimate aim of a force read-out for individual molecules within the whole IS will demand the combination of various imaging techniques, as was shown in first attempts by increasing the spatiotemporal resolution  or addreAnother major limitation to overcome is the handling and storage of large amounts of data generated during image acquisition. New concepts to increase the computational power for data analysis will have to be instated. The increasing complexity of microscopy systems and generated data will have to be matched by well trained and skilled operators and machine learning/artificial intelligence-based analysis algorithms, respectively.Immune signaling has been imaged at high speed , high reDynamic techniques (<1 s per image) are constantly pushing the spatial resolution limits. Parallelized RESOLFT, which relies on photoswitchable fluorescent proteins, now achieves 80 nm 3D resolution in entire cells at 1–2 frames per second . Light-sHigh-resolution SMLM techniques (<10 nm) have traditionally suffered from particularly slow imaging speed. However, improvements in analysis pipelines through deep-learning approaches ,130, optAnother aspect holding the field back is availability of state-of-the-art techniques and analysis tools. As mentioned earlier, it took ∼7 years to develop a turn-key commercial LLSM. Fortunately, the microscopy field has been embracing democratization of designs and low-cost solution to bring new techniques to the masses. 3D printed microscopes ,136 offeWhile dynamic imaging is key, there is much left to be learned from static approaches. Expansion microscopy has recently been used to map out receptors on T cells , which hDespite these technical developments, there is something inherently difficult about imaging cell–cell interactions and synapse formation and there are only a handful of studies out there taking advantage of the latest methodologies. This partially stems from the move into dynamic 3D super-resolution imaging being far from trivial. The relatively fast restructuring of the cell membrane and the reorganization of membrane proteins present a complex challenge involving multiple unanswered questions, even with the availability of high-speed high-resolution techniques. Where exactly is the membrane or glycocalyx? Where are the proteins in relation to the membrane? Is the cell currently signaling? Is the cell under stress? Furthermore, the basic definition of detecting signaling is itself challenging: does it correspond to calcium flux, expression of certain proteins or formation of signaling complexes? Complementary labeling approaches have the potential to define where the interactions occur, which can provide a helpful cue , but theAdvanced imaging is key for visualizing the spatiotemporal regulation of immune signaling.We summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling.We discuss the limitations of current technologies and share insights on how to overcome these limitations to visualize immune signaling with unprecedented details.


The TMG analyses suggest that runners already suffered peripheral fatigue before the VK and that 20–30 min are enough for muscular recovery after the race. In summary, our data suggest that participants experienced a pre-competition fatigue status. Further longitudinal studies are necessary to investigate the mechanisms underlying fatigue during trail running races, while training periodization and tapering strategies could play a key role for minimizing pre-competition fatigue status.Background: Understanding fatigue mechanisms is crucial for exercise performance. However, scientific evidence on non-invasive methods for assessing fatigue in trail running competitions is scarce, especially when vertical kilometer trail running races (VK) are considered. The main purpose of this study was to assess the autonomic nervous system (ANS) activity  and the state of muscle activation  before and after a VK competition. Methods: A cross-sectional pilot study was performed. After applying inclusion/exclusion criteria, 8 recreational male trail runners  volunteered to participate and were assessed for both central  and peripheral  fatigue before and after a VK race. Results: After the VK, resting heart rate, RMSSD ( The main findings of our investigation were: (1) as expected, there was a significant increase in the SNS activity after the competition, which lasted up to 45–60 min; (2) simultaneously, post-exercise PNS activity was significantly reduced; and (3) time-related variables and Dm levels presented by our runners suggest pre-competition peripheral fatigue.To the best of our knowledge, this is the first study evaluating central  and peripheral  fatigue before and immediately after  a VK trail running race in recreational trail runners. Although trail running is an emerging topic, the great majority of studies performed in the past few years have focused on metabolic, biomechanical and neuromuscular parameters. However, no studies have simultaneously assessed central and peripheral fatigue via non-invasive methods, especially when uphill trail running is considered .As expected, HRV parameters assessed after the competition showed significantly decreased values when compared to pre-competition levels. However, one interesting point is that when values of the variables related to the modulation of PNS  are compThe analyses of the pre-competition HRV time-domain variables  traditionally related to PNS activity ,51 showe= 0.89–1.35), indicating a clear downregulation of the PNS  that are beyond the 90th percentile, which is related to a high level of sympathetic stress . FurtherTaken together, in addition to the fatigue generated by the VK, both time domain and nonlinear measures may represent a sensitive downward modulation in the PNS of our runners and a lack of autonomic balance  prior to the race. This interpretation could suggest that a supercompensation status was not attained, which could be linked with fatigue. In this regard, some authors have suggested that recreational endurance runners who do not properly recover between training sessions, or those experiencing psychological stress or autonomic neuromuscular fatigue, are at higher risk of developing the so-called “overtraining syndrome”, which impairs endurance performance and leads to long-term fatigue . Moreovep = 0.018; d = 0.85), TrVM  and TsGM , when the pooled data of both legs were considered. These variations in Td, Tr and Ts might be connected to metabolic changes in myoplasmic Ca2+ [Regarding peripheral fatigue, despite having analyzed six variables by TMG  in four muscle groups , there were statistically significant differences only in TdRF  makes it difficult to evaluate a large number of participants. Future longitudinal studies are guaranteed for investigating the mechanisms underlying fatigue in endurance trail running events, especially when uphill running  is considered. In this regard, it would be of great interest to assess recovery variables over subsequent days after the competition to analyze recovery status evolution.The present study demonstrates that a VK race affects the ANS system, downregulating the PNS and upregulating the SNS. However, regarding peripheral fatigue, only small changes in the contractile capacity of specific muscle groups were detected. In addition, the pre-race measurements of HRV could suggest trail runners experienced a lack of recovery or non-functional overreaching before the race. Furthermore, while the neuromuscular stimulus of the competition did not seem to infer a great peripheral fatigue in these athletes, central fatigue significantly increased after the race. Considering the link between both central and peripheral fatigue, training periodization and new tapering strategies are called to play a key role in minimizing pre-competition fatigue status, thus favoring running performance. In this regard, monitoring HRV during the preparation period has been shown to be an effective strategy to avoid pre-competition fatigue.


Hepatoblastoma (HB) is the most common malignancy within the rare cohort ofpediatric primary liver tumors. It may arise sporadically or in association withgermline mutations in specific genetic syndromes. Histogenesis recapitulatesfetal hepatic development, however, this tumor can exhibit a markedlyheterogeneous appearance both macroscopically and under the microscope.Histologic subtypes are classified based on morphologic appearance, withadditional discrimination based on emerging molecular and immunohistochemicalfeatures. Numerous diagnostic pitfalls exist from clinical presentation throughto ancillary testing; at all stages, the surgical pathologist must be discerningand open to collaboration with colleagues of different specialties. Problematicareas include the adequacy of tissue sampling, correlation of histology withradiologic appearance and alpha feto-protein (AFP) serology, forming adiagnostic consensus within the pediatric pathology community and choosing ashrewd immunohistochemical panel. This review discusses the sequence of eventsleading up to histologic assessment, and the nuances of microscopic evaluation.Along the way, pitfalls are highlighted, providing a tool for the surgicalpathologists to support their individual approach. The incidence of HB appears to be increasing. Epidemiological data arelimited, however a steady increase of 4.3% per year between 1992 and 2004 has beendocumented in children younger than 19 years. This trend seems to continue into recent literature. Feng etal illustrate an annual percentage increase of 2.2% between 2000 and 2015. Various explanations have been proposed: cases may be revealed by screeningyoung children with the genetic propensity to develop HB, using abdominal ultrasoundand alpha feto-protein (AFP) serology;4 another likely contributor isthe tremendous advance in neonatal medicine, improving the survival of infants whobear dual risk factors for HB development, prematurity and very low birth weight of <1500g.Hepatoblastoma (HB) is the most common primary liver tumor of pediatric populations;while sporadic cases in adults and older children have been reported, theoverwhelming majority occur in children younger than three years. Its striking heterogeneity can be explained by its origin, arising from theaberrant division of both primary hepatoblasts and human fetal liver multipotentprogenitor cells. Hepatoblasts are hepatic stem cells responsible for sequencedprogression from fetal to fully differentiated adult liver tissue. Liver multipotentprogenitor cells are poorly differentiated at the outset and have the potential todifferentiate into a variety of tissue types, from those native to the liver, to fatand bone. Distinct morphologic subtypes exist, classified according to their tissuecomponents. Further categorization is described according to molecular features, asadvances in immunohistochemical techniques allow the discrimination ofmorphologically similar tumors. While the majority arise sporadically, up to a third of HB cases areassociated with a genetic syndrome or congenital anomaly. Among those most often implicated are Beckwith-Wiedemann syndrome, familialadenomatous polyposis, Edward syndrome (trisomy 18) and Down syndrome (trisomy21).79 In synchrony with researchteasing out the histologic subtypes, attention is paid to the unique gene expressionprofile of tumors, which may be helpful in stratifying their behavior. Inparticular, the beta-catenin pathway is strongly implicated the genesis andprogression of HB, and thus represents a tantalizing therapeutic target.11A tumor of embryonal origin, HB, recapitulates the hepatic development of fetal life.In order to procure an accurate and timely diagnosis for this young patientpopulation, it is important to be aware of common diagnostic pitfalls. This reviewdiscusses histopathological diagnosis, particularly since case numbers in mostdepartments are too low to form a reliable internal database for comparison. Theinterface between clinical medicine and histology is also explored, as each reliesupon the other to influence therapeutic decisions.12 Rarely, precocious puberty orfrank virilization may prompt investigation due to ectopic sex hormone of beta hCGproduction by tumor cells. Laboratory workup reveals anaemia in the majority (70%)and thrombocytosis in approximately 50%, thought to be due to thrombopoietinproduction by tumor cells. An overwhelming majority of patients have raised serum AFP at presentation:while helpful as an adjunct to clinical suspicion, AFP levels are physiologicallyraised at birth and may not normalize for the first 6 months of life.12 Additionally, AFP levels maynot be raised in tumors comprising poorly differentiated cells, such as the smallcell undifferentiated histologic subtype.14 However, an awareness of thelimitations of investigative modalities prevents an over-reliance on individualparameters or laboratory values. At all times, it is prescient to considerdifferential diagnoses when evaluating the clinical history. Liver tumors are adiverse group of neoplasms with radically different management protocols andprognoses. In the context of a large, aggressive hepatocellular tumor with highserum AFP levels, a newer entity should be born in mind: hepatocellularmalignant neoplasm, not otherwise specified (HCN-NOS) is a subset oftumors described at the 2014 COG liver tumors symposium. The discussion surrounding this consensus classification is explored furtherin the text, however an awareness of the spectrum of differential diagnoses is vitalbefore slide review.Patients with HB most commonly present with abdominal distention or a palpable massin the right upper quadrant. A vague constellation of symptoms is often associated,including weight loss, anorexia, nausea, vomiting, and diffuse abdominal pain.Jaundice may present in a slim minority, approaching 5% resulting from biliaryimpingement by the tumor mass.13 In children, CT and MRI scan aid in distinguishing HB fromother tumors of childhood, such as mesenchymal hamartoma and pediatrichepatocellular carcinoma (HCC), though in this context MRI is preferred due to itssuperior discernment of mixed soft tissues.16 MRI may reveal an epithelialtumor component, vascular invasion and the presence of fibrotic bands. Althoughconsidered a soft sign, intra-tumoral calcifications on CT add weight to adifferential diagnosis of HB, since these are present in approximately 50%. Ultrasound is clinically easier to perform in the pediatric population sinceanaesthesia is not required; a tumor mass and its internal components can be mappedusing ultrasound, however, this modality lacks the refined image quality of MRI. To monitor recurrence post-treatment, Fluorodeoxyglucose positron emissiontomography (FDG-PET) can be used, particularly to identify earlyrecurrence.5Unfortunately, due to the low FDG avidity in cells of pure fetal type, these tumorsmay be challenging to distinguish from normal background liver, and recurrence maynot be readily identified. In such circumstances, imaging should be taken in theclinical context of serum AFP levels, which fall post-treatment and tend to risewith recurrence and repetitive CT surveillance.18Radiologic imaging is essential for identification of tumors, pre-operative planningand monitoring for recurrence. Congenital HB may be detected as early as the startof the third trimester by maternal ultrasound, presenting as a single, solid,echogenic lesion.1921 As preoperative chemotherapyis increasingly becoming the standard of care, core needle biopsy and fine-needleaspiration are often utilized in the first instance, to provide tissue for paraffinpreparation and immunohistochemistry.13The gold standard of diagnosis remains histological examination of the tumor mass,supported by clinical, biochemical and radiological assessment.21 The Collegeof American Pathologists emphasizes the difficulty in distinguishingwell-differentiated hepatocellular malignancy from regenerative changes and benignproliferations.21 FNA has been described in the diagnostic workup of mesenchymalhamartoma: HB is a major differential for the slightly less common mesenchymalhamartoma and has its own distinctive cytological features, such as clusters oftumor cells showing acinar and trabecular patterns, small round tumor cells with ahigh nuclear to cytoplasmic ratio, hyperchromatic nuclei with prominent nucleoli,and the presence of extramedullary haemopoiesis.22 Due to the heterologousnature of both HB and mesenchymal hamartoma, and the narrow sampling potential ofFNA, these two lesions with vastly different clinical trajectories can be mistakenfor one another. FNA may miss the spindle cell mesenchymal element classicallyassociated with mesenchymal hamartoma. In addition, mesenchymal hamartoma may beassociated with increased serum AFP, further obfuscating the clinicaldistinction.23 Taking this into account, FNA is not considered adequate fordiagnosis of HB. The current recommendation for diagnosis of HB is biopsy ifsurgical resection in the first instance is not an option. Examples of lesions notamenable to primary resection include large vessel invasion or those that aremultifocal.16 The formerwill warrant pre-surgical chemotherapy and the latter may remainunresectable.15 Needle biopsy optimally results in 4 to 6 liver cores, however,more is always considered better. The biopsy may take place percutaneously or via a more invasive laparoscopyor laparotomy, guided by characteristics of the tumor such as vascularity which makeit inaccessible to less invasive biopsy methods. Notably, a diagnosis of purewell-differentiated fetal type HB can only be made on a resection specimen, whereall tumor cells are seen to be of distinctive fetal morphology, not on biopsy aloneor following chemotherapy.19 This safeguards against the omission of a smaller, lessdifferentiated subtype rendering the sample a mixed tumor with a different clinicalpath.Fine needle aspiration (FNA) is rarely utilized in the diagnostic approach to liverlesions. However, it may be used alongside radiology when a benign lesion issuspected. It is a tempting modality due to its less invasive sampling method.However, it is not generally regarded as providing sufficient evidence fordiagnosis, mainly when dealing with a mixed or undifferentiated pattern.25In contrast to the stepwise accumulation of somatic mutations often implicated inadult malignancies, pediatric tumors appear to develop more abruptly, pointing tohighly penetrant somatic mutations in undifferentiated cell lines or geneticpredisposition. The latter has been described in association with germline mutationsin specific genetic syndromes, which account for approximately 15% of HBcases.27Myriad cytogenetic changes have been identified in HB; those failing to maintainnormal chromosome number appear to be key to tumor development. In addition,germline and somatic mutations compound developmental factors, such as low birthweight and prematurity, to influence HB induction and growth.28 Wnt signaling is intrinsic to embryonic development, promotingorganized cellular proliferation and differentiation.29 It is fitting that the mostfrequently reported mutation in HB is in CTNNB1, a gene encodingthe beta-catenin protein, playing an important role in cell communication andadhesion.26AXIN1 and AXIN2, downstream components of the Wntpathway, contribute to beta-catenin degradation.28 Zhang et al(2019) describe that all beta-catenin mutations are tumorigenic when co-expressedwith mutant YAP; they also provide context for the strikingbiological diversity of HB, by describing the influence of myriad beta-cateninmutation identities on tumor characteristics such as growth rate, metabolicprofiles, and diverse histological appearance.30 N6-Methyladenosine (m6A) is amodification of RNA with rampant involvement in normal cellular processing andmalignant disease alike. It has been implicated in the development of a variety ofcancers via its governance of gene expression. Genes associated with m6A formationand function feature prominently in HB, underscoring their role in its promotion andclinical course.31 Such genes include METTL3, which consequentlyinfluences CTNNB1 as a downstream target, alongside its heterodimerpartner METTL14.28 While the sea of geneticaberrations associated with HB can be overwhelming to behold, the pathologist isurged to remain aware of new discoveries, which are frequent. The focus of researchis often the prediction of tumor behavior, tumor response to current therapies andidentification of novel therapeutic targets, all of which have a tangible impact onpatients. One example from recent literature is the described relationship betweenMETTL3 expression and the clinicopathological characteristics of patients with HB.Increased METTL3 expression was associated with frequent recurrence and poor prognosis. Such developments serve to better stratify patient risk and inform managementdecisions.Mutations implicated in the development of HB often involve the Wnt signalingpathway, more specifically, relating to the beta-catenin protein, a transcriptionalcofactor.33 Regarding a shared genetic aetiology, the RETproto-oncogene is a likely culprit. Pathogenic mutation of RET isthe most common genetic aberrance identified in patients with Hirschsprung diseaseand has been extensively studied, implicating over 100 unique mutations of various types.RET exerts its effects through a variety of pathways some of which,like PI3/AKT, have rarely been described as driving HB. Pathogenic mutations of RET are diffusely involved in anumber of solid organ malignancies and cancer syndromes, by virtue of the role ofRET as a mediator of cell proliferation, survival, migration,apoptosis and differentiation.33Several case reports describe the co-occurrence of HB with other developmentaldisorders, such as Hirschsprung disease. In this context, it is noted thatHirschsprung disease may be associated with syndromes predisposing towardscancer. The rarity of pediatric liver cancers has consistently limited largebiological studies. Interobserver variability is commonplace, even among the world'sexperts. Within the consensus classification, a significant sticking point involvedidentifying the presence of a minor (<50%) small cell component, or thecoexistence of HB with pediatric HCC. In establishing the diagnosis of such a rare malignancy with the security itis due, consultation for expert second opinion is consistentlyrecommended.35A number of collaborative efforts internationally has elaborated correlation betweentumor biology, clinical decision making, and patient outcome; expansion of thePediatric Liver Tumors Consensus Classification (PLTCC) incorporated histopathologyinto the Children's Oncology Group (COG) protocols in order to stratify risk andtreatment decisions.15 Whilepediatric HCC tends to present in an older age group, there is significantmorphologic overlap with HB. A misdiagnosis is critical, as chemotherapeutic regimens utilized withsuccess in HB have no impact on the activity of pediatric HCC. Like HB, progress in the field of bespoke pediatric HCC management has beenlimited by the heterogeneity of the tumors and the paucity of cases and samples forstudy.37Within the cohort of pediatric liver tumors, it is imperative to distinguish not onlybetween HB subtypes, but between different entities which may present similarly toclinicians and pathologists alike. Pediatric HCC is a small but significantcontribution to this cohort, making up approximately 20% of malignant pediatricliver tumors. This constellation of appearances which preclude exact classification hasbeen termed hepatocellular malignant neoplasm, not otherwise specified(HCN-NOS). Prior to description of this entity, a series of seven highly aggressive andchemo-resistant epithelial liver tumors was inducted into the literature by Prokuratet al Similar to cases assessed in formation of the consensusclassification, Prokurat et al noticed a mixture of poorlydifferentiated cells, HCC-like cells and HB-like cells at varying stages ofdifferentiation, often occurring within the same tumor mass.38 As a resultof these observations, this recalcitrant group of neoplasms was termedtransitional cell tumors of the liver. While such tumors may represent a transitional lineage with a commonprecursor, there are myriad other possibilities such as chemotherapy-induced changeto previously identified tumors, or a novel neoplasm of true transitional cellorigin.36 As such, the consensus classification removed the potentiallyobfuscating title of transitional cell tumor in favor of HCN-NOS.Within the consensus classification, a group of tumors resisted furtherclassification, demonstrating a variety of morphologies. Some cases exhibitedfeatures of both pediatric HCC and HB within the same tumor. Tumor cells exhibit a variety of appearances, from primitive blastema throughpoorly differentiated embryonal hepatocytes to fetal hepatocytes. Regardinghistological assessment, the variability of cytology and tumor architecture, bothwithin and between cases, is a potential pitfall even for an experienced pathologist. The rarity of HB means that even larger centers do not receive thesespecimens with a frequency necessary to develop an internal database ofexperience.15 To combat this, a systematic central histopathological reviewof pediatric liver tumors is encouraged, based on the work of cooperative groupssuch as COG. The subsequent international PLTCC developed a classification systembased on a cohort of 50 pediatric liver tumor cases, which stratified treatmentdecisions and served patient interests based on internationally sharedexperience.15 The description or rare and novel histologic subtypes, such asthe presence or absence of teratoid features within the mixed epithelial andmesenchymal subtype, provided a much-needed framework for diagnosticians worldwide.In addition to the interpretation of heterogeneous histology, accurate and timelydiagnosis of HB was challenged by the lack of immunohistochemical stains, lack of orinsufficient clinical history, and sampling limitations in the form of small orlimited biopsies.15 In this regard, the pathologist relies upon the expertise ofher clinical colleagues to provide sufficient primary and ancillary diagnosticmaterial.15 Considering these challenges of rarity and consensus, HBs havecertain identifying qualities macroscopically and microscopically, which will bediscussed here.HB is so named for its histological appearance, chiefly the hepatic epithelial tissueresembling immature fetal or embryonal liver.18 Macroscopic extension into the vena cava identified in over half. Even at this early stage, the pathologist should be mindful of differentialdiagnoses. If the tumor is seen to arise in the left lobe of the liver, an uncommonbehavior for the majority of primary liver tumors, the fibrolamellar variant ofpediatric HCC should come to mind. This tumor is an important consideration as it portends a poor prognosis, andotherwise closely resembles the appearance of HB macroscopically. On sectioning, the cut surface of a HB has a bulging appearance. The lesionmay be partially encapsulated and involve cystic change. Areas of necrosis and haemorrhage are frequently observed. The color of thetumor surface varies depending on its histologic subtype: the better-differentiatedfetal HB is tan-brown in color, owing to its similarity to the normal liver. Othersubtypes and mixed subtypes are more variegated, ranging from pale-tan to white togreen.19Recapitulating radiological findings, HBs are commonly seen grossly as a large,solitary mass (80%) localized to the right lobe of the liver in the majority(60%). This ensures that the tumor is properly subclassified, and smaller mixedareas are not omitted. This becomes especially important when unfavorablehistopathologic features, such as a small undifferentiated component.14 When making the first cut inthe block selection process, the vascular margin should be submitted first, toprevent carry-over of tumor and an artificially positive margin.During gross examination of a resection specimen, margins are routinely inked and thespecimen is photographed and mapped . Keeping25 Arrest orhijack of the differentiation pathway of liver multipotent progenitor cells andprimary hepatoblasts explains the markedly heterologous potential of HB.39HB is comprised of both epithelial and stromal components: the former can be eitherembryonal or fetal and the latter may exhibit a mixture of connective tissue andheterologous elements such as cartilage, bone and skeletal muscle.40 Mixed subtypes occur, the most common of which is mixedfetal/embryonal. The presence of a mesenchymal component in addition to anepithelial component of any subtype defined another mixed pattern, a mixedepithelial/mesenchymal tumor.. Within the mixed epithelial/mesenchymal subtype, a further division isdescribed, based on the presence or absence of heterologous elements representativeof all germ layers. These minor subtypes are named teratoid and non-teratoid,respectively , Société InternationaleD’oncologie Pédiatrique (SIOP), College of AmericanPathologists (CAP), United Kingdom Children's Cancer StudyGroup (UKCCSG), and the Canadian Association of Pathologists– Association canadienne des pathologistes (CAP-ACP)recommend establishing a process of centralized case review.An individual pathologist or center does not routinely viewenough cases to develop a reliable internal resource bank,and consultation for expert opinion is consistentlyrecommended.- Although consensus is not always achieved, each new casehas the potential to add valuable knowledge to a slendercollection of cases in the circulating literature.What about pediatric HCC?- In slightly older children, pediatric HCC encroaches intothe differential list. In particular, the macrotrabecularpattern of HB represents a diagnostic challenge, as the twocan be morphologically similar in the narrow window of acore biopsy.- There is significant overlap between theimmunohistochemical and genetic profile of the twoentities.- Beta-catenin is generally less intense in a nucleardistribution in pediatric HCC when compared with HB.- HB is favored in the presence of glutamine synthetase andcyclin D1 positivity.- HB is more often associated with chromosome 1pabnormalities and trisomy of chromosomes 2, 20 and 8 thanpediatric HCC.Is your immunohistochemical panel bespoke?- The routine immunostains recommended to support HBdiagnosis are: beta catenin, glypican 3, glutaminesynthetase, and INI1.- INI1 is of particular importance in differentiating theprognostically poor small cell undifferentiated subtype frommalignant rhabdoid tumors.- Based on the clinical context and radiological appearance,the panel of immunostains can be adapted to rule out commondifferential diagnoses, such as pediatric HCC andmesenchymal hamartoma, although currently no panel of stainscan HCC from HB with absolute confidence.- In the context of well-differentiated fetal HB, distinctionfrom normal liver may be achieved usingimmunohistochemistry, as it may look extremely similar tonormal neonatal liver.HB is a challenging diagnosis for the pathologist and her clinicalcolleagues, owing to its rare and heterogeneous nature. The clinical course is oftennon-specific until the later stages of disease, and biochemical assessment isvariably fruitful. Serum AFP levels represent an important tumor marker for HB butare non-specific, complicated further by the lack of expression in theprognostically unfavorable small cell undifferentiated subtype.7 Many pitfalls may beencountered on the path from clinical presentation through to histopathologicalassessment, all of which must be accounted for in the formulation of a diagnosis. Ascases continue to emerge and make their way into our current body of knowledge,tumor behavior will be more clearly defined. Facets of the beta-catenin signalingpathway show promise as therapeutic targets and perhaps as prognosticindicators.30 The management of HB involves radical and life alteringtherapy, such as chemotherapy, tumorectomy and transplantation. By identifying pitfalls as each stage of the diagnostic process, an accurateand timely diagnosis may be achieved, the importance of which cannot beunderstated.By distilling the sequence of investigation and diagnosis relating to HB, certaincommon pitfalls come to light. The following potential trips are of particularimportance to the surgical pathologist, who must synthesize abundant clinical,histological and ancillary information prior to reaching a conclusion:


Can a novel methodology using collaborative research coordinated online be successfully applied to the development of a guideline for the diagnosis and treatment of a common infectious disease, pyogenic osteomyelitis?This consensus statement and systematic review using a novel WikiGuidelines methodology addresses 7 questions regarding the management of osteomyelitis, resulting in the establishment of 2 clear recommendations  and 5 clinical reviews that outline a present lack of adequate, hypothesis-confirming data.These results suggest that this novel, egalitarian methodology enables a clear separation of established care standards based on hypothesis-confirming evidence from practice preferences that are based on lower quality or no evidence. This consensus statement and systematic review from the WikiGuidelines Group, an online collaborative research initiative, examines standards of evidence for pyogenic osteomyelitis care and offers clinical recommendations. Traditional approaches to practice guidelines frequently result in dissociation between strength of recommendation and quality of evidence.To construct a clinical guideline for pyogenic osteomyelitis management, with a new standard of evidence to resolve the gap between strength of recommendation and quality of evidence, through the use of a novel open access approach utilizing social media tools.This consensus statement and systematic review study used a novel approach from the WikiGuidelines Group, an open access collaborative research project, to construct clinical guidelines for pyogenic osteomyelitis. In June 2021 and February 2022, authors recruited via social media conducted multiple PubMed literature searches, including all years and languages, regarding osteomyelitis management; criteria for article quality and inclusion were specified in the group’s charter. The GRADE system for evaluating evidence was not used based on previously published concerns regarding the potential dissociation between strength of recommendation and quality of evidence. Instead, the charter required that clear recommendations be made only when reproducible, prospective, controlled studies provided hypothesis-confirming evidence. In the absence of such data, clinical reviews were drafted to discuss pros and cons of care choices. Both clear recommendations and clinical reviews were planned with the intention to be regularly updated as new data become available.Sixty-three participants with diverse expertise from 8 countries developed the group’s charter and its first guideline on pyogenic osteomyelitis. These participants included both nonacademic and academic physicians and pharmacists specializing in general internal medicine or hospital medicine, infectious diseases, orthopedic surgery, pharmacology, and medical microbiology. Of the 7 questions addressed in the guideline, 2 clear recommendations were offered for the use of oral antibiotic therapy and the duration of therapy. In addition, 5 clinical reviews were authored addressing diagnosis, approaches to osteomyelitis underlying a pressure ulcer, timing for the administration of empirical therapy, specific antimicrobial options , and the role of biomarkers and imaging to assess responses to therapy.The WikiGuidelines approach offers a novel methodology for clinical guideline development that precludes recommendations based on low-quality data or opinion. The primary limitation is the need for more rigorous clinical investigations, enabling additional clear recommendations for clinical questions currently unresolved by high-quality data. Pyogenic osteomyelitis occurs at a rate of approximately 20 cases per 100 000 person-years, with rates rising among patients with diabetes and older patients, as well as those with prosthetic joints.15 In the modern era, radiography, surgical methods, and antibiotics have revolutionized its management. However, these successful interventions have resulted in long-standing diagnostic and therapeutic paradigms that have guided treatment despite lacking strong evidence, including the need for diagnostic x-rays and intravenous-only antibiotic therapy for all patients.16 Recent studies have begun to challenge these dogmas.18 This guideline focuses on data regarding management of pyogenic osteomyelitis in adults  1 properly conducted, adequately powered randomized controlled trial (RCT); and (2) at least 1 other concordant, prospective, controlled clinical study—either a second RCT, a quasi-experimental pre-post study, a pragmatic nonrandomized trial, or a carefully conducted historically controlled study. In the absence of such data, the charter requires provision of clinical reviews that discuss care choices. However, recognizing the core ethical and clinical principle of “first do no harm,” authors could recommend against the routine provision of unsubstantiated care as part of clinical reviews. We also sought to incorporate principles of high value care  and health care quality .19Instead, the group sought to incorporate the “humility of uncertainty”Drafting members participated in reviews for 7 questions regarding the diagnosis and management of pyogenic osteomyelitis. For each question, members conducted their own literature review using PubMed, including all years and languages, with key words that varied by the question being asked. Articles were assessed for quality and inclusion by criteria specified in the charter. References from identified articles were also searched for potential inclusion. When divergent opinions on article interpretation or clinical practice existed among the authors, we did not attempt to force consensus; rather, in accord with the charter, we sought to transparently highlight those diverging opinions by discussing care alternatives. For answers based on more than 1 relevant RCT, meta-analysis was conducted using Review Manager 5.4.1 (Cochrane Collaboration).The consortium that established the WikiGuidelines Charter consisted of 63 participants from 8 countries: Australia, Canada, Colombia, Saudi Arabia, Spain, Switzerland, the United Kingdom, and the US. These participants included physicians, pharmacists, and microbiologists with expertise in general internal and hospital medicine, pediatrics, infectious diseases, orthopedic surgery, pharmacology, and medical microbiology.The participants addressed 7 questions regarding the diagnosis and management of pyogenic osteomyelitis but found data sufficient to establish clear recommendations for only 2: oral antibiotic therapy for pyogenic osteomyelitis and duration of therapy. In contrast, 5 questions were addressed with clinical reviews in the absence of high-quality data: diagnosis of pyogenic osteomyelitis, management of osteomyelitis underlying pressure ulcers, appropriate timing of empirical therapy, rational selection of antimicrobial options, and use of serial biomarkers or imaging studies to evaluate therapeutic response.Staphylococcus aureus) can be helpful to target with therapy and potentially obviate the need for bone biopsy. If available, bone biopsy for histopathology is highly accurate if positive, but cannot rule out osteomyelitis if negative. Culture of biopsy specimens of the affected bone may help identify etiology and target antimicrobial therapy.Based on observational studies, we do not recommend the routine use of plain x-rays  or computed tomography scans (inadequate sensitivity) for all patients with a possible diagnosis of osteomyelitis  as they Based on observational studies, plain x-rays have low sensitivity and specificity for diagnosing DFO . The proThere is no established, accurate referent standard diagnostic test for PJI. Certain tagged white cell scans are the most accurate imaging studies for PJI ; however21 Therefore, it may be preferable to avoid the routine use of antibiotic therapy for osteomyelitis underlying a pressure ulcer unless deep bone biopsy confirms osteomyelitis and surgical wound closure is planned, or the patient has accompanying sepsis syndrome or local soft tissue infection. Irrespective of antibiotic use, a multimodal therapeutic approach includes nutritional optimization, wound debridement and care, pressure off-loading, and psychosocial management.Observational studies indicate that imaging and inflammatory biomarkers are not diagnostically accurate for osteomyelitis underlying a pressure ulcer and we do not recommend their routine use for this purpose. Antibiotics have not been shown to be of benefit (and may be of harm) in the absence of surgical wound closure, but osteomyelitis may increase the risk of surgical flap failure.Some observational studies suggest that administration of antibiotics prior to bone biopsy or surgical management may modestly decrease yield of bone cultures for patients with osteomyelitis, including DFO and PJI. Thus, presuming other microbiological methods  have not already established a microbial etiology, it is reasonable to consider deferring antimicrobial therapy initiation until bone and/or joint microbiological samples are obtained for clinically stable patients. However, other studies are not concordant, and histopathology results are unlikely to be affected by prior short-term antibiotics. Decisions regarding the delay of empirical therapy therefore balance potential harm due to the risk of progression of life-threatening infection  or impending spinal cord compression against the potential benefit of microbiological data.S aureus, and gram-negative bacilli (S aureus (MRSA) or Pseudomonas aeruginosa depends on the presence of specific risk factors (addressed below). In all cases, local susceptibility patterns, patient-specific risk factors, and prior culture data influence the choice of antibiotic selection. Culture results can be used to tailor empirical therapy when possible.Based on data from observational studies, if antibiotic therapy cannot be delayed until culture availability, it is reasonable to empirically cover aerobic gram-positive cocci, especially  bacilli . Many prBased on culture data from observational studies, inclusion of empirical anti-MRSA coverage depends on local prevalence and patient-specific risk factors, such as known colonization status , prior positive cultures, and health care exposure. In a setting with low MRSA incidence, no known MRSA colonization or prior positive cultures, and minimal health care contact, it is reasonable to withhold empirical MRSA coverage.P aeruginosa; (3) gangrenous wounds; (4) had a recent surgical procedure ; or (5) specific sites of infection particularly associated with pseudomonal infection .Based on culture data from observational studies, routine use of empirical antipseudomonal therapy for osteomyelitis is unnecessary. Such agents are added in the presence of specific risk factors, including patients with chronic wounds who have: (1) been exposed to multiple prior courses of antibiotics; (2) previously had cultures positive for Outcome data related to antibiotic bone penetration are limited for osteomyelitis. Thus, theoretical bone penetration  for patients who: (1) are clinically stable ; (2) have adequate source control ; (3) are likely to absorb oral medications from a functioning gastrointestinal tract; (4) have an available regimen used in published osteomyelitis studies to cover likely target pathogens; and (5) have no psychosocial reasons that preclude the safe use of oral therapy. There is no required minimum duration of IV lead-in; patients may be switched to oral therapy when all the above criteria are met, even at the empirical therapy stage. Specific drug options and doses are discussed in the detailed review section (Based on 8 concordant RCTs comparing intravenous (IV) to oral therapy35  and 9 RC section .In the absence of RCTs, observational studies have generally found that neither serial inflammatory biomarkers  nor routinely repeated imaging accurately predict long-term treatment success for osteomyelitis or PJI for individual patients, nor have they been shown to meaningfully alter treatment decisions beyond clinical observation. Thus, following inflammatory biomarkers and repeated imaging may not offer benefit or contribute to high value care in most patients. Nonetheless, repeated imaging may be useful for patients who are clinically failing therapy to inform source control attempts, identify mechanical complications such as pathological fracture, and/or to trigger reconsideration of the initial diagnosis.44 and concordant observational studies, we recommend a maximum of 6 weeks of antibiotic therapy for hematogenous or contiguous pyogenic osteomyelitis (including DFO), assuming adequate source control  and no retained prosthetic implant ; however, confirmatory data are desired. Based on observational studies and 1 small RCT, it is reasonable to refrain from antibiotic use after total resection of infected bone if the treating physicians are confident that all infected bone has been resected. If administered, we do not recommend exceeding 2 to 5 days of therapy if there is no complicating soft tissue infection.43 participating experts unanimously agree that 12 is preferred to 6 weeks of antibiotics for PJI treated with debridement, antibiotics, and implant retention (DAIR). Some experts also clearly preferred 12 weeks of antibiotics for PJI treated with prosthetic exchanges. However, others believed that equipoise remains between 6 vs 12 weeks for these patients, particularly if S aureus is not the etiologic pathogen, or for 1-stage exchanges or 2-stage revisions with negative cultures prior to implantation.Based on the Duration of Antibiotic Treatment in Prosthetic Osteo-articular infection (DATIPO) RCT,Duration of therapy for other infected implants is not clear. A reasonable strategy, without evidence for or against, may be to treat with antibiotics until the bone heals sufficiently enough that the implants can be removed, such as in cases of fracture. Finally, chronic oral suppressive therapy may be considered for patients for whom the risks and benefits of curative surgery is deemed unacceptable; however, available data have not defined the risks and benefits of this approach well to this point.Based on the results of recent studies, the current approach to pyogenic osteomyelitis and PJI management can increasingly incorporate newer diagnostic and therapeutic concepts. Such changes include recognizing the low value and high cost and burden that plain x-rays incur if routinely ordered for all patients with possible osteomyelitis, reducing or eliminating the routine ordering of low-value, low-accuracy blood biomarkers , increasing adoption of oral step-down therapy, and limiting the duration of therapy to the shortest established to be necessary for optimizing cure in RCTs . These changes incorporate considerations of high value care and implementation in LMIC and resource-constrained settings, and thus are applicable across diverse care environments.The main limitation of this study was that the establishment of only 2 clear recommendations highlights the need for additional high-quality studies of osteomyelitis. In particular, studies are needed regarding new approaches to diagnostics; to elucidate the comparative effectiveness of various antimicrobial options, including adjunctive rifampin or anaerobic therapy; to identify which patients are more likely to relapse after completion of therapy; to further clarify antibiotic durations of therapy; to define the role and optimal methodologies of surgical management; and to define the role of nonantimicrobial adjunctive strategies . We also seek to incorporate authors from LMIC countries in future revisions to ensure the WikiGuidelines are broadly applicable to these settings.WikiGuidelines represent a novel approach to guideline construction, clearly delineating evidenced-based recommendations from opinions based on lower-quality data. Resulting changes in management of pyogenic osteomyelitis include recognizing the low value and high burden that plain x-rays incur if routinely ordered for all patients, reducing the routine ordering of low value, low accuracy blood biomarkers, increasing adoption of oral therapy, and limiting the duration of therapy to the shortest necessary for optimizing cure.These guidelines are based on published data available as of March 1, 2022. Clinicians who believe other evidence should be considered may contact any of the authors to initiate possible revisions, which the authors intend to complete in close to real-time. The authors understand that no clinical trial can extrapolate to all possible patient care scenarios. Thus, we expect that these guidelines should not establish medicolegal standards of care or replace clinician judgment for individual patients.


Hypoxia and faulty vasculature are well-known hallmarks of cancer and in addition to being associated with poor prognosis in patients, these hallmarks are also known to contribute to therapy resistance. In recent years, therapeutics that alleviate hypoxia and promote normalization of vasculature are being explored for cancer therapy. In addition to being hypoxic, cancers such as non-small cell lung cancers exhibit elevated oxidative phosphorylation. Therapeutic strategies that can normalize vasculature and reduce oxidative phosphorylation could greatly benefit the landscape of cancer therapeutics. Here, we highlight a heme-targeting therapeutic strategy that demonstrates significant tumor growth inhibition in non-small cell lung cancer mouse models using multi-spectral optoacoustic tomography. The tumor microenvironment (TME) is often highly heterogeneous and influences invasion, metastasis, and cancer progression as well as response to many types of therapy. Tumor vasculature and oxygenation are important aspects of TME and faulty tumor vascular oxygenation has been identified as a marker for poor prognosis in cancer.Hypoxia arises from poor oxygen supply due to inefficient vasculature as well as increased oxygen demand due to rapid growth. Hypoxia is associated with poor prognosis, increased genomic instability, elevated metastatic potential, and resistance to chemotherapy and radiotherapy . HypoxiaCancer cells exhibit upregulated glycolysis, which has led to the assumption that oxidative phosphorylation (OXPHOS) is downregulated in all cancers. However, there is increasing evidence of elevated levels of OXPHOS in many types of cancer –14. CertThere is overwhelming evidence pointing to the reliance of cancer cells on mitochondrial dysfunction and oxidative metabolism for their growth and progression. This suggests that targeting OXPHOS and mitochondria, in general, can be an effective strategy to treat cancer. However, to treat genetically and metabolically diverse cancers, it is essential to investigate novel therapeutic avenues that would be effective against a wide variety of cancers. Targeting OXPHOS via limitation of heme is one such promising approach , 20.Y. pestis) binds to heme strongly, inhibits heme uptake, decreases mitochondrial heme levels, and diminishes OXPHOS and ATP generation in NSCLC cells. HSP2 has significantly suppressed subcutaneous and orthotopic NSCLC tumor xenografts in mice [Heme is central to oxygen utilization and serves as a prosthetic group or cofactor for many OXPHOS proteins. HSP2 or HeSP2 (heme-sequestering peptide 2) and CycT (cyclopamine tartrate) are two heme targeting agents that effectively inhibit OXPHOS, and have been shown to suppress lung tumor growth and progression in human tumor xenograft mouse models , 20 Fig. HSP2 (g in mice . Many im in mice  and Ghos in mice  (Figure These heme-targeting drugs are effective at reducing tumor burden in autochthonous mouse models of lung cancer . Heme-ta