Most cancer-related deaths are a result of metastasis, and thus the importance of this process as a target of therapy cannot be understated. By asking ‘how can we effectively treat cancer?’, we do not capture the complexity of a disease encompassing >200 different cancer types — many consisting of multiple subtypes — with considerable intratumoural heterogeneity, which can result in variable responses to a specific therapy. Moreover, we have much less information on the pathophysiological characteristics of metastases than is available for the primary tumour. Most disseminated tumour cells that arrive in distant tissues, surrounded by unfamiliar cells and a foreign microenvironment, are likely to die; however, those that survive can generate metastatic tumours with a markedly different biology from that of the primary tumour. To treat metastasis effectively, we must inhibit fundamental metastatic processes and develop specific preclinical and clinical strategies that do not rely on primary tumour responses. To address this crucial issue, Cancer Research UK and Cancer Therapeutics CRC Australia formed a Metastasis Working Group with representatives from not-for-profit, academic, government, industry and regulatory bodies in order to develop recommendations on how to tackle the challenges associated with treating (micro)metastatic disease. Herein, we describe the challenges identified as well as the proposed approaches for discovering and developing anticancer agents designed specifically to prevent or delay the metastatic outgrowth of cancer. Most cancer-related deaths are attributable to metastasis, but few treatments are specifically designed to disrupt this process. In this Position Paper, representatives of the joint Cancer Research UK and Cancer Therapeutics CRC Australia Metastasis Working Group describe the challenges associated with discovering and developing anticancer agents designed specifically to prevent or delay the metastatic outgrowth of cancer and provide guidance on how these challenges might be overcome. Metastasis is associated with a poor patient prognosis and is the foremost cause of cancer-related death, with approximately 90% of patients who succumb to cancer dying of metastatic disease.Metastasis is inherently complex, with different distant sites having a distinct and specific extracellular matrix and cellular composition compared with that of the originating site, and therefore metastases must be considered biologically different from the primary tumour.The standard cancer drug discovery and development pathway, including that for molecularly targeted and immunotherapies, generally ignores the ability of experimental medicines to inhibit metastasis.A wealth of potential preclinical targets for anti-metastatic drug discovery and development have already been identified but remain to be validated using appropriate preclinical models that reflect the pathogenesis of metastatic disease in patients.Despite some successes in the treatment of bone metastases, following extensive analyses in preclinical models, multiple late-stage failures in clinical development have resulted in anti-metastatic drug development efforts being deprioritized by the pharmaceutical industry.Successful development of effective anti-metastatic therapies will require the regulatory agencies to work together with researchers, drug developers and statisticians to redefine the clinical development paradigm in order to encourage development of this complex but high-potential category of oncology drugs. Despite the advent of effective immunotherapies within the past decade, the majority of patients with advanced-stage and/or high-risk cancers continue to die as a direct result of metastatic disease or owing to complications of its treatment3. Indeed, improvements in the survival of patients with cancer over time have not equally benefited those with metastatic disease4.Metastasis of cancer to distal sites is associated with poor patient prognosis and is the foremost cause of cancer-related death6, with confirmatory improvements in clinical outcomes, ignoring the ability to inhibit metastasis. Only after clinically meaningful tumour responses and/or improvements in patient survival have been demonstrated in the metastatic setting will the drug be tested in adjuvant trials, with the aim of preventing or delaying the development of overt metastatic disease. Consequently, a paucity of preclinical discovery and thus clinical development exists for agents targeting the biological mechanisms underlying the metastatic process.Nevertheless, most standard-of-care treatments and new molecularly targeted therapies, including immunotherapies, were developed on the basis of initial evidence of anticancer activity — either direct or via immune system engagement — obtained in preclinical studies with tumorigenesis and/or primary growth, not metastatic activity, as the main end points. Similarly, clinical drug development generally relies on the demonstration of tumour shrinkage according to the radiological Response Evaluation Criteria for Solid Tumors (RECIST)An urgent need remains for novel therapeutic strategies and agents that prevent the establishment of and/or tissue colonization by metastases, which ultimately lead to organ failure, morbidity and death. In addition, new strategies must be developed to facilitate clinical testing of therapeutic agents with an anti-metastatic mechanism of action. To assist the cancer drug discovery and development community in addressing this critical issue, Cancer Research UK (CRUK), Cancer Research Technology (CRT) and Cancer Therapeutics CRC Australia (CTx) formed a Metastasis Working Group with representatives from academia, industry, government and regulatory bodies in order to develop recommendations on how to surmount the challenges associated with treating metastatic disease. This article provides an overview of these challenges and describes the Metastasis Working Group recommendations on best practices for the discovery and development of anticancer agents designed specifically to circumvent metastasis, with consideration given to their implementation in clinical trials.7. Indeed, the signalling pathways driving metastasis can vary between primary and secondary tumours and between metastases that arise at different sites8. The target of translational research efforts is often an occult population of tumour cells disseminating from the primary tumour, sitting dormant in a sanctuary site or constituting a micrometastasis in a distant organ. Selection for clinical testing of candidate anti-metastatic agents that might have limited or no effect on conventional preclinical outcomes, such as primary tumour growth, is another key challenge. In addition, validated biomarkers that can be used to increase the efficiency of mechanistic experiments and accelerate drug development are rarely available.The development of new effective medicines that interrupt the primary causes of metastasis is a daunting but important challenge. Mechanistically, metastatic tumour cells are genetically unstable, and in most cancers no single dominant pathway is likely to control metastasis10. Tumour cells might become invasive early in cancer development, and thus prevention of dissemination from a primary tumour is unlikely to be a clinically successful strategy owing to the presence of pre-existing but undetected metastases; however, prevention of secondary metastases is a plausible rationale for intervention. Metastatic dissemination is a multistage process, and various points of intervention have been identified and credentialed at the preclinical level metastases before patient diagnosis is acknowledged to be poorly characterized8. The distant tissue site will also have a distinct and specific extracellular matrix and cellular composition compared with that of the tumour tissue from which the metastatic cell originated. Metastases must therefore be considered biologically different from primary tumour cells, at least in the early stages of outgrowth. Studies specifically designed to examine the biological development of metastases are not common, and if we hope to target metastases effectively, we must gain a more complete understanding of the underlying biology. This issue is complex, although some successes have already been achieved, for example, in understanding the processes of pancreatic cancer metastasis17.The metastatic cell is a single genetic entity derived from a mass of cells possessing extensive genetic heterogeneity and consequent plasticity18. Tumour cell dormancy also alters chemotherapeutic efficacy, either because the non-dividing cells are more resistant to such treatment19 or because they are protected by cellular and extracellular components of their microenvironment22  between removal or successful therapy of the primary tumour and subsequent clinical relapse with disseminated disease. Metastatic dormancy can be achieved by many means. Tumour cells can exit from the cell cycle or balance their proliferation and apoptosis. Host cells can limit angiogenesis or alter anticancer immune responses, resulting in immunoediting (with an equilibrium between immune elimination and escape of tumour cells)The optimal means of selecting patients with a predictable risk and rate of disease progression for enrolment in a clinical trial of an anti-metastatic agent remain largely unknown. This challenge is compounded by the inability to reliably quantify the prevalence and extent of occult metastatic disease at enrolment. Because adjuvant clinical trials are often conducted only after positive results have been obtained in early phase clinical studies involving patients with advanced-stage (metastatic) disease and require considerable funding, large numbers of patients and long follow-up durations to capture the primary outcome measure ), new non-conventional clinical development approaches are needed. The clinical trial design might vary depending on the patient population, the dosing schema , the primary end points and the available biomarkers of activity. In most cases, such trials will also need to be initiated without a history of regulatory approval of the experimental agent.24. Subsequent studies enabled a more nuanced understanding of the metastasis-promoting as well as the metastasis-limiting effects of the MMPs and revealed that several other proteases have overlapping functions25. The preclinical models used had limited relevance to the clinical testing scenario, with the experimental design typically involving initiation of MMP inhibitor treatment soon after tumour cell inoculation, to form either a primary tumour or experimental metastases. In this setting, the MMP inhibitors were shown to be effective26; however, in the clinic, MMP inhibitors were tested in patients with advanced-stage disease, usually with drug-resistant metastatic disease. As a result, these phase II and phase III clinical trials of MMP inhibitors failed to show strong signals of efficacy28. In addition, the early drugs had broad spectrum anti-protease activity and were characterized by serious adverse effects in patients26. Other examples of anti-metastatic agents for which promising preclinical activity has not been translated into clinical efficacy include cilengitide (targeting αvβ3 and αvβ5 integrins on angiogenic blood vessels)29, and dasatinib and saracatinib (targeting SRC and BCR–ABL1)4. These late-stage failures in clinical development have resulted in major financial losses, leading to anti-metastatic drug development being deprioritized by the pharmaceutical industry.Several past efforts to target the mechanisms of tumour metastasis have resulted in failure. Matrix metalloproteinase (MMP) inhibitors were developed as anti-metastatic agents on the basis of their capacity to inhibit tissue invasion by tumour cells early in the metastatic cascade and their cytostatic activity in preclinical models30. Denosumab is a humanized monoclonal antibody targeting RANKL that has been shown in preclinical experiments to bind with its target in the bones of healthy transgenic mice expressing chimeric mouse–human RANKL31. In initial clinical trials in the metastatic setting, traditional tumour growth inhibition or survival end points were not used as outcome measures with this cytostatic agent; instead, a reduction in skeletal-related events (SREs) was the primary end point33. Deleterious SREs, such as a bone fracture from expansion of an existing metastasis or a new metastasis, were essentially direct readouts of the extent of metastatic burden. Accordingly, patients with breast or prostate cancers were enrolled because they are prone to developing bone metastases. Significant reductions in the incidence of SREs in patients treated with denosumab compared with those who received standard care were observed for both types of cancer35. In men with bone-metastatic castration-resistant prostate cancer (CRPC), the median time to first on-study SRE with denosumab was 20.7 months versus 17.1 months with the bisphosphonate zoledronic acid 34. In women with advanced-stage breast cancer, denosumab was superior to zoledronic acid in delaying the time to first on-study SRE  and also the time to first and subsequent on-study SREs 35. Denosumab was then compared with placebo in adjuvant trials and delayed initial bone metastasis in patients with CRPC by a median of 4.2 months36. However, in placebo-controlled trials involving patients with breast cancer, adjuvant denosumab was associated with only a minor37 or no38 reduction in the DFS or overall survival (OS). The reasons for this disparity between SREs and OS outcomes are not entirely clear but might reflect a shift in bone metabolism that reduces the incidence of SREs without direct effects on cancer progression.Conversely, successes have been achieved in the treatment of bone metastases, following extensive analyses in preclinical models, either with antibodies targeting receptor activator of NF-κB ligand  or with a class of drugs known as bisphosphonates. Both of these therapies interrupt the ‘vicious cycle’ of bone metastasis. The bone metastatic vicious cycle is a specific example of tumour cell–microenvironment interactions that are likely to occur at other metastatic sites . In bone, tumour cells produce factors that activate osteoblasts to produce RANKL, which activates osteoclasts that subsequently degrade bone, releasing a host of growth factors that stimulate metastatic colonization by tumour cells39. In addition, bisphosphonates have broader anticancer activity against metastatic lesions in visceral organs, possibly through inhibition of angiogenesis or through inhibition of M2-like macrophages in other tissues40. A meta-analysis of clinical trials using bisphosphonates, supported by earlier preclinical data, has demonstrated reduced bone metastases and prolonged OS, at least in postmenopausal women with early stage breast cancer41. No benefit of bisphosphonate treatment was observed for premenopausal women41.The value of bisphosphonates in reducing bone resorption and bone metastasis through direct targeting of osteoclasts has been demonstrated in many preclinical studies43, targeted manipulation of genes to alter metastasis in preclinical models46, functional genomic screens49 and drug repurposing efforts50. In addition to functional preclinical experiments, evidence that the target is associated with metastasis in the human disease is required. For the development of a targeted therapy, such as a small molecule, a peptide or an antibody, knowledge of the biological activity of the target is essential for supporting development of a functional biomarker. Targets with strong correlations with metastasis but not passing the functional tests might serve as biomarkers of a response.Many targets with known or proposed roles in metastasis could be candidates for drug development Table . These p52. For numerous cancer types, the primary lesion can be well controlled by standard therapies  — the challenge is to control the onset and growth of secondary lesions.Effective candidate identification relies on preclinical models that accurately recapitulate the disease pathogenesis in patients and, specifically, the particular process being targeted . Many commonly used preclinical tumour models are better suited for testing agents that have a direct antitumour effect, often on primary tumours, rather than clinically relevant effects on metastasis52. If a molecularly targeted therapy is to be tested, an ideal preclinical model is one in which the target molecule promotes one or more steps in the metastatic process within that preclinical model. For example, inducible expression of the transcription factor Twist-related protein 1 in mice can drive epithelial-to-mesenchymal transition and result in increased metastasis of squamous cell carcinoma53. Other examples include prevention of metastases through targeting of SRC expression in orthotopic mouse models of human pancreatic adenocarcinoma54 or dual pharmacological inhibition of MET and VEGFR2 (ref.55) in various tumour models and tumour cell lines, or transgenic mice in which mammary tumour development and metastasis are driven by the expression of HER2 (ref.56) or E545K-mutant PIK3CA57. Examining drug efficacy in models with complex genetics, including in both metastatic driver and passenger pathways, can also be informative by reflecting the genomic complexity of patient tumours.No single preclinical model exists that wholly reflects metastasis in patients with cancer. Several different preclinical models of the type of cancer under study should be used when testing the anti-metastatic activity of a new drug to account for the diversity of the disease in patients58. As well as influencing tumour growth directly, these changes alter the capacity of the immune system to recognize and attack tumour cells59. These factors must be taken into account when selecting preclinical models in order to ensure that interventions are effective for the tumour type and/or organ of metastatic involvement58. For example, in a mouse model of prostate cancer, androgen ablation (by castration) was demonstrated to result in bone loss and enhanced the growth of disseminated tumour cells (DTCs) in the bone; however, detrimental exacerbation of bone metastasis could be overcome by administering bisphosphonates together with androgen ablation60.The fact that host tissues dictate the extent to which a tumour can metastasize is becoming increasingly evident. The tumour microenvironment is complex, varies extensively in different organs and is influenced by tumour–host cell interactions, physical and metabolic changes, and secreted cytokines, chemokines and growth factors61. Mammals other than mice, such as rats62, are used occasionally, as are non-mammalian hosts such as zebrafish63, Drosophila64 and the chick embryo chorioallantoic membrane65.No in vitro test adequately models the entire metastatic process; therefore, in vivo modelling is essential. Generally, metastasis models consist of either mouse tumours in a syngeneic host, thereby allowing for full engagement of the immune system, or human tumours engrafted into immunosuppressed hostsKrasG12D/+; LSL-Tp53R172H/+; Pdx1-Cre KPC GEMM used for preclinical studies of both pancreatic cancer prevention and therapy66). When murine transplantable tumour models are used, cells can be introduced, preferably at orthotopic sites, to initiate primary tumour growth and subsequent spontaneous metastasis. A major advantage of such mouse metastasis models is the presence of matched stromal tissues that can recapitulate growth factor signalling and anticancer immune responses. Given the obvious and critical role of the host immune system in regulating metastasis67, models of murine tumours in immunocompetent syngeneic hosts should be included in the preclinical testing of any new therapeutic whenever possible.Genetically engineered mouse models (GEMMs) use mice bearing oncogenes that initiate a primary tumour , which in some cases progress to metastatic disease . The advent of even more severely immunocompromised mice, such as non-obese diabetic–severe combined immunodeficient (NOD–SCID) and NOD–SCID–IL-2-receptor γ-chain-mutant (NSG) mice, has enabled a higher proportion of tumours to be established in such models, with more circulating tumour cells (CTCs) and metastases. However, in patients, tumours develop despite the presence of a competent immune system by evolving mechanisms to escape immune detection and/or destruction; therefore, various methods of incorporating a human immune system into these severely immunocompromised mice are the subject of active research61. CTCs isolated from 10 ml of blood have also been used to generate patient-derived CTC xenograft models (CDXs) and have several advantages over PDX models generated using tumour biopsy samples. These advantages include the development of tumours with a molecular profile generally similar to that of the primary tumour and single CDXs that respond to chemotherapy in the same way as the donor patient’s tumour; the ability to generate models for patients with tumours that are not amenable to biopsy sampling or surgery; and the use of a population of tumour cells that has already gained an invasive behaviour, reflecting intra-patient heterogeneity, and that can be used as a surrogate to study metastasis (reviewed in ref.71). Nevertheless, CDXs do have drawbacks, such as lack of a functional immune system, and can be challenging to establish for some tumour types.Metastasis assays can also involve established cancer cell lines or tissues recovered directly from patients undergoing tumour biopsy sampling or resection. As opposed to a cell line that has drifted genomically during long-term culturing, patient-derived xenografts (PDXs) have the important advantage of more closely reflecting the genomic profile of the original tumour. PDXs are also reported to metastasize to the same organs as metastases in the donor patient73 Given the increasing importance of immunotherapy, and the immune contributions to responses to standard therapies, use of syngeneic, immune-proficient mice is advisable. Indeed, multiple aspects of the microenvironment are contributors to the metastatic cascade and ideally should be considered in preclinical models.Site of injectionSubcutaneous models should be avoided. For models of spontaneous metastasis, orthotopic injection of tumour cells is mandatory.Site of metastasisThe sites of metastasis in the mouse model should reflect the characteristic sites of dissemination associated with the human disease being studied.Prior chemotherapy or radiation therapy, or concurrent therapyAny new therapeutic must be given in the context of approved standard treatments. The model can be used to assess whether the new therapy should replace the standard treatment, be combined with it or be used sequentially.Likely combination therapiesThe functional redundancy of the metastatic process ultimately mandates that drug combinations be developed.Oral or intravenous dosingWhen long-term drug administration is required, as in most metastasis prevention studies, oral dosing is imperative unless agents with long half-lives, such as monoclonal antibodies, are under development. Other routes of administration should be considered only if they are used clinically for the cancer type under study.PharmacokineticsCollaborate with a pharmacologist, collect serum samples and generate pharmacokinetic data.Other end pointsWhenever possible, use end points in animal studies that closely reflect clinically relevant end points, such as disease-free survival, quality of life and overall survival, rather than tumour growth curves alone.45 as primary end points, with survival as a secondary end point. Less often, other potentially important end points, such as rates of cell proliferation and apoptosis, microenvironmental alterations or immune infiltration are reported. This approach enables investigation of novel therapies but also the development of drug resistance that is associated with metastasis. Chemoresistance is often considered to be caused by gene mutations that negate the cytotoxic effects of therapy. Surprisingly, diverse metastasis pathways have been functionally implicated in chemoresistance in mouse models , enabling kinetic studies of their development. Standard metastasis models use imaging, histological counts and/or a quantitative measure of metastatic burden87. Likewise, in patients with breast cancer participating in the AZURE trial (zoledronic acid in combination with standard adjuvant therapy), a significant improvement in DFS  and OS  was obtained only in women who had been postmenopausal for >5 years at the commencement of the trial88.Another consideration when developing anti-metastatic therapies is whether to target tumour cells directly or indirectly by modifying the tumour microenvironment to be more suppressive to tumour growth. A common argument for targeting host cells is their greater genomic stability, which might make the development of drug resistance mechanisms less likely. However, the tumour microenvironment is complex and differs between each organ in which metastases have been established. Although not always adequate to predict metastatic events, preclinical models can help to understand the different host microenvironments and can be used to test therapies focused on site-specific metastasis. The unique microenvironment of bone provides the best example, relating to the aforementioned application of bisphosphonates and denosumab. In preclinical models of breast cancer bone metastasis, responses to bisphosphonates were found to be dependent on menopausal status: ovariectomized mice  had a greater tumour burden in bone than control mice  but responded well to the therapy, whereas tumour burden in control mice was unaffected94.Development of pharmacodynamic markers indicative of drug activity must also be incorporated into preclinical testing. In clinical trials, traditional end points of radiological tumour responses and survival are increasingly inadequate. Thus, knowledge of whether the drug hits its intended target and how the tumour cells and/or tumour microenvironment respond is of growing importance. When designing experiments in animals, adequate consideration should be given to analyses that are achievable in patients — that is, liquid and/or tumour biopsy-based assessments and imaging. Circulating cell-free tumour DNA (ctDNA), tumour-derived exosomes or CTCs are increasingly common sources of pharmacodynamic markers that can be tested in mice and patients95. Any correlations must be confirmed at the protein level in tumour samples from a large number of patients, typically using tissue microarrays. For example, in preclinical models of spontaneous mammary tumour metastasis to bone, restoration of interferon regulatory factor 7 (IRF7) expression in the tumour cells did not alter primary tumour growth but did inhibit bone metastasis44. In a retrospective analysis of data from 855 samples from primary breast cancers, high expression of an IRF7 pathway gene signature was associated with reduced bone metastasis of breast cancer  but had no prognostic value in predicting metastasis to visceral organs44. In tissue array analyses, IRF7 protein was detected in 56% of primary tumours compared with 17% of distant metastases and in only 11% of bone metastases44. Prospective analyses can provide very strong evidence for potential therapeutic targets or biomarkers, but in the absence of the relevant cohorts for prospective analysis, appropriately designed prospective retrospective analyses can also generate good levels of evidence96.Evidence of the activity of a prospective target using preclinical models is necessary but is not sufficient: assessment of relevance using clinical samples is imperative. Clinical evidence can be obtained retrospectively by analysing publicly available databases for the transcript of interest and assessing its prognostic and/or predictive value in a specified cohort of patients with cancer that has appropriate follow-up data. Such databases could also be used to identify potential predictive biomarkers for later study in clinical trials. Supplementing analyses with additional information, such as data on inactivation or mutations in genes specifically associated with metastasis, could also help build evidence for a particular target. If adopted, this approach must be used with caution, given that genetic alterations recorded in large databases might provide less robust targets for anti-metastatic agents than for other anticancer therapies because many metastasis pathways involve alterations in gene expression rather than mutationsAssociations with non-coding RNA species can be assessed similarly through in situ hybridization in tissue samples. Metastatic biopsy samples are rarer than primary tumour specimens but are important for analyses of biomarker expression in the development of anti-metastatic therapies. Data must be interpreted with consideration of the patient’s prior treatments (or lack thereof) in terms of consistency with the proposed clinical setting in which the therapy will be developed.The desired pharmacological profile used to guide drug screening necessitates a shift from that typically accepted in an acute tumour shrinkage model to one of chronic, ideally oral administration — with the exception of a monoclonal antibody, for which monthly infusions might be considered feasible — with an appropriate risk–benefit profile. Optimization of structure–activity relationships can be used to select lead drug candidates with a pharmacokinetic profile appropriate for chronic use. For example, drugs that have good oral absorption characteristics and effective distribution to target tissues and are poor substrates for metabolic (cytochrome P450) enzymes in the liver  are suited to simple once daily oral administration over months or years.99. For the example of brain metastasis, drug properties that increase blood–brain barrier permeability and reduce the potential for membrane efflux transporter elimination might be a priority. Toxicological structure alerts (toxicophores) need to be avoided in the development of any drug that is to be dosed chronically, particularly in elderly or frail patients who are likely to present with comorbidities, including reduced renal and liver function, or in patients who have previously been treated with multiple lines of anticancer therapy and are therefore likely to have a poor performance status. Avoidance of drugs that have high risk of hERG voltage-gated potassium channel blockade, which is associated with potentially fatal cardiotoxicity, is a special case in point100. Other pharmacological features associated with on-target and off-target toxicities also need to be avoided, particularly the generation of reactive metabolites that can cause carcinogenicity and nonspecific cytotoxicity.In the cancer setting, specific characteristics of the target disease, such as the frequency of metastasis to the brain, must also be consideredDesigning clinical trials and identifying end points that reflect the prevention of metastatic disease and can meet regulatory authority expectations for evidence of clinically significant benefit are arguably the most critical barrier to the development of new anti-metastatic agents. Measuring a clinically meaningful outcome in a realistic time frame, without requiring the recruitment of overwhelming patient numbers, is a key aim. The paucity of putative anti-metastatic agents being tested in clinical trials bears testament to these barriers.102 and adjuvant tamoxifen for breast cancer103. This scenario has led to a drug development pathway in which therapies likely to be effective in eliminating micrometastases are progressed to the adjuvant setting only after they first demonstrate antitumour effects in patients with advanced-stage disease. Such an approach also assumes that the biology of the metastases is similar to that of the primary tumour, which is not universally true. This drug development paradigm is further reinforced by the need to manage the very large risks associated with trials in the adjuvant setting  by first demonstrating activity and acceptable toxicity in trials involving smaller numbers of patients with more rapidly attainable end points. In other words, demonstrated efficacy in patients with pre-existing metastases has traditionally been the obligate gateway to adjuvant trials of interventions for metastasis prevention.Chemotherapies, hormone therapies, molecularly targeted agents, immunotherapies and various combinations thereof have produced responses and prolonged progression-free survival (PFS) and OS in the metastatic setting but are insufficient to achieve cure in most patients. Some of these same treatments have prevented or delayed the development of overt metastatic disease in some  patients when administered to those without detectable metastases but in whom micrometastases are suspected. Examples include the use of combination chemotherapy in the perioperative setting for bladder cancerThe ability to delay or prevent metastases has the potential to enormously improve the survival durations of patients with cancer and could even lead to cures. However, the opportunity presented by anti-metastatic drugs cannot be explored adequately using conventional drug development pathways because drugs without cytotoxic or clinically meaningful cytostatic effects in the patients with overt metastatic disease will never advance to adjuvant trials. This point is central to the rethinking of clinical trial designs for metastasis prevention, with the clinical use of potential anti-metastatic drugs falling into three possible scenarios.104, biochemical techniques (such as rising serum PSA levels in patients with prostate cancer105) or molecular assays for tumour-derived DNA . However, the predictive power of some of these tests is debatable, with questions remaining regarding their sensitivity and specificity107. Prospective randomized clinical studies in specific disease settings are required to examine the utility of these detection methods and thereby validate their use in guiding treatment decisions. Patients with micrometastasis can be identified across many, if not most, types of cancer, providing important new opportunities for anti-metastatic therapy. Occult micrometastatic disease might either be actively growing or dormant and how this difference might affect the choice and timing of therapy remains unclear. The detection of rising PSA levels in patients with no other evidence of disease after prior treatment of prostate cancer can be used as a platform for metastasis prevention (NCT03119857), although many trials simply use PSA metrics as a primary end point of treatment efficacy.A practical definition of micrometastatic disease is that which is suspected to be present at the time of treatment of the primary tumour but is not evident using conventional imaging and clinical examination. Micrometastases can sometimes be detected by other methods, such as in bone marrow aspirates using flow cytometry108. Several clinical trials  are now testing the combination of local radiotherapy with immunotherapy to exacerbate the damage to overt metastatic lesions outside the radiation field110. If this hypothesis is proven, then the abscopal effect could potentially be harnessed for the management of micrometastatic disease to effect cures in future trials.In general, the aim of cancer surgery is to remove the primary tumour in order to obtain optimal local disease control and prevent the subsequent development of metastatic disease. Often, however, tumour resection is not feasible — despite the absence of metastatic disease — because of the anatomical location of the tumour or the likely inability of the individual patient to tolerate the proposed surgery . In practice, management of these patients presents a substantial clinical challenge. The use of drugs to prevent metastatic spread in addition to therapy to achieve local disease control  is a potential approach to overcoming this challenge. This strategy could also be an alternative to tumour resection for patients in whom the morbidity and mortality associated with surgery would be better avoided. Another option for such patients arises from the emerging realization of the abscopal effects of radiotherapy — that local radiotherapy can lead to immune-mediated destruction of tumours outside the irradiated field111, and those with breast ductal carcinoma in situ can undergo mastectomy112. Even for some pre-cancerous conditions, the established organ-wide risk of pre-invasive disease  can warrant preventive surgery  or regular surveillance with a view to major surgery (such as Barrett oesophagus). In some cases, complete removal of all at-risk tissue is not possible . Indeed, existing treatments are already used to reduce the risk of progression in patients with pre-invasive neoplasia  immunotherapy for those with urothelial carcinoma in situ) or a known risk of developing cancer . However, these approaches often have a disappointing effect on survival outcomes114, and the agents themselves might not target the pathways that most potently drive the invasive phenotype. Furthermore, continuous long-term administration of potentially toxic agents can be required to maintain the chemopreventive effect, which might not be feasible; for example, chemoprevention with retinoids in patients with pre-malignant head and neck tumours has been limited by poor tolerability 115. Consequently, the opportunity to develop rationally targeted, more efficacious and better tolerated systemic therapies in such patient populations is attractive. In this scenario, anti-invasion therapies might be the most useful anti-metastatic approach.Metastasis is rare in the absence of an invasive primary tumour; thus, a strong clinical rationale exists for eliminating cancer in its pre-invasive state to prevent disseminated disease. Patients with discrete pre-invasive lesions might undergo major local therapy; for example, patients with non-invasive tumours of the bladder often undergo cystectomyA dominant problem in each of the preceding scenarios is that intervention is likely to be required years in advance of the clinically important event (predominantly metastatic relapse or death). This latency presents substantial challenges for the design of statistically powered clinical trials that meet the regulatory standards for evidence. The main implications are both economic and clinical. Economically, the costs of trials with large cohorts and long follow-up durations are prohibitive, and the predicted return on investment is restricted by the potentially limited time remaining on a patent. Indeed, many current adjuvant therapies have come into routine use only after or near the time of patent expiry. For this reason, such studies are rarely industry funded. Clinically, the imperative is to make more rapid progress than this scenario allows.The end points traditionally used in oncology clinical trials present an additional challenge. End points for determining antitumour efficacy in patients with advanced-stage disease continue to be based on conventional outcome measures including the objective response rate (ORR) based on reductions in tumour dimensions on cross-sectional imaging of established metastases, PFS and/or OS. Adjuvant trials typically have a primary end point of DFS. These indicators of efficacy either present a specific challenge to the timely development of anti-metastatic therapy or are not applicable in the absence of lesions that are detectable on imaging. Biomarkers that enable the detection of disease progression earlier than is possible through imaging of new metastatic lesions are clearly desirable; however, the use of such markers might require prospective validation, compounding the time and financial requirements.With these challenges in mind, several ways forward are discernible. Owing to the plethora of potential drug candidates and the high costs of late-phase clinical studies, early clinical development must be maximized as follows. Anti-metastatic agents are unlikely to be substantially different from any other candidate anticancer drug in this regard, but early go versus no-go decisions will need to be based on robust mechanistic and other pharmacodynamic biomarkers rather than on the typical evidence of objective tumour responses in patients with advanced-stage disease. Given the essential favourable safety profile of these anti-metastatic drugs, efficient early phase clinical studies could feasibly be conducted in healthy volunteers; thus, the identification of biomarkers that are evaluable in non-malignant tissues will be of crucial importance.113 and the use of a reduction in the numbers of polyps in the development of celecoxib for chemoprevention in patients with familial polyposis coli116. Early approval, on the basis on surrogate end points, will then need to be followed by confirmation of the clinical benefit in registry-type post-marketing studies.The appropriateness of the novel end points will need to be supported by community-derived data providing compelling evidence that regulatory authority requirements will be met. By their nature, the novel end points are likely to be context-specific. Nevertheless, examples are available of accepted surrogate end points that have been used successfully in clinical development of drugs targeting pre-invasive pathways. These examples include the use of progression from early stage to stage T2 disease in establishing the role of intravesical BCG for the treatment of high-risk non-muscle-invasive bladder cancer117. However, this end point was controversial118 because the use of conventional imaging (with isotope bone scans and CT) to establish the metastasis-free state will certainly result in failures to identify metastases that might be detectable using novel imaging modalities, such as whole-body MRI119 or 68Ga-prostate-specific membrane antigen-PET120. The use of these novel imaging techniques is unlikely to have changed the outcome of the trial ; however, if patients had disease staging using these alternative, more sensitive imaging modalities, the control intervention  would have been inappropriate for the subset of patients with upstaging to metastatic disease, and thus the true therapeutic value of apalutamide might have been overstated. Hence, completion of dedicated studies to identify the true incidence of metastatic disease in particular patient populations might be necessary before such end points can be used to explore drugs with the specific aim of preventing metastasis.In February 2018, the FDA also accepted the surrogate end point of metastasis-free survival in the first registration of apalutamide for the treatment of non-metastatic CRPC121. Similar end points are being explored in patients randomly assigned to a metastasis preventive strategy or control treatment after surgery for liver-limited metastatic colorectal cancer . Novel liquid biopsy assays of circulating tumour components, including ctDNA and CTCs, could also offer important opportunities for the development of biomarkers as new surrogate end points or intermediate markers to reduce the risk and accelerate the progress of anti-metastatic drug development (see the ‘Regulatory and registration pathways’ section)126.Secondary prevention studies of metastasis are ongoing. In one such study (NCT03190967), patients with brain-metastatic breast cancer are being randomly assigned to receive metastasis prevention with the antibody–drug conjugate trastuzumab emtansine alone or in combination with metronomic temozolomide, with the primary objective of extending survival without new brain metastasis128; however, few studies have been completed prospectively, have incorporated independent validation cohorts or have used methodologies that can be accurately repeated and certified in diagnostic laboratories. The magnitude of metastatic risk is also an important consideration. Whether clinicians will feel comfortable designing a trial in a population with a 30% or 50% risk of metastasis over a defined period is debatable. The discovery of novel biomarkers that can be used to estimate the risk of micrometastases is a research area that has not been adequately addressed to date, although the use of ctDNA-based approaches to molecularly define minimal residual disease (MRD) might provide opportunities in this regard. Subgroups of patients with an especially high risk of early distant metastases are clearly identifiable. Examples include those with limited-stage small-cell lung carcinoma129, locally advanced pancreatic ductal adenocarcinoma130 or colorectal cancer and resectable liver metastases131. These disease settings are attractive both commercially and clinically, although reliance on such populations for early go versus no-go decisions holds the risk that effective anti-metastatic drugs will be prematurely discarded. Specifically, these high-risk populations might not display any degree of micrometastatic dormancy. Furthermore, these patients do not present an opportunity to explore drugs targeting pre-invasive targets.Selecting populations at particularly high risk of having an early clinical event at a somewhat predictable rate for inclusion in first proof-of-concept trials — similar to the enrolment of patients at high risk of metastatic recurrence in the aforementioned studies of secondary metastasis prevention strategies — is a key approach to accelerating the development of anti-metastatic drugs. A wealth of literature is available describing prognostic factors that can help to identify such patientsAnother way to improve the efficiency of late-stage drug development is to include only patients with disease in which the aberrant pathway being targeted is known to be active. This approach has other clear advantages to patients in that it avoids exposure to the toxicities of such drugs in those who are unlikely to benefit and enables their prioritization to receive other treatments. Indeed, this concept of precision medicine is rapidly becoming mainstream in all areas of anticancer drug development. To enable application of these principles to the development of anti-metastatic drugs, a full understanding of the link between the therapeutic target and the relevant subpopulation is vital, given that metastases are likely to have more complex and different drivers than those of the primary tumour. Heterogeneity between individual metastases adds an additional layer of complexity. Moreover, clinically applicable diagnostics should be developed in parallel with the drugs themselves. For some patients, however, time is of the essence in deciding on the next therapy; thus, a requirement to schedule biopsy sampling and evaluate the specimen in an accredited laboratory might present difficulties, particularly in the secondary metastatic setting, in which the oncologist and patient want to decide quickly on the next line of therapy . Conversely, in the postoperative adjuvant setting, this time delay might not be clinically relevant while the patient recovers from the primary surgical management. Similarly, when the aim is to evaluate the novel agent in a maintenance setting, the patient first needs to complete conventional postoperative systemic anticancer therapy.Drugs that specifically target the processes of metastasis have the potential to transform the care of the majority of patients with solid tumours, although such agents are unlikely to be used as their sole therapy. One can envisage that treatment of the primary tumour and standard-of-care adjuvant therapy will remain the initial interventions, followed by potentially lifelong maintenance therapy to prevent the growth of pre-existing (micro)metastases and/or to prevent further spread of metastases. Thus, one challenging but necessary aim of clinical trials will be to establish the optimal combination and sequencing of these therapies with other modalities including surgery, radiotherapy, chemotherapy, molecularly targeted therapy and/or immunotherapy. The need to address the long-term toxicities of these drugs given the likely chronic duration of therapy is also essential.Many promising mechanistic biological targets exist for metastasis prevention strategies Table ; howevern = 37) were based on ORRs, with the remaining 4 approvals based on PFS using assessment criteria defined by indication-specific working groups132. The RECIST alone were used in the majority of studies that led to accelerated approvals. The RECIST and working group-derived criteria are standardized, well-defined and provide a rapid readout of objective tumour responses or disease progression5, whereas assessment criteria appropriate for anti-metastatic treatments are not standardized or clearly defined. These limitations are particularly problematic because the existing criteria designed for assessing objective responses or progression of solid tumours are unlikely to be of any use in assessing anti-metastatic agents that might have little effect on primary tumours; additionally, in most cases, the patient will not have any detectable disease at the time of treatment with such drugs.A primary disincentive for anti-metastatic agents is the de facto use of time-to-event end points, such as PFS or OS, to assess efficacy in early stage clinical research. These end points present a far greater logistical and financial hurdle than the short-term ORR end points used to assess almost all newly registered oncology agents. In the period of 2006–2016, the FDA approved 180 oncology drugs , including 41 accelerated approvals: most accelerated approvals (P = 0.028) in 1,432 men with CRPC at high risk of developing bone metastases, the FDA denied an application for expansion of denosumab use to an anti-metastatic indication in this disease133. The regulator cited that it was “unclear whether an improvement in bone-metastasis-free survival alone in patients with CRPC at high risk of bone metastases is an adequate measure of clinical benefit in support of new labelling claims for a new patient population” (ODAC Briefing Document BLA 125320/28 Denosumab (XGEVA), 2012, FDA)133. This example underscores the risk involved in developing anti-metastatic agents and highlights the need to complete robust validation of putative new end points concurrently with the clinical development of such agents135.In the absence of regulatory precedent or standardization, individual product developers will be required to explore new surrogate end points without certainty that regulators will accept their clinical relevance. Fortune rarely favours the brave in drug development. Even with a significant increase in biologically relevant bone-metastasis-free survival durations -registered duplex ctDNA blood test for colorectal cancer recurrence has been available in the USA since 2016 for surveillance of patients after tumour resection140.Plasma ctDNA profiling has demonstrated promise in detecting MRD and discriminating patients with and without eventual clinical recurrence following surgery and/or adjuvant therapy, notably in breast cancerThe lack of fixed criteria for drug developers is an unacceptable disincentive, the effects being sadly evident in the lack of any new chemical entity initially registered for the prevention of metastasis. We urge regulatory agencies to work with researchers, drug developers and statisticians to identify and define guidelines for surrogate end points in order to encourage development of this under-represented category of oncology drugs.Given that some anti-metastasis agents might be used in a preventive setting, the safety profile of such an Investigational Medicinal Product (IMP) will have to be much cleaner than those of standard anticancer agents. Proceeding to clinical trials even in the absence of adequate preclinical efficacy models might be acceptable, although a strong rationale will be needed to support long-term administration of the IMP in a healthier population of patients with cancer. The acceptable safety profile will be different for IMPs aimed at suppressing and/or eradicating established metastatic lesions. The benefit–risk assessment should include a detailed discussion of standard treatments, the temporal relationship with standard therapy , proposed safety monitoring and risk-minimization strategies; the potential for drug–drug interactions and risks associated with possible delaying of standard therapy should be addressed.141). Companion diagnostics can be developed in parallel to the IMP and used for exploratory purposes. Once validated, the diagnostic can be used to dictate patient eligibility for trial participation and/or treatment assignment. In the USA, the FDA has provided guidance on validation of biomarkers for use in clinical development142, and in the European Union (EU), the European Medicines Agency has released guidance for biomarker analysis of clinical trial samples143. For marketing any test in the EU, Conformité Européene (CE) marking is required, indicating that the product meets EU safety, health or environmental requirements and compliance with EU legislation.Development of new biomarkers as surrogate end points should be guided by correlations with previously validated end points and/or clinically relevant parameters  must have the same role as in the human setting. Multiple models should be used.Experimental conditions should be designed to reflect the standard of care in the clinical setting and the desired route of treatment administration.Careful consideration should be given to the involvement of the immune system and role of chemoresistance within the models used.Drug discovery and preclinical developmentAnti-metastatic drugs are likely to be given via the oral route repeatedly over a prolonged period of time (perhaps years), necessitating a shift from the risk–benefit profile of traditional anticancer agents towards one with a considerably lower level of risk; the absorption, distribution, metabolism and excretion (ADME) profile of the drug should reflect this necessity.Drugs that have liver metabolism liabilities and/or common drug–drug interactions, undergo enzymatic hydrolysis or are substrates of membrane efflux transporters should be avoided.The pharmacokinetic parameters of the drug must be optimized to enable high levels of target exposure, including a high dissolution rate and good solubility and permeability.Lead clinical drug candidates should be tested in several different and, ideally, genomically complex models to account for diversity of the disease in patients.Pharmacodynamic markers need to be developed that reflect drug activity and can be translated to the clinical setting.Toxicology readouts should generally be cleaner than that often accepted for anticancer agents; for example, the drug should not result in blockade of hERG voltage-gated potassium channels nor generate reactive metabolites that can lead to downstream carcinogenicity or nonspecific toxicity.Clinical developmentGiven the potential ethical issues surrounding the testing of experimental agents with an anti-metastatic mechanism, specifically relating to the very limited possibility of clinical benefit in patients with advanced-stage cancer, studies involving healthy volunteers should be considered as a first step in clinical testing.Early clinical development  should be focused on demonstrating proof of biological concept using validated pharmacodynamic markers relevant to the target and drug being tested; if healthy volunteers are enrolled in clinical trials, these markers must be measurable in non-malignant tissues and/or in blood.Any companion diagnostics that might have to be developed in parallel to the experimental therapeutic and Conformité Européene (CE) marked (indicating a product that meets European Union (EU) safety, health or environmental requirements and complies with EU legislation) can be used to guide treatment and/or trial enrolment decisions.Depending on the mechanism of action of the agent being tested, window of opportunity studies could also be considered during early phase clinical development to enable direct assessment of the biological mechanism in established primary or metastatic tumours.Alternate surrogate measures of clinical benefit, beyond traditional radiological criteria based on tumour shrinkage, are needed and will be dependent on the disease and setting being investigated; potential examples include end points based on time to appearance of new lesions and/or secondary lesions or levels of circulating tumour cells or cell-free tumour DNA.Initial proof-of-concept clinical studies can be conducted in selected patient populations at high risk of an early clinical event, such as a new metastasis. Thus, before and during clinical development, expert advice from oncologists with experience working with such patient groups should be sought. Moreover, the aberrant pathway under assessment should be confirmed as being active in this population.In light of the points above, clinical development timelines longer than those associated with traditional anticancer drugs should be anticipated; early approval based on surrogate outcomes should be a key goal, when possible.Regulatory pathwaysEnsure that a strong rationale is provided in regulatory submissions to support the long-term administration of the anti-metastatic agent in a population of healthier patients with cancer.Given the current absence of regulatory precedent or standardization, exploration of new surrogate end points should be discussed with the appropriate regulator before embarking on and during clinical drug development.If approval is given on the basis of short-term surrogate end points, a requirement to complete ‘sales revenue-funded’ phase IV confirmatory studies with larger cohorts, longer follow-up durations and more traditional end points, such as overall survival, should be anticipated.We urge regulatory agencies to work together with researchers, drug developers and statisticians to define guidelines on surrogate end points to encourage development of this complex but high-potential category of oncology drugs.Supplementary Box S1


Human male reproductive disorders are common and may have a fetal origin — the testicular dysgenesis syndrome (TDS) hypothesis. In rats, experimentally induced TDS disorders result from disruption of fetal androgen production/action specifically in the masculinization programming window (MPW). MPW androgen action also programs longer anogenital distance (AGD) in male versus female rats; shorter male AGD is correlated with risk and severity of induced TDS disorders. AGD thus provides a lifelong, calibrated readout of MPW androgen exposure and predicts likelihood of reproductive dysfunction. Pregnant rat exposure to environmental chemicals, notably certain phthalates , pesticides or paracetamol, can reduce fetal testis testosterone and AGD and induce TDS disorders, provided exposure includes the MPW. In humans, AGD is longer in males than females and the presumptive MPW is 8–14 weeks’ gestation. Some, but not all, epidemiological studies of maternal DEHP (or pesticides) exposure reported shorter AGD in sons, but this occurred at DEHP exposure levels several thousand-fold lower than are effective in rats. In fetal human testis culture/xenografts, DEHP/DBP do not reduce testosterone production, whereas therapeutic paracetamol exposure does. In humans, androgen production in the MPW is controlled differently (human chorionic gonadotrophin-driven) than in rats (paracrine controlled), and other organs  contribute to MPW androgens, essential for normal masculinization, via the ‘backdoor pathway’. Consequently, early placental dysfunction, which is affected by maternal lifestyle and diet, and maternal painkiller use, may be more important than environmental chemical exposures in the origin of TDS in humans. Reproductive disorders in newborn boys  and in young men  are remarkably common, are inter-related, are probably increasing in incidence ,2 and poA radical change in thinking occurred with the discovery in 2008 in laboratory rats of the so-called MPW . This dein utero by ultrasound [As in rodents, human males have a longer AGD than females at birth/during childhood and adulthood  Figure . This setrasound . Thus, itrasound ,10  rather than anogenital distance (AGD) is the currently preferred measurement postnatally  by the fetal testis, as anything that perturbs this function during the MPW is likely to reduce AGD and ultimate reproductive organ size/function ,10,40,41in utero [Various pesticides or fungicides  can also inhibit fetal testis testosterone production dose-dependently in rats ,9 as canin utero .The findings described in rats above have prompted numerous epidemiological studies in humans, examining whether exposure to any of the ‘anti-androgenic’ chemicals during pregnancy is associated with altered AGD or occurrence of hypospadias or cryptorchidism at birth in sons. Results from these epidemiological studies are quite mixed and are too complex and numerous to discuss in detail here . in utero exposure in rats are generally far higher than the levels of exposure that occur in the epidemiological studies [A particular issue with the association studies involving exposure to phthalates or pesticides, but not painkillers, is that the doses of individual compounds that induce effects via  studies . For exa studies , and in  studies ,68,69, a studies . Even hi studies ,72. Diff studies , but thiin vitro culture or as fully functioning ex vivo xenografts in immune compromised mice, there is no suppression of testosterone production, whereas there is in the rat under equivalent conditions [in utero might be compromised by DEHP and DBP exposure. However, this also means accepting that the human fetal testis is hugely more sensitive to these chemicals than is the rat, which is the opposite of the direct evidence [in vivo evidence do not support this hypothesis. First, some women with inflammatory bowel disease (IBD) have been exposed to 50–100 times more DBP than the normal population because the pharmaceutical  which they took to control their IBD, including throughout pregnancy, had an enteric coating containing DBP in some cases [in utero exposure of pregnant marmoset monkeys to 500 mg/kg/day monobutyl phthalate (the active metabolite of DBP) during the period of the presumptive MPW did not induce any reproductive disorders in the resulting male offspring as it would in rats [in vitro or in utero [An alternative way to reconcile the dose disparity for rat-human findings is to assume that the human fetal testis is several thousand-fold more sensitive than the rat to DEHP and DBP in terms of their ability to suppress testosterone production. However, when fetal human testes are exposed to DEHP (or DBP) at doses equivalent to 500 mg/kg/day in rats, either in nditions . As thesevidence ,77. Moreme cases . Studiesme cases . Second, in rats . Finallyin utero ,77.Perhaps the biggest concern in extrapolating rat (or mouse) fetal testis testosterone effects to the human is that the regulation (and importance) of testosterone production by the fetal testis is fundamentally different in the rat and human . RemarkaAn important consequence of the LH receptor-dependence of testosterone production by the human fetal testis during the presumptive MPW is that this also means there is parallel dependence on normal hCG exposure , becauseDirect information about the fetal masculinization process in the 1st trimester in human males is obviously limited and most insight has come from cases of masculinization disorders. Such disorders have been associated with specific gene mutations, most of which impinge either on the sexual differentiation process  or on teIt has long been accepted that normal masculinization of the fetus is absolutely dependent on testosterone produced by the fetal testis — and now we know that this probably has to occur during a specific period, the MPW. This understanding has derived primarily from experimental studies in rodents, because direct information is difficult to obtain for the human. Hopefully this brief review has shown that whilst the rodent experimental evidence appears highly relevant to man, there are fundamental differences between man and rodents that limit its seamless translatability. Chief among these is the basic difference in regulation of testicular steroidogenesis during the presumptive MPW in man (hCG–LH receptor-driven) versus rodents , but other less crystallized differences may be functionally more important. For example, the fact that androgen production in the presumptive MPW in human males is doubly dependent on placental function, via hCG (testis effects) and progesterone (backdoor pathway effects) production, but perhaps also via other presently unknown pathways. These pathways are clearly important as placental dysfunction in early pregnancy is unequivocally associated with greater risk of masculinization disorders  in human males .An important implication of the experimental animal–human differences is that the maternal lifestyle or environmental factors that might negatively impact androgen exposure during the MPW in humans could be fundamentally different from what does this under experimental conditions in rodents. So far, most human  studies have simply followed a similar path to rodent experimental studies, focusing on environmental chemicals such as phthalates and pesticides, when searching for potential causes of impaired androgen exposure during the MPW. However, perhaps this is looking under the wrong lamp post. Arguably, more effort should be directed at identifying what external factors might impair early placental development and function, especially maternal diet and lifestyle which are known to be influential in this regard  Figure , and whiImportance of the field: Approximately 1 in 6 young men has a reproductive disorder, especially a low sperm count, and growing clinical and experimental evidence points to these disorders comprising a TDS that may be linked to deficient androgen exposure during early fetal life. Identification of the fetal MPW, provides a focus for research aimed at identifying modifiable factors in early pregnancy that have potential to disrupt androgen production or action during the MPW leading to TDS.Summary of the current thinking: Sufficient androgen exposure in the MPW is essential for programming normal masculinization and later reproductive tract/organ development, though normal androgen exposure postnatally  is also important to fulfill this programming. Inappropriate exposure to certain environmental chemicals  in pregnancy has been the main focus of animal and human epidemiological research to identify maternal factors that might perturb androgen production in the MPW. However, direct studies of chemical effects on human fetal testis testosterone production raise questionmarks about the importance of these effects in utero.Comment on future directions: Regulation of androgen production in the human fetal male during the presumptive MPW differs fundamentally from that in rodent experimental models, and highlights the importance of normal placental development and function for normal androgen exposure. Future studies need to take more account of this, and of maternal dietary and lifestyle factors that are known to impact early placental development, although further evaluation of exposure to pharmaceuticals (e.g. paracetamol) alone or in combination with environmental chemical mixtures, and their effects on fetal testis function may also prove important.


In contrast with medical imaging diagnostics powered by artificial intelligence (AI), in which deep learning has led to breakthroughs in recent years, patient outcome prediction poses an inherently challenging problem because it focuses on events that have not yet occurred. Interestingly, the performance of machine learning–based patient outcome prediction models has rarely been compared with that of human clinicians in the literature. Human intuition and insight may be sources of underused predictive information that AI will not be able to identify in electronic data. Both human and AI predictions should be investigated together with the aim of achieving a human-AI symbiosis that synergistically and complementarily combines AI with the predictive abilities of clinicians. The central premises are that 1) complex health data contains predictive information that ML can effectively extract and transform into a predictive algorithm and 2) accurate prediction of patient outcomes can facilitate early, preventative intervention and more efficient health care resource allocation through identification of high-risk patients. For example, predicting which intensive care unit patients are likely to develop sepsis can prompt early initiation of fluid resuscitation, vasopressor therapy, or antibiotics, which can reduce damage from insufficient organ perfusion  and clasmission) . This chInterestingly, while human and AI predictions are often directly compared in medical imaging research -8, patiethey generate on a regular basis. Third, the implicit assumption is that humans cannot accurately predict patient outcomes because analysis of complex, high-dimensional clinical data may be required; moreover, recall bias is rampant in the human mind.There are several possible reasons why human performance is more frequently studied in medical imaging than in patient outcome prediction. First, radiologists are trained to analyze, interpret, and classify images, whereas most other medical specialists are not trained to directly predict patient outcomes. While accurate prognostic information can certainly be helpful in any medical specialty, it is usually generated by empirical risk scoring systems such as the Framingham Risk Score  or AcuteHowever, there is no reason to rule out the possibility that human clinicians can outperform AI in patient outcome prediction, at least in some clinical scenarios. While AI can only access information that can be recorded in the form of electronic data, human clinicians interact face-to-face with their patients and have access to both clinical and contextual information. The qualitative information collected via clinicians’ five senses can be critical in patient outcome prediction; however, this information is mostly absent in EHRs, if it is possible to record it at all. Although some qualitative observations can be recorded in EHRs as free-text notes, such as nursing notes, these data are logged in a limited, inconsistent fashion. Human intuition and insight may well be the most underused resources in patient outcome prediction.While the performance of ML-based patient outcome prediction models appears impressive on paper, the most accurately predicted cases tend to be “easy” cases where the likely outcomes are already obvious to human clinicians . This fuOn the other hand, AI easily outperforms humans in processing, analyzing, and finding patterns in complex, high-dimensional data . As demoThe bottom line is that both AI and humans can make unique contributions to patient outcome prediction, and they should help each other to maximize predictive performance. Patient outcome prediction research should aim for human-AI symbiosis, where the respective predictive abilities of AI and human clinicians are combined in a synergistic and complementary way . Given tAnother way for AI and humans to work together is via the human-in-the-loop model, where humans directly inform machines on how to learn from the data at hand by providing guidance based on human intuition and knowledge. The term “interactive machine learning”  was coinAI and human prediction performance may vary across different types of patients. Complex patterns in data can be more predictive than human intuition in certain patient subgroups, and the opposite may be true in other subpopulations. An investigation of how AI and human predictions can be optimally combined for different types of patients could directly contribute to advancing precision medicine. A better understanding of the respective predictive powers of AI and humans in various clinical scenarios can also help increase human trust in AI . This can facilitate evidence-based adoption of AI technology.why an algorithm arrives at a given conclusion; this requires transparency, traceability, and causality. The active field of explainable AI has been producing useful methods, such as SHapley Additive exPlanations (SHAP) [For human clinicians to completely trust AI, it is necessary to understand s (SHAP) , that cas (SHAP) . TherefoOne major roadblock to the proposed human-AI symbiosis is the need to collect a large number of human predictions in a variety of clinical scenarios, which is labor-intensive and adds to clinicians’ workloads. Seamlessly integrated electronic prediction collection platforms  can minimize this burden and enable large-scale prediction collection.Once predictive performance is optimized via human-AI symbiosis, the next important step is to formulate clinical guidelines so that the predictive information is actionable. This is a crucial step, as accurate predictions alone will not lead to any real impact; rather, the combination of accurate predictions and appropriate interventions by clinicians will have a greater effect ,26.The ultimate goal of patient outcome prediction is to improve patient outcomes and decrease health care costs through early intervention and efficient use of health care resources. To prove that this goal has been met, we will need to perform randomized clinical trials of AI-driven patient care , such as


Varicella-Zoster virus (VZV) is a human herpesvirus that causes varicella (chickenpox) as a primary infection, and, following a variable period of ganglionic latency in neurons, it reactivates to cause herpes zoster (shingles). An analysis of VZV infection in cultures of neural cells, in particular when these have been obtained from induced pluripotent stem cells (iPSCs) or neural stem cells consisting of highly purified neuronal cultures, has revealed much data that may be of neurobiological significance. Early studies of VZV infection of mature cultured neural cells were mainly descriptive, but more recent studies in homogeneous neural stem cell cultures have used both neuronal cell markers and advanced molecular technology. Two general findings from such studies have been that (a) VZV infection of neurons is less severe, based on several criteria, than that observed in human fibroblasts, and (b) VZV infection of neurons does not lead to apoptosis in these cells in contrast to apoptosis observed in fibroblastic cells. Insights gained from such studies in human neural stem cells suggest that a less severe initial lytic infection in neurons, which are resistant to apoptosis, is likely to facilitate a pathological pathway to a latent state of the virus in human ganglia. The virus then remains in a latent state primarily in neuronal cells in several types of peripheral ganglia throughout the nervous system including the dorsal root ganglia (DRG), cranial nerve ganglia, particularly the trigeminal ganglia (TG), and also various autonomic ganglia including, as recently recognised, those located in the enteric nervous system ,2,3. Fol herpete ,6, but a herpete  and incl herpete  but this herpete . A detaiVZV has a double-stranded DNA genome the size of which is almost 125,000 base pairs and contains 68 open reading frames (ORF), three of which are duplicated . The virIn this overview we will consider some studies of acute and persistent VZV infection of human neural cells, focussing mainly on in vitro data. After considering some previous reports of viral infection of mature human and animal neural cells, we will then discuss some previous reports of VZV infection of neural stem cells. In practice these latter studies have mainly been in cell cultures obtained by differentiating induced human pluripotent stem cells (iPSCs), or embryonic stem cells into neurons, and these will be the main focus of this review.Early studies of VZV infection of cultured human neural cells relied primarily on morphological methods of cell identification  as they A later study of heterogeneous populations of both central and peripheral neural cell cultures obtained from human fetal nervous tissue did used specific cell markers for both neurons and glial cells in culture. Thus Assouline et al.  used cel+ (positive) astrocytes and 20% GFAP− (negative) fibroblastic cells. Using two-fluorochrome immunofluorescence, it was shown that both a cpe and viral antigen expression (VZV immediate–early protein 62 and gpI proteins) were detected in the cells a week after infection. However, it was found that astrocyte GFAP expression was both altered and diminished as the VZV infection progressed indicating a downregulation of GFAP expression by the virus itself. Detailed investigation showed that this downregulation was mediated by early rather than late events in the viral replication cycle and did not appear to be due to virally induced shut-off of host cell protein synthesis. These results were also observed using the multipassaged VZV Ellen strain, and the findings were thought to possibly represent a form of biological uncertainty phenomenon in that VZV itself was capable of modifying the result of the actual method of identifying the cell type that is was infecting. This finding of a VZV-mediated downregulation of GFAP was recently confirmed in an in vivo study of human VZV encephalitis [The human astrocyte was also the main focus of a later study in which the cell free VZV OKA strain was used to infect dissociated cell cultures of human fetal brain . The culphalitis .A study also indicated that VZV may have unpredictable effects in a very different neural cell culture system which is felt to be relevant to the current discussion . The aimThere are at least four reasons why knowledge of the infection of neural stem cells may have potential importance in neurobiology and human neurological diseases. These are (a) to gain a better understanding of the biological basis of the VZV-neuron interaction using the various in vitro model systems, (b) they provide a mechanism of obtaining nearly pure terminally differentiated neuronal cultures which can be investigated for their susceptibility to viral infection, (c) they may give potential clues as to how VZV may interact with the developing fetal nervous system with clear relevance to congenital birth defects, and (d) they could provide a system in which to investigate the potential in vitro efficacy of therapeutic anti-viral agents. The discussion will include both VZV infection of embryonic neural stem cells and induced pluripotent stem cellsThe intrinsic problems in studying VZV latency mentioned above inspired the search for new model systems, including neuronal stem cells, induced pluripotent stem cells (iPSCs) and neuronal organoid-like structures to make it possible to work with a human pathogenic virus like VZV in a relevant neuronal context in vitro. However, the number of existing neuronal stem cell derived and IPSC-derived neuronal models to study the virological and molecular aspects of VZV infection, replication and latency is relatively sparse, and these complex model systems remain challenging and often imperfect for mimicking true neuronal VZV infection and latency in humans.To provide an additional way to study the VZV-host relationship in neurons, Pugazhenti and co-workers  developeClearly, there are major differences between VZV infection of neuronal cells compared to other cell types, such as fibroblasts, which is also reflected in the differences between these tissues during the natural history of human infection with VZV, this being closely related to the ability of VZV to be neurotropic and establish neuronal latency. Several research groups independently have found that VZV-infected neurons remain viable for over 2 weeks, whereas fibroblasts tend to show cpe much earlier.Although it has been demonstrated that neurons derived from pluripotent human embryonic stem cells (hESC) can be infected by VZV , it was One important unresolved question in the field and also complicating in vitro studies of VZV infection is related to VZV permissiveness in neurons and the unusual high level of viral cell association compared to most other herpesviruses. Using hESCderived neurons infected with cell-free wild-type VZV, it was demonstrated that neurons are highly permissive for VZV infection, and around 100 times more permissive compared to early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts . SpecifiSome of the problems associated with working with terminally differentiated human neurons in vitro, i.e., their slow replication, relative short life span, and heterogeneity was addressed in a study by Goodwin et al. who described an experimental model of normal human neural progenitor cells in tissue-like assemblies or small three- dimensional organoids . These oAn important aspect that complicates and hampers studies and interpretation of VZV infection of in vitro-derived neurons is that these cultures are heterogenous containing cell types that may have different susceptibilities to VZV infection, establishment and maintenance of latency, and viral reactivation . TherefoThere have been several published studies of acute VZV infection of induced pluripotent stem cells (iPSCs). These cells are derived from fibroblasts which have been experimentally dedifferentiated in vitro to mimic embryonic stem cells which are subsequently induced to differentiate into neuronal cells . These sLee et al.  devised Subsequent studies have analysed the VZV-neuronal cell interaction in detail. Grose et al.  generateAnother study carried out around the same time was that of Yu et al.  which inTwo further studies, both published in the same year, were reported from the Colorado research group which provided more insights into the growth of VZV in induced pluripotent stem cells containing almost homogenous (>95%) populations of neuronal cells. Thus, Baird et al.  studied A recent study  also useThere are several key issues in the VZV-neural cell interaction which have been discussed here that remain to be better understood. First, why are non-neuronal cells such as glial cells more susceptible to acute lytic VZV infection than neurons? A clue may be found in the recent work described by Li Puma et al.  where asOverall, many important aspects related to VZV tropism, replication, and latency in mature neurons, IPSC-derived neurons, and neural stem cells remain incompletely understood. Current and future studies should focus on addressing these questions using physiologically relevant models, such as freshly isolated post-mortem human sensory ganglia, together with investigations and comparisons of viral infection and cellular fate in neural progenitor cells, IPSC-derived neurons, astrocytes, and glia cells. Moreover, implementing experimental systems with neuronal three-dimensional cell assemblies or organoids should allow studies resembling the pathophysiological processes and immune responses occurring in human ganglia. Finally, genetic engineering by CRISPR/Cas9 technologies to modify or delete specific host genes involved in virus-cell interactions as well as using viral mutants should provide further insights into host-pathogen interactions in the human nervous system. Collectively, such approaches should provide deeper insights into the cellular and viral mechanisms governing VZV infection and establishment of latency in neurons with important parallels and relevance to VZV neuroinfection and disease in humans.


Exercise is recommended for the healthy population as it increases fitness and prevents diseases. Moreover, exercise is also applied as an adjunct therapy for patients with various chronic diseases including cancer. Childhood cancer is a rare, heterogeneous disease that differs from adult cancer. Improved therapeutic strategies have increased childhood cancer survival rates to above 80% in developed countries. Although this is higher than the average adult cancer survival rate of about 50%, therapy results often in substantial long-term side effects in childhood cancer survivors. Exercise in adult cancer patients has many beneficial effects and may slow down tumor progression and improve survival in some cancer types, suggesting that exercise may influence cancer cell behavior. In contrast to adults, there is not much data on general effects of exercise in children. Whilst it seems possible that exercise might delay cancer progression or improve survival in children as well, there is no reliable data yet to support this hypothesis. Depending on the type of cancer, animal studies of adult cancer types show that the exercise-induced increase of the catecholamines epinephrine and norepinephrine, have suppressive as well as promoting effects on cancer cells. The diverse effects of exercise in adult cancer patients require investigating whether these results can be achieved in children with cancer. Thanks Physical activity and structured exercise programs not only help to prevent many chronic diseases, but exercise can also be an effective additive treatment strategy . This miWhilst the effects of exercise on adult cancer patients are increasingly characterized , far lesExercise not only improves fitness and prevents disease but is also an effective treatment strategy for many chronic diseases . In theiGeneral Effects: Exercise has widely been shown to influence quality of life as well as physical health. Examples for general effects of exercise training are an increase of cardiorespiratory fitness, increased muscular mass or a decreased cancer-induced fatigue  is still understudied and robust evidence regarding specific and health-beneficial exercise programs is lacking . In adulMuch of the early research in exercise oncology has focused on the general effects of exercise in cancer patients. In contrast, recent basic research studies aim to identify molecular and cellular mechanisms by which physical activity or exercise training might directly change cancer cell behavior. These studies all investigate adult cancer types.Observational studies in animal models suggest that exercise has direct effects on cancer cells. Generally, the exercising muscles and other organs will alter the molecular and cellular composition of blood and thereby potentially influence tumor cells. Additionally, effects of exercise may be directly mediated by the nervous system, a part of the tumor microenvironment gaining increasing attention in cancer research –48. A reIn humans, exercise not only increases the concentration of many metabolites e.g., lactate , proteinThe molecular mechanisms by which exercise can affect adult cancer cell behavior have been discussed extensively in several recent reviews. Direct cancer effects in adult patients include influence on proliferation, signal transduction, cancer metabolism, inflammation, and cancer-immune system interactions , 22, 23.The exercise-induced stress hormones epinephrine  and norepinephrine  not only regulate many acute adaptations to exercise but can also affect cancer cell signaling and behavior. The stimulation of catecholamines by exercise was first described in the early 1950s . GeneralIn addition to humans, Pedersen et al. demonstrated that voluntary running of mice before but not after injection of B16, melanoma cells reduced the growth and volume of the developing tumors. Moreover, there were more natural killer cells in the xenotransplant tumors of the mice that had exercised. Further experiments demonstrated that this was dependent on catecholamines as receptor blockade with the unselective β-blocker propranolol prevented this effect . In a fuKRAS-mutant mice thereby significantly increasing incidence of pancreatic adenocarcinomas. Moreover, they also reported observational data that pancreatic adenocarcinoma patients treated with unselective beta-blockers for other reasons survived significantly longer than patients that did not receive this specific medication  already originate during early embryonal development , 73. TheHW and IL developed the concept of the review and were the main writers of the manuscript. SK and PW contributed equally to the concept, collected and analyzed data, and contributed to the writing. MS and BR contributed to the concept and revised the manuscript. All authors read and approved the final manuscript.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.


Acute liver failure is a rare syndrome with significant morbidity and mortality, particularly in absence of transplantation as a rescue therapy. An important mechanism contributing to mortality is hyperammonemia which drives cerebral edema and raised intracranial pressure. Multiple therapies for managing hyperammonemia have been trialed. Continuous renal replacement therapy is effective in treating hyperammonemia in other disease states (notably inborn errors of metabolism). Its efficacy in acute liver failure has been suggested but further investigation is required to prove this. The objective of this systematic review will be to determine the efficacy of continuous renal replacement therapy in patients with acute liver failure and its effect on mortality and transplant-free survival.MEDLINE, EMBASE, Web of Science, and Cochrane Database will be searched. Identified studies will include all patients with acute liver failure in a critical care unit treated with continuous renal replacement therapy. Primary outcome will be effectiveness of ammonia clearance and mortality. Patients treated with any other modality of ammonia lowering therapy (such as plasma exchange or Molecular Adsorbent Recirculating System) will be excluded. Narrative synthesis of the identified studies will occur and if clinical homogeneity is identified, data will be pooled for meta-analysis using a DerSimonian-Laird random effects model.We present a protocol for a systematic review seeking to establish a link between transplant-free survival in acute liver failure and the use of continuous renal replacement therapy. Given the anticipated paucity of literature on this subject, both narrative and quantitative syntheses are planned.CRD42019122520, registered April 16, 2019.(PROSPERO)  Acute liver failure (ALF) is defined by the American Association for the Study of Liver Disease (AASLD) as a new coagulopathy  and any degree of encephalopathy in a patient with new onset liver failure < 26 weeks) [6 weeks . Its defMortality and morbidity in acute liver failure are driven by multiple mechanisms, including vasodilator shock and multi-organ failure. An important complication of ALF is hepatic encephalopathy which is with associated intracranial hypertension . In contMultiple strategies have been trialed to decrease serum ammonia and improve cerebral edema including the use of hypertonic saline , 20, tarThe use of CRRT for clearance of hyperammonemia and improvement in cerebral edema is best described in the pediatric literature where it is used for management of inborn errors of metabolism , 24. HowCRRT also plays an important role in management of acute kidney injury (AKI). According to one study, this can occur in up to 70% of patients with ALF, and 30% requires renal replacement therapy, increasing both morbidity and mortality . Thus, iThe primary objective of this systematic review will be to determine the efficacy of CRRT in patients with acute liver failure and its effect on mortality and transplant-free survival. Our hypothesis is that the use of CRRT in patients with acute liver failure may lead to improved cerebral edema and improved transplant-free survival. The main research question being address is “In patients with acute liver failure, does the use of continuous renal replacement therapy improve transplant-free survival compared to usual care?” This will help guide future research inquiries including observational and experimental trials.The present protocol has been registered within the PROSPERO database (registration ID: CRD42019122520). The present study protocol is being reported in accordance with the reporting guidance provided in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) statement   of interest, and study designs. Given the anticipated paucity of high quality randomized controlled trials, all study designs, including randomized trials, cohort studies, and case control studies, will be included. Case reports and series will not be including given the general lack of a comparator arm in such studies. Studies from all languages will be considered, so long as English translation is available. Data presented from conference abstracts will also be included as well as that published in journals. The inclusion criteria for this study are adult patients (over 18 years of age) admitted to an intensive care unit with a diagnosis of acute liver failure. Any etiology of acute liver failure will be considered. The intervention sought is the use of continuous renal replacement therapy while the comparator is usual care without renal replacement therapy. Acute liver failure will be defined in accordance with the AASLD guidelines as liver disease of less than 26 weeks’ duration and the presence of coagulopathy  as well as any degree of encephalopathy [Case studies and case series will be excluded, as will any existing reviews, meta-analyses, commentaries, or opinion pieces. Exclusion criterion for this review is the presence of chronic liver failure more than 26 weeks’ duration prior to ICU admission. Studies will also be excluded if another form of ammonia lowering therapy  was used in addition to CRRT.The studied intervention is the use of continuous renal replacement therapy, employed at any point during critical illness. All forms of veno-venous renal replacement therapy (including hemofiltration and hemodiafiltration) will be considered. The comparator is usual care which includes all drugs and supportive care used in the management of acute liver failure. In keeping with exclusion criteria above, other forms of ammonia lowering therapy (such as MARS and plasma exchange) are not considered to be usual care.For the acute liver failure group, the primary outcome of interest is transplant-free survival and mortality at 21 days. Secondary outcomes are reduction in serum ammonia levels over 72 h following admission or initiation of CCRT, persistence of multi-organ failure, degree of metabolic encephalopathy (using West Haven grade over 72 h), hospital or ICU length of stay, and use of life sustaining therapies.Selected studies will be assessed by two independent authors using the Newcastle Ottawa Scale for observational studies (likely the majority of identified studies) . Any incIdentified studies will be reviewed by two independent authors. Where differences exist, consensus will be sought through discussion. First, titles and abstracts will be screened for eligibility criteria. Once primary studies are screened, included studies will proceed to full-text review and will be assessed based on criteria outlined above. Authors of selected studies will be contacted if necessary to provided missing and relevant data. A flow chart showing studies included or excluded at each stage of the process will be provided.Data will be extracted using a standardized form generated a priori by the authors. Extracted data will include wherever available a number of variables. With respect to patient characteristics, age, etiology of liver failure, stage of encephalopathy, number of patients study inclusion and exclusion criteria, and geographic location will be recorded. Frequency, dose, and duration of continuous renal replacement therapy will be extracted from the intervention. Any co-interventions used  in either the intervention or comparator group will be recorded. With respect to outcomes, the method and timing of assessment and any adverse reactions will be included. Finally, outcomes related to mortality, transplant-free survival, and ammonia levels pre and post treatment will be extracted. Where reported, number and modality of required life support interventions and ICU and hospital length of stay will be extracted. At a study level, any reported funding sources, the process through which consent was obtained and presence of ethics board approval, will be recorded.Data will first be analyzed qualitatively in the form of summary table and narrative synthesis. This will be used to assess heterogeneity of interventions and outcomes in order to gauge the feasibility of meta-analysis. Heterogeneity will be evaluated using the I-squared statistic. If sufficient clinical homogeneity is identified among the studies, data will be pooled for meta-analysis using a DerSimonian-Laird random effects model. Binary outcomes  will be pooled using risk ratios while continuous outcomes  will be pooled using mean differences. All estimates will be reported with 95% confidence intervals.Clinicaltrials.gov will be searched for evidence of unpublished or abandoned trials, which will be assumed to be negative. If any individual meta-analysis contains more than 8 studies, small study bias (publication) bias will be evaluated using a funnel plot. Finally, outcome measures extracted from these studies will be gauged using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) classification.The primary anticipated source of meta-bias will be a publication bias given the small number of observational trials on this subject. Acute liver failure is a rare form of liver failure with nonetheless important impacts on morbidity and mortality. Although survival can be as high as 75%, this is heavily influenced by the use of liver transplant as a rescue therapy, a procedure associated with both immediate and chronic complications. Transplant-free survival is significant lower, at around 55% , 5. TranPreviously studied investigational therapies, including MARS and hypertonic saline, have not clearly shown a mortality benefit , 20. TheAdditional file 1:. PRISMA-P 2015 ChecklistAdditional file 2:. Sample MEDLINE search strategy