Prion diseases are fatal and incurable neurodegenerative disorders affecting both humans and animals. The development of in vitro cellular models from naturally susceptible species such as ruminants can be very useful for the study of prion disease mechanisms and the discovery of potential therapies. Our study shows for the first time how the culture, in the form of three-dimensional spheroids of ovine mesenchymal stem cells derived from bone marrow in growth and neurogenic conditions, makes these cells more permissive to prion infection, mimicking the prion toxicity occurring in these diseases. This three-dimensional system appears as a potential in vitro model for studying prion diseases in a microenvironment approaching in vivo conditions.Sc) in scrapie-infected oBM-MSCs and spheroids were measured by ELISA. In neurogenic conditions, monolayer cells and spheroids maintained the levels of PrPSc over time. In growth conditions, however, oBM-MSCs showed decreasing levels of PrPSc throughout time, whereas spheroids were able to maintain stable PrPSc levels. The presence of PrPSc in spheroids was also confirmed by immunocytochemistry. Altogether, these results show that a 3D culture microenvironment improves the permissiveness of oBM-MSCs to scrapie infection in growth conditions and maintains the infection ability in neurogenic conditions, making this model of potential use for prion studies.In neurodegenerative diseases, including prion diseases, cellular in vitro models appear as fundamental tools for the study of pathogenic mechanisms and potential therapeutic compounds. Two-dimensional (2D) monolayer cell culture systems are the most used cell-based assays, but these platforms are not able to reproduce the microenvironment of in vivo cells. This limitation can be surpassed using three-dimensional (3D) culture systems such as spheroids that more effectively mimic in vivo cell interactions. Herein, we evaluated the effect of scrapie prion infection in monolayer-cultured ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) and oBM-MSC-derived spheroids in growth and neurogenic conditions, analyzing their cell viability and their ability to maintain prion infection. An MTT assay was performed in oBM-MSCs and spheroids subjected to three conditions: inoculated with brain homogenate from scrapie-infected sheep, inoculated with brain homogenate from healthy sheep, and non-inoculated controls. The 3D conditions improved the cell viability in most cases, although in scrapie-infected spheroids in growth conditions, a decrease in cell viability was observed. The levels of pathological prion protein (PrP Cell cultures have proven to be useful tools for a variety of applications in research. There are currently many established cell lines and primary cultures available and widely used for different purposes. The two-dimensional 2D) platforms in which flat monolayer cells are cultured are most commonly used for research in cell-based assays . These 2D platforTo overcome these drawbacks, three-dimensional (3D) cell culture platforms have emerged as a promising approach. Although the optimal 3D condition requirements vary between cell types, and the characteristic features of cells in 3D cultures differ in accordance with their types, these 3D culture systems have proven to be more realistic for translating cell-based assay findings to in vivo applications due to their ability to closely mimic the behavior of in vivo cells . There aSpheroids are spherical-shaped multicellular aggregates that can be formed from several types of cells, including mesenchymal stem cells (MSCs) . They arLRRK2 G2019S mutation have also been created to successfully model LRRK2-associated sporadic PD [In the field of neurodegenerative diseases, 3D cell culture platforms appear as good models to reproduce different features of neurodegeneration linked to these diseases. In Alzheimer’s disease (AD), 3D hydrogel-embedded human neural stem cells ,15, neurradic PD .C) called PrPSc [Prion diseases, or transmissible spongiform encephalopathies (TSEs), are neurodegenerative disorders caused by a pathological misfolded protein derived from an innocuous cellular prion protein  cultured in growth conditions and subjected to neurogenic differentiation . In this culture . These rPRNP gene. This sheep belonged to a flock from the Center of Encephalopathies and Emerging Transmissible Diseases , maintained for research purposes. After the sedation with xylazine and the local anesthesia with lidocaine, bone marrow aspirate was collected from the humeral head as previously described [A bone marrow sample was obtained from an adult female rasa aragonesa sheep of two years of age who carried the ARQ/ARQ genotype for the escribed . All proMSC isolation from the bone marrow aspirate (3 mL) was performed following the previously described protocol ,37,38. TAll the subsequent experiments were performed using this oBM-MSC culture. The number of technical replicates used in each experiment is shown in The minimal criteria to characterize MSCs are their plastic-adherence capacity in standard culture conditions and their ability to differentiate to mesodermal lineages  in vitro .® Osteogenesis Differentiation Kit, Gibco, Life Technologies, Waltham, MA, USA) and chondrogenic  differentiation. For adipogenic differentiation, basal medium supplemented with 1 μM dexamethasone, 500 μm 3-Isobutyl-1-methylxanthine (IBMX), 200 μM indomethacin, and 15% of rabbit serum was used [Adipogenic, osteogenic, and chondrogenic differentiation was evaluated in vitro. The differentiation into mesodermal lineages was performed using specific commercial differentiation kits for osteogenic  were used. In each well, 45,000 cells were seeded to form a unique spheroid per well. The cells were seeded in droplets, and afterwards, 200 μL of basal medium was added into each well. To ensure a correct formation of the spheroids, the plates were incubated for 5 days at 37 °C with 5% CO2 and differentiated into neuron-like cells using a HyClone AdvanceSTEMTM Neural Differentiation Kit (ThermoFisher Scientific). After 24 h of culture under neurogenic conditions, cells showed a neuron-like morphology, which was monitored by optical microscopy. The pick of neurogenic differentiation was observed at 72 h, as previously described [Ovine BM-MSCs were seeded at 5000 cells/cmescribed . To mainFor the neurogenic differentiation of spheroids, after they were stabilized, the same neural induction kit was used as that for the 2D cultures, following the same guidelines for medium change.To verify the correct neurogenic differentiation, neuron-like cells and spheroids were stained with 1% Cresyl Violet Solution (abcam) to detect Nissl bodies, which are granular structures present in neuronal cell bodies and composed of rough endoplasmic reticula rich in RNA . The staNEFM (neurofilament medium chain), MAP2 (microtubule-associated protein 2) and TUBB3 (tubulin beta 3 class III) [HPRT (hypoxanthine phosphoribosyltransferase) and G6PD (glucose-6-phosphate dehydrogenase) were used as housekeeping genes [The expression of a set of neuronal markers was assessed by real-time quantitative PCR (RT-qPCR) in both neuron-like cells and spheroids. These markers were ass III) . HPRT (hng genes .TM II kit (ThermoFisher Scientific) was used. RNA from each of the two technical replicates was retrieved from two wells of a P6 culture plate.For total RNA extraction from 2D cell cultures and further retrotranscription to cDNA, the Cells-to-cDNATM RNA Miniprep kit . Two technical replicates were analyzed. The quality and quantity of these RNA samples were checked with a NanoDrop spectrophotometer , and a Qubit 4.0 fluorometer . cDNA was then obtained using the qScriptTM cDNA SuperMix (QuantaBio). All procedures were performed following the manufacturer’s instructions.Total RNA was extracted from 42 pooled spheroids using the Direct-zolTM Green Master Mix  in a QuantStudio 3 Real-Time PCR instrument (Applied Biosystems). A dissociation curve was performed after every RT-qPCR reaction to confirm the amplification of a single amplicon. The primer design in different amplicons avoided genomic DNA amplification. The comparative quantification of the results was standardized by the 2−∆∆Ct method [HPRT and G6PD housekeeping genes as a normalizer.Gene expression was quantified by RT-qPCR using the Fast SYBRt method , using tSc in the CNS tissues was confirmed with two rapid diagnostic tests  as previously descried [Two different inocula were created using central nervous system (CNS) samples: one from a healthy sheep (negative control) and one from a classical scrapie-infected sheep, both carrying the ARQ/ARQ genotype. The inocula were preserved at the tissue bank of the CEETE, University of Zaragoza. The presence/absence of PrPdescried . CNS homdescried .2, and each spheroid was generated from 45,000 oBM-MSCs. For each condition, three groups were established: positive (infected with scrapie inoculum), negative , and control (without inocula). For infection, basal medium was substituted by inocula diluted 1:10 in DMEM medium  for the 2D oBM-MSCs and spheroids in growth conditions, and in HyClone medium for the 2D oBM-MSCs and spheroids subjected to neurogenic differentiation. Cells and spheroids were maintained in this medium for 48 h to analyze cell viability and prion replication ability. Afterwards, the medium was changed twice a week.The effect of prion infections and their ability to replicate prions were investigated in 2D oBM-MSC cultures and spheroids in growth and neurogenic conditions. Ovine BM-MSCs were seeded at 5000 cells/cmSc of 2D oBM-MSCs and spheroids inoculated with scrapie-infected brain homogenate in growth and neurogenic conditions, the amount of pathogenic protein was quantified by ELISA at 2, 5, and 8 dpi using the EEB-Scrapie HerdCheck kit (IDEXX) and following the manufacturer’s recommendations. A previous study conducted by our group showed that this kit was suitable for the sensitive detection of PrPSc in oBM-MSC cultures [2 in 6-well plates, and the retrieval of the cells was performed by means of trypsinization and subsequent centrifugation to obtain 3 technical replicates per condition and time (dpi). Spheroids formed from 45,000 cells were also collected, and 3 spheroids/technical replicates were analyzed per condition and time (dpi).To test the ability to replicate PrPcultures . oBM-MSCSc in spheroids was also confirmed by immunocytochemistry at 5 dpi. Spheroids in growth and neurogenic conditions were fixed in 4% paraformaldehyde and stained with 0.2% eosin. Each spheroid was then included in a 1% agarose matrix; subsequently, the matrices containing the spheroids were embedded in paraffin. Afterwards, paraffin-embedded spheroids were cut to obtain 3-micrometer-thick slices. Hematoxylin–eosin staining was performed to evaluate the integrity of the spheroids after their inclusion in paraffin.The presence of PrPFor immunocytochemistry, after deparaffination and rehydration, spheroid sections were digested with 4 µg/mL of proteinase K for 15 min at 37 °C, then subjected to antigen retrieval with citrate buffer (pH 6.0) for 10 min at 96 °C in a PTLink (Dako). Afterwards, endogenous peroxidase activity was blocked using a precast solution . Sections were then incubated for 30 min at room temperature with the primary antibodies L42  and F89 . Omission of the primary antibody served as a background control for nonspecific binding of the secondary antibody. Subsequently, the sections were incubated with an anti-mouse enzyme-conjugated EnVision polymer  for 30 min at room temperature. Diaminobenzidine  was used as the chromogen. The spheroid sections were assessed and photographed using a Zeiss Axioskop 40 optical microscope  and AxioVision Rel.4.7 software.2, and 8 technical replicates were analyzed for each condition and time (dpi). The MTT assay was carried out as previously described [An MTT assay was performed to evaluate cell viability and early prion toxicity in oBM-MSCs and neuron-like cells at 2, 5, and 8 days post-inoculation (dpi). Inoculum removal was performed at 2 dpi considering the day of infection with the inocula as day 0. Cell viability in oBM-MSCs and neuron-like cells was studied in positive, negative, and control cultures at 2, 5, and 8 dpi, coinciding with the day of inoculum removal, 2 dpi. Cells were seeded in 96-well plates at 5000 cells/cmescribed .The viability after prion infection was also analyzed in spheroids in both growth and neurogenic conditions by MTT assay at 2, 5, and 8 dpi in 3 conditions . Spheroids were formed from 45,000 cells per spheroid in 96-well Nuclon Sphera low-attachment plates (ThermoFisher Scientific), and 4 technical replicates were analyzed for each condition and each time point (dpi). An MTT assay was performed using the same reagents as in oBM-MSCs and neuron-like cells, but the incubation times were different: spheroids were incubated at 37 °C for 24 h with MTT solution, and for 4 h at room temperature while protected from light with HCl solution .t-test, defining p < 0.05 as a statistically significant difference.In both cases, differences in cell viability were assessed with Student’s To characterize oBM-MSCs, their ability to differentiate into adipocytes, chondrocytes, and osteocytes was assessed.Cells under adipogenic conditions presented red cytoplasmatic lipid droplets a,b. In cAs shown in NEFM, MAP2, and TUBB3.The differentiation of oBM-MSCs into neuron-like cells was assessed by Nissl bodies staining in cells subjected to neurogenic differentiation, as well as by analyzing the expression of three neuronal markers: TUBB3 and MAP2 displayed higher expression levels in neuron-like cells in comparison to the cells in growth conditions. NEFM, conversely, showed lower expression levels in cells subjected to neurogenic differentiation (The presence of Nissl bodies in the soma of neuron-like cells was confirmed using Cresyl Violet staining . Regardintiation . OverallNEFM, TUBB3, and MAP2 neuronal markers.The evaluation of neurogenic differentiation in spheroids was carried out in the same way as in oBM-MSCs: Cresyl violet staining was performed along with the expression analysis of Together with the presence of Nissl bodies, spheroids under neurogenic differentiation conditions displayed a kind of prolongation on its contour that could be associated with neuronal morphology . MoreoveSc signal was measured after the infection with scrapie brain homogenate in oBM-MSCs and neuron-like cells at 2, 5, and 8 days post-inoculation. As shown in Sc in oBM-MSCs cultured in 2D decreased over time, whereas in neuron-like cells, the levels were stably maintained throughout the study period, with a slight decrease at 8 dpi.The PrPSc detected by ELISA were also analyzed in scrapie-infected spheroids in growth and neurogenic conditions. Unlike 2D oBM-MSCs, spheroids in growth conditions showed stable levels of PrPSc over time (Sc signal was found at 5 dpi, but later, t 8 dpi, PrPSc levels were recovered a (Sc in scrapie-infected spheroids was also confirmed by immunocytochemistry (The levels of PrPver time a. In sphovered a b. The prhemistry .The effect of prion infection on cell viability was assessed at three infection times. oBM-MSCs infected with scrapie-positive inoculum showed increased viability compared to the cells infected with negative inoculum and the non-inoculated controls at 2 dpi, whereas at 5 dpi, a significant decrease in viability was observed in scrapie- and negative-infected cells compared to the controls. At 8 dpi, the levels of viability in the cells infected with positive and negative inocula were recovered, and were similar to the non-inoculated controls a.In neuron-like cells, an initial increase in cell viability was also observed in scrapie-infected cells along with a significant decrease at 5 dpi. Conversely to oBM-MSCs, this decrease was maintained at 8 dpi b.Cell viability was also assessed in spheroids in both growth and neurogenic differentiation conditions.In growth conditions, spheroids infected with the negative inoculum displayed increasing cell viability over time. The ones infected with positive inoculum, however, showed an increased cell viability at 5 dpi, but afterwards, at 8 dpi, the viability decreased greatly a.In neurogenic conditions, spheroids infected with the negative inoculum also showed high levels of cell viability at the three infection times. Contrary to what was observed in spheroids in growth conditions, spheroids infected with scrapie-positive inocula displayed increments in cell viability over time b.Sc and possible prion toxicity that could affect cell viability.Three-dimensional in vitro culture systems have arisen as a novel approach for cell cultures. These systems are able to more effectively mirror the microenvironment and interactions of the cells in vivo, making them useful tools for creating in vitro disease models that reliably reproduce disease-inherent characteristics. Our group previously evaluated the effect of scrapie prion infection in oBM-MSCs cultured in 2D in basal and neurogenic conditions . In ordeTUBB3, NEFM, and MAP2, which would explain the slight increase in the expression of some neurogenic markers between basal and neurogenic conditions. This fact implies that the expression analysis of neuronal markers should be complemented with other techniques to evaluate the neural differentiation of MSCs, such as morphological changes and specific stains [MAP2 was much lower in differentiated spheroids than in 2D cultures; variations in the expression levels of MAP2 and TUBB3 between 2D and 3D cultures have also been described in the neural induction of neural progenitor cells obtained from human iPSC [Mesenchymal stem cells were the first to be characterized. Their plastic adhesion ability and the tri-lineage differentiation confirmed the nature of these cells ,39. The c stains ,46, as wman iPSC .Sc levels in 2D cultures of oBM-MSCs infected with scrapie throughout the duration of the culture, whereas neuron-like cells displayed steady PrPSc levels over time. These results confirm that oBM-MSCs cultured in two-dimensional conditions are less permissive to scrapie infection compared to neuron-like cells, which seem to be able to uptake and maintain scrapie infection over time.Murine bone marrow-derived MSCs can be infected and propagate the Fukuoka-1 human prion strain ,49, and Sc over time. On the other hand, like neuron-like cells, spheroids in neurogenic conditions also took up and propagated PrPSc levels. These results indicate that the 3D microenvironment makes oBM-MSCs more permissive to prion infection when cultured in growth conditions, and maintains the ability of neuron-like cells to absorb and propagate scrapie prions.Regarding 3D in vitro models for prion diseases, only human cerebral organoids have been described to uptake and propagate sCJD prions . Within Sc could be different, which would affect the reaction of MSC to infection. Nevertheless, the early response to scrapie infection was the same in both studies, an increment of cell viability, which means a higher proliferation potential either in growth or neurogenic conditions when cultured on plates after being in contact with scrapie tissues. The decrease in cell viability observed afterwards could be a consequence of prion toxicity.Although primary neuronal cultures show toxicity after prion propagation in vitro that varies in a strain- and neuronal type-specific manner ,56, muriOvine MSC spheroids inoculated with brain homogenates of healthy and scrapie-infected sheep displayed different viability patterns than the monolayer-cultured cells. The negative inoculum increased the viability of spheroids in both growth and neurogenic conditions; however, this increase was not observed in spheroids infected with scrapie, which remained at viability levels similar to the controls. Compared to monolayer-cultured MSCs, human MSC spheroids show higher cell survival and enhanced cell yield and stemness ,63,64. TSc profile generated in both oBM-MSCs and spheroids in growth and neurogenic conditions.In conclusion, oBM-MSC-derived spheroids in growth and neurogenic conditions seem to be able to uptake and propagate scrapie prions and mimic prion toxicity, making this three-dimensional approach a potential in vitro model to study prion disease mechanisms and therapeutics in a more in vivo-like environment. Nevertheless, further studies using a larger number of cultures are still necessary to confirm the reproducibility of this in vitro model, along with the analysis of the PrP


Continuous androgen deprivation therapy ± first-generation non-steroidal antiandrogen was previously the standard-of-care for patients with metastatic castration-sensitive prostate cancer (mCSPC). Treatment intensification with novel hormonal therapy (NHT) or taxane chemotherapy is now approved and guideline-recommended for these patients.Physician-reported data on adult patients with mCSPC from the Adelphi Prostate Cancer Disease Specific Programme were analyzed descriptively. We evaluated real-world treatment trends for patients with mCSPC in 5 European countries  and the United States (US), looking at differences between patients initiating treatment in 2016-2018 and in 2019-2020. We also investigated treatment trends by ethnicity and insurance status in the US.This study found that most patients with mCSPC do not receive treatment intensification. However, greater use of treatment intensification with NHT and taxane chemotherapy was observed in 2019-2020 than in 2016-2018 across 5 European countries. In the US, greater use of treatment intensification with NHT in 2019-2020 than in 2016-2018 was observed for all ethnicity groups and those with Medicare and commercial insurance status.As the number of patients with mCSPC who receive treatment intensification increases, more patients who progress to metastatic castration-resistant prostate cancer (mCRPC) will have been exposed to intensified treatments. Treatment options for patients with mCSPC and mCRPC overlap, suggesting that an unmet need will emerge for new therapies. Further studies are needed to understand optimal treatment sequencing in mCSPC and mCRPC. This study evaluated real-world metastatic castration-sensitive prostate cancer (mCSPC) treatment trends between 2016 to 2020 across a large, diverse patient population. The study also investigated whether treatment trends vary by ethnicity and insurance status. In this real-world study of adult patients with metastatic castration-sensitive prostate cancer (mCSPC), greater use of treatment intensification with novel hormonal therapy was observed in 2019-2020 than in 2016-2018 across 5 European countries and the United States. Across Europe, greater use of taxane chemotherapy was also observed in the later time period. As the number of patients with mCSPC who receive treatment intensification increases, more patients will have been exposed to a greater number of intensified treatments, suggesting that an unmet need will emerge for new therapies in metastatic castration-resistant prostate cancer.1 When therapies such as docetaxel (taxane chemotherapy), abiraterone, apalutamide, or enzalutamide  are added to ADT, this is often referred to as treatment intensification.2Continuous androgen deprivation therapy (ADT) with or without a first-generation nonsteroidal antiandrogen (NSAA) is commonly used to treat patients with metastatic castration-sensitive prostate cancer (mCSPC) and was previously the standard-of-care for these patients.3-6; LATITUDE and STAMPEDE (abiraterone)7-11; TITAN 12-14; and ARCHES and ENZAMET .16 These trials demonstrated improved progression-free survival (PFS) and overall survival (OS) compared with placebo in patients with mCSPC treated with ADT. In addition, NHTs demonstrated similar18 or even improved9 quality-of-life compared with placebo in these patients. Prostate cancer guidelines therefore recommend treatment intensification for patients with mCSPC.19-22Pivotal randomized clinical trials of treatment intensification in patients with mCSPC have been published from 2015 onwards, including CHAARTED and STAMPEDE (docetaxel)23; docetaxel in October 201924; apalutamide in December 201925; and enzalutamide in March 202126 for patients with mCSPC. In the US, the Food and Drug Administration (FDA) approved abiraterone in February 201827; apalutamide in September 201928; and enzalutamide in December 201929 for patients with mCSPC. In the US, use of docetaxel in patients with mCSPC is currently off-label.30 However, off-label prescribing is common in oncology31 and is evident in real-world studies of patients with mCSPC.32-43 Research is also emerging on the use of triplet therapy in mCSPC,45 and this may already be making its way into real-world practice.The timing of different therapy approvals varied between Europe and the United States (US). The European Medicines Agency (EMA) approved abiraterone in October 201747 as well as patient preferences and physician perceptions.48-50 There are claims-based US and Canadian data showing that most patients do not receive treatment intensification for mCSPC32-43; however, it is important to keep in mind that these claims-based studies began before most FDA approvals of NHTs in mCSPC. To our knowledge, no real-world data currently exist on the utilization of mCSPC treatment intensification in Europe.Ultimately, real-world treatment utilization may depend on a number of country-specific and region-specific factors including treatment availability, regulatory approval, reimbursement, and insurance status (particularly in the US),The objective of our study was to evaluate real-world mCSPC treatment trends from 2016 to 2020 across a large, diverse patient population in 5 European countries  and the US. We also investigated whether treatment trends vary by ethnicity and insurance status in the US. Our recent time period allows us to capture treatment trends after the approval of NHTs for use in mCSPC.51-53Data from the Adelphi Prostate Cancer Disease Specific Programme (DSP) were utilized and subsequently analyzed for the purpose of this study. The DSP is a point-in-time, physician-conducted extraction of medical chart data. The surveys are conducted in routine clinical practice and the data collected describe patient demographics and clinical characteristics, prostate cancer disease management including treatment history, the burden and impact of prostate cancer, and associated treatment effects from the perspective of the physician. The DSP methodology has been previously published and validated.Physicians in the UK, France, Germany, Spain, Italy, and the US reported information for their patients with metastatic prostate cancer attending a physician’s appointment between January and August 2020.A geographically diverse sample of physicians was identified by local fieldwork agents using physician panels and publicly available lists. All physicians self-identified as oncologists or urologists . All physicians had personal responsibility for prescribing decisions for patients with prostate cancer and were seeing 2 or more patients with mCSPC and 2 or more patients with mCRPC per month.At the time of data collection, patients included in this study were males aged ≥18 years, currently diagnosed with metastatic prostate cancer, not known to have ever enrolled in a prostate cancer clinical trial, and were receiving treatment for metastatic prostate cancer (any line). The data we analyzed included patients with mCSPC at the time of data collection or who had mCSPC previously and progressed to metastatic castration-resistant prostate cancer (mCRPC). All patients who initiated an mCSPC treatment between 2016 and 2020 were included in the study.For the purpose of this study, we define ADT as medical castration with or without a first-generation NSAA. Medical ADT included ketoconazole, chlormadinone, cyproterone, buserelin, degarelix, goserelin, leuprorelin, triptorelin, and histrelin. First-generation NSAA included bicalutamide, flutamide, and nilutamide.In the real-world setting, treatment intensification may include agents that are not currently approved for use in mCSPC. For the purposes of this study, treatment intensification is therefore defined as any other systemic therapy in addition to ADT, including NHT, taxane chemotherapy, taxane-based combinations, and other regimens such as radiotherapy and immunotherapy, regardless of the treatment’s indication or approval status. NHTs included abiraterone, apalutamide, darolutamide, and enzalutamide. Taxane chemotherapy included docetaxel, cabazitaxel, and paclitaxel.Participating physicians completed an attitudinal survey with questions on physician and practice characteristics. Following this, physicians completed detailed electronic patient record forms (ePRFs) for their next eligible, adult patients treated for mCSPC (4 patients) or mCRPC (4 patients). The number of patients per physician was limited to allow for a varied representation.The ePRFs collected detailed information on patient demographics and clinical characteristics, patient management, and treatment history at the time of data collection. Information was collected on patients with mCSPC at data collection as well as patients with mCRPC at data collection who had historical mCSPC treatment information available. Ethnicity was identified by physicians and was not self-identified by patients.54The Eastern Cooperative Oncology Group Performance Status Scale (ECOG) was used to assess performance status, which scores from 0 (fully active) to 4 (completely disabled).The Adelphi DSP was submitted to and obtained exemption from the Western Institutional Review Board, study protocol number AG8741.55 and as such it did not require ethics committee approval. Each survey was performed in full accordance with relevant legislation at the time of data collection, including the US Health Insurance Portability and Accountability Act 1996,56 and the Health Information Technology for Economic and Clinical Health Act.57 Data were collected in such a way that patients and physicians could not be identified directly.Data collection was undertaken in line with European Pharmaceutical Marketing Research Association guidelinesData were analyzed descriptively using IBM SPSS Data Collection Survey Reporter Version 6 or later . For continuous variables, we reported mean and SD, and/or median and range. For categorical variables, frequency and percentage distribution were reported.Treatments initiated across all lines of mCSPC therapy in each country were reported based on year of treatment initiation and split into 2 time periods: 2016-2018 and 2019-2020. Our analysis looked at the differences between the 2 time periods. While patients appeared in each time period only once, patients could initiate multiple lines of therapy within each time period. This resulted in some answers totaling more than 100% if different options were chosen across treatment lines.Time to treatment intensification was calculated only among patients who received treatment intensification in mCSPC and was measured both from the date of mCSPC diagnosis to the initiation of treatment intensification and from the time of first-line mCSPC treatment initiation to the initiation of treatment intensification.Across all countries, 328 physicians (278 in Europe and 50 in the US) provided data on 1560 patients (1321 in Europe and 239 in the US) with mCSPC  and 2. TAcross the 5 European countries, at the time of mCSPC diagnosis, the median (range) age was 70 (44-88) years. Most patients (83%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 at the time of mCSPC diagnosis. Ninety-three percent of patients were White/Caucasian, 3% were Afro-Caribbean, and 4% were other ethnicities. At the time of data collection, the median (range) age was 72 (45-90) years. Eighty-eight percent of patients had bone metastases and 19% of patients had visceral metastases. Most patients (84%) were treated by an oncologist and 16% were treated by a urologist . Overall, just under half (47%) of patients were treated at a community hospital and just over half (53%) were treated at an academic medical center. In Germany and Italy, most patients  were treated at a community hospital. However, in the UK, France, and Spain, most patients  were treated at an academic medical center .In the US, at the time of mCSPC diagnosis, the median (range) age was 68 (49-89) years. Most patients (88%) had an ECOG performance status of 0 or 1. Sixty-seven percent of US patients were White/Caucasian, 21% were African American, and 13% were other ethnicities . Fifty-one percent of US patients were in the Northeast. At the time of data collection, the median (range) age was 69 (50-90) years. Sixty-nine percent of patients had bone metastases and 30% of patients had visceral metastases. Three-quarters (75%) of patients were treated by a medical oncologist and the remaining quarter (25%) was treated by a urologist. Just over half (52%) of patients were treated at a community hospital and just under half (48%) were treated at an academic medical center.In total, 58% of US patients had Medicare insurance, 33% commercial, 6% Medicaid, and 3% other/no health insurance. A greater percentage of African American patients had Medicare insurance (69%) than White/Caucasian patients or patients of other ethnicity (both 55%), as well as other/no health insurance . A smaller percentage of African American patients had commercial insurance (14%) than White/Caucasian patients or patients of other ethnicity . A smaller percentage of White/Caucasian patients had Medicaid (4%) than African American patients or patients of other ethnicity , greater use of treatment intensification (NHT and taxane chemotherapy) for patients with mCSPC was observed in 2019-2020 than in 2016-2018. Germany saw the largest change in the use of treatment intensification and Italy saw the smallest change. The greatest overall use of treatment intensification in 2019-2020 was seen in Spain, with 29% of patients receiving taxane chemotherapy, 29% receiving abiraterone, and 11% receiving enzalutamide. In the UK, treatment intensification with enzalutamide was seen more than treatment intensification with abiraterone. In France, Germany, Spain, and Italy, treatment intensification with abiraterone was seen more than treatment intensification with enzalutamide. The UK had the highest percentage of patients who received treatment intensification with taxane chemotherapy relative to France, Germany, Spain, and Italy .Median (range) time from mCSPC diagnosis to treatment intensification remained at 0 (0-72) months for 2016-2018 and 2019-2020. Mean (SD) time from mCSPC diagnosis to treatment intensification in 2016-2018 was 4.1 (11.0) months, and in 2019-2020 was 3.1 (8.1) months. Median (range) time from first-line mCSPC treatment initiation to treatment intensification remained at 0 (0-69) months for the 2 time periods. Mean (SD) time from first-line mCSPC treatment initiation to treatment intensification in 2016-2018 was 3.1 (9.5) months, and in 2019-2020 was 0.8 (4.8) months .In the US, the use of treatment intensification with enzalutamide was 14% and with abiraterone was 10% in 2016-2018. In 2019-2020 the use of treatment intensification with abiraterone was 21%, 19% with enzalutamide, 7% with apalutamide, and 2% with darolutamide. Treatment intensification with taxane chemotherapy was 16% in 2016-2018 and 10% in 2019-2020. Treatment intensification with other agents was 19% in 2016-2018 and 5% in 2019-2020 . SimilarMedian (range) time from mCSPC diagnosis to treatment intensification remained at 0  months for 2016-2018 and 2019-2020. Median (range) time from first-line mCSPC treatment initiation to treatment intensification remained at 0  months for 2016-2018 and 2019-2020. Mean (SD) time to treatment intensification following mCSPC diagnosis in 2016-2018 was 3.2 (7.5) months, and in 2019-2020 was 2.5 (6.4) months. Mean (SD) time to treatment intensification following first-line mCSPC treatment initiation in 2016-2018 was 0.4 (2.9) months, and in 2019-2020 was 0.3 (1.5) months .In Europe, treatment intensification with a NHT, taxane chemotherapy, or both was 46% for urologist-treated patients and 45% for oncologist-treated patients. In the US, treatment intensification with a NHT was 68% for oncologist-treated patients and 35% for urologist-treated patients . Disease32-43 this study found that most patients with mCSPC do not receive treatment intensification, despite clinical trial evidence3-18 and guideline support19-22 for these therapies. However, our study showed an overall trend toward increasing treatment intensification and decreasing time to treatment intensification across 5 European countries and the US between 2016-2018 and 2019-2020. Of note, to our knowledge, this is one of the first studies to analyze treatment intensification trends in Europe. This is also one of the first studies to investigate treatment intensification trends in both Europe and the US as a single study.In concordance with other real-world studies,23 docetaxel (2019),24 and apalutamide (2019).25 Enzalutamide was later approved in 2021 for use in patients with mCSPC.26Across 5 European countries, a greater percentage of patients with mCSPC receiving NHT and taxane chemotherapy was seen in 2019-2020 than in 2016-2018. This is reflective of the EMA approval dates of abiraterone (2017),27 apalutamide (2019)28, and enzalutamide (2019) for mCSPC.29 Greater use of abiraterone during 2019-2020 than in 2016-2018 may also be driven by the approval of generic abiraterone in the US during the later time period.58 Other US and Canadian claims-based studies also show similar trends for NHT38-41 despite covering time periods prior to at least some NHT approval dates. Use of treatment intensification regardless of approval is a trend also seen in our study in both Europe and the US for enzalutamide and docetaxel, respectively.In the US, we observed more NHT use, but less use of taxane chemotherapy and other agents, in 2019-2020 than in 2016-2018. These results are not unexpected given the FDA approval dates of abiraterone (2018),59 All institutions in this study implemented some changes in the delivery of treatment, including 45% of institutions that reported switching patients to oral therapies. In the US, we observed less taxane chemotherapy use and more NHT use in 2019-2020 than in 2016-2018, although in Europe, we observed more taxane chemotherapy and NHT use in 2019-2020 than in 2016-2018. One might speculate that, without the pandemic’s influence, more patients would have received taxane chemotherapy relative to oral treatments such as NHT.The period from which our data are drawn overlapped with the COVID-19 pandemic, which likely had an impact on treatment trends. For context, a global survey of 129 healthcare professionals across 17 different countries  found that 60% of institutions cancelled or postponed systemic anti-cancer therapy for oncology patients due to reasons related to the pandemic .61 In contrast, our study of mCSPC patients in the US found that treatment trends appeared largely similar across ethnicities; more treatment intensification with NHT was observed for all ethnicity groups in the later time period than in the earlier time period. Additionally, although use of ADT ± first-generation NSAA , was observed as lower in 2019-2020 than in 2016-2018 for the overall population as well as in African American and other ethnicity patient groups, greater use of this treatment regimen for White/Caucasian patients was observed in 2019-2020 than in 2016-2018. In a US, multi-institutional, retrospective analysis of 107 Black patients with mCSPC (of whom 87% were diagnosed with stage 4 disease in or after 2015), 27% received ADT ± first-generation NSAA and 73% received treatment intensification inclusive of 20% ADT + chemotherapy, 45% ADT + NHT, and 8% ADT + chemotherapy + NHT.62 Our analysis of US African American patients found similar treatment trends. However, our numbers of patients by US ethnicity were small, so the value of any observations of treatment patterns by ethnicity is limited. Future studies with larger sample sizes across ethnicities should further explore differences in treatment trends.There is some real-world evidence on racial disparities in metastatic prostate cancer in the US that suggests treatment underutilization is of greater concern for Black than White patients.Our study also investigated how US insurance status affects real-world treatment trends. We found that, while less use of ADT ± first-generation NSAA  was observed overall and in patients with Medicare in 2019-2020 than in 2016-2018, greater use of this regimen was observed in 2019-2020 than in 2016-2018 for patients with commercial insurance. The numbers of patients with Medicaid and other/no health insurance were too small to assess.When looking at the entire study period, treatment intensification rates in Europe appeared similar across physician specialties. However, we observed more treatment intensification overall in oncologist-treated patients than in urologist-treated patients in the US, with no urologists in this study prescribing taxane chemotherapy for their patients with mCSPC.63 In a US survey of 100 patients with metastatic prostate cancer, 54% agreed with the statement “Avoiding financial trouble due to treatment of my prostate cancer is very important to me.”48 Twenty-one percent of respondents did not agree with the statement “I rely on my doctor to tell me how to treat my prostate cancer.”48 A US claims database analysis of commercially insured patients with mCRPC who received treatment intensification found that patients with a low household income were, paradoxically, more likely to receive a more expensive treatment .63 The authors concluded that qualification for patient assistance programs and thus being shielded from higher out-of-pocket costs may have been a factor in this.It is important to recognize that physicians are not the only decision-makers responsible for treatment utilization. Patient preferences based on aspects of their quality-of-life, treatment accessibility, and medical expenses impact the treatments they receive.Physician selection in the DSP is a potential bias as it is influenced by willingness to take part and may not be representative of the overall population of physicians treating prostate cancer. Selection also excluded US physicians working at sites such as the Veterans Health Administration, and therefore these patients were not included in this study. Another limitation results from the fact that patients with more frequent visits are more likely to be included in the sample than patients with less frequent visits to their physician. These patients may therefore not fully represent the overall population of patients with mCSPC. However, the systematic approach to recruitment intends to reduce selection bias.Although the overall sample size for this study was large, sample sizes per country were small; future studies with larger sample sizes per country should investigate this topic further. Additionally, reimbursement for different medications varies widely by country and will therefore influence the treatment trends of each country studied. Reimbursement in European countries may vary by regions within countries; however, this level of analysis was not possible.Sample size also meant that we were unable to analyze more granular, annual treatment trends; thus, we chose the 2016-2018 and 2019-2020 time periods presented in this study. To be selected for this study, patients with mCSPC during 2016-2018 had to continue medical visits with their physician until 2020, which may have led to differences in baseline characteristics between the 2 time periods. Additionally, sample size is not the same for all patient baseline demographics and disease characteristics. This is because the DSP allowed physicians to state “don’t know” in response to some questions so as not to limit collection.Our study used descriptive analyses only and we were therefore unable to make conclusions based on comparisons between any patient groups or between the 2 study periods.This study found that most patients with mCSPC do not receive treatment intensification despite the availability of these therapies. However, we observed greater use of treatment intensification with both NHT and taxane chemotherapy in 2019-2020 than in 2016-2018 across 5 European countries. In the US, more treatment intensification with NHT was observed for all ethnicity groups, as well as those with Medicare and commercial insurance status, in 2019-2020 than in 2016-2018.As the number of patients with mCSPC who receive treatment intensification increases and time to treatment intensification decreases, more patients who progress to mCRPC will have been exposed to intensified treatments. As therapies for patients with mCSPC and mCRPC overlap, patients who progress to mCRPC may need new treatment options. This suggests an unmet need will emerge for new therapies in mCRPC and highlights the need for further studies to understand optimal treatment sequencing in mCSPC and mCRPC.oyad045_suppl_Supplementary_MaterialClick here for additional data file.


The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) in tumor tissue has been related to the prognosis in various malignancies. Meanwhile, neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammation marker also has been associated with the prognosis in them. However, few reports have investigated the relationship between pulmonary metastases from sarcoma and these biomarkers.We retrospectively recruited 102 patients undergoing metastasectomy for pulmonary metastases from uterine leiomyosarcoma at Okayama University Hospital from January 2006 to December 2019. TILs and TLSs were evaluated by immunohistochemical staining of surgically resected specimens of pulmonary metastases using anti-CD3/CD8/CD103/Foxp3/CD20 antibodies. NLR was calculated from the blood examination immediately before the most recent pulmonary metastasectomy. We elucidated the relationship between the prognosis and these factors. Because we considered that the status of tumor tissue and systemic inflammation were equally valuable, we also assessed the impact of the combination of TILs or TLSs and NLR on the prognosis.As for TILs, CD3-positive cells and CD8-positive cells were correlated with the prognosis. The prognosis was significantly better in patients with CD3-high group, CD8-high group, TLSs-high group, and NLR-low group, respectively. The prognosis of CD8-high/NLR-low group and TLSs-high/NLR-low group was significantly better than that of CD8-low/NLR-high group and TLSs-low/NLR-high group, respectively.CD3-positive TILs, CD8-positive TILs, TLSs, and NLR are correlated with the prognosis, respectively. The combination of CD8-positive TILs or TLSs and NLR may be the indicators to predict the prognosis of patients with pulmonary metastases from uterine leiomyosarcoma.The online version contains supplementary material available at 10.1245/s10434-023-14176-x. However, effective chemotherapy or molecular-targeted drugs are yet to be developed. When recurrent lesions can be resected completely, surgical resection can be the best strategy.4 After the first pulmonary metastasectomy (PM), sarcomas often relapse in the lungs. It was reported that the repeat of metastasectomy could bring good prognosis.5Uterine sarcomas are rare tumors, accounting for 3–7% of uterine malignancies and are less than 1% of all malignancies from female genital organs.6 Immunotherapy provides the unprecedented opportunity to effectively treat, and even cure, several previously untreatable malignancies.7 An anticancer, immune response enables cancer cells to be killed by a series of stepwise events, including trafficking of T cells to tumors, infiltration of T cells into tumors, and recognition of cancer cells by T cells.8 The introduction of immune-checkpoint inhibitors for the treatment of numerous different types of cancer has created enormous interest among cancer immunologists and oncologists.9 In recent years, the researches of antitumor immunity have achieved a remarkable development. For an anticancer, immune response to lead to effective killing of cancer cells, a series of stepwise events mentioned above must be initiated and allowed to proceed and expand iteratively.8 Cytotoxic T cells, natural killer cells, B cells, and dendritic cells play important roles in these steps.11 Tumor-infiltrating lymphocytes (TILs) are thought to be an important indicator, reflecting the local immune-related tumor microenvironment (TME).12 Many studies have reported that TILs are associated with a good prognosis.16Immunotherapy has taken a center stage as a novel cancer treatment approach.17 TLSs share similar structural and functional characteristics with secondary lymphoid organs. TLSs are usually located in the invasive margin or in the stroma rather than the tumor core. TLSs can be identified as CD20-postive, B-cell follicles by immunohistochemistry.18 The prognostic impact of TLSs has been widely explored. Most reports have indicated that TLSs are associated with positive immunoreactivity and favorable clinical outcomes in most types of malignancies.21Tertiary lymphoid structures (TLSs) are ectopic lymphoid organs that can develop at sites of chronic inflammation, such as those associated with infection and autoimmunity, but also form within the TME.22 Systemic inflammatory responses, including the high neutrophil-to-lymphocyte ratio (NLR), are related to tumor development and progression and have been shown to be associated with outcomes in patients with various malignancies.22 We previously reported the NLR immediately before the most recent PM could be a novel independent prognostic factor for patients who underwent surgical resection for pulmonary metastases from various sarcomas.23Conversely, as the tumor grows, nonspecific, inflammatory responses caused by malignant cells or surrounding tissue tend to increase the peripheral blood neutrophil count and reduce the lymphocyte count.The purpose of this study was to investigate whether TILs, TLSs, and NLR were correlated with the prognosis of patients with pulmonary metastases from uterine leiomyosarcoma.This retrospective study was approved by Ethics Committee, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences and Okayama University Hospital, Okayama, Japan . Oral consent was given wherever possible from the study patients regarding the analyses using the surgically resected specimens, as there is no invasiveness for specimen collection. For deceased patients, we posted information-disclosing documents with the opportunity for the patients or family members of the deceased to opt out of this study on the website of Ethics Committee. Regarding the patients for whom we had obtained consent of the secondary use of specimen for the comprehensive gene analyses targeting various malignancies , we also posted information-disclosing documents with the opportunity to reject this study. We maintain a database of the patients undergoing PM for pulmonary metastases from various primary sarcomas in Okayama University Hospital. Using our updated database, we conducted a retrospective review of a total of 256 patients who underwent PM between January 2006 and December 2019. Of the 256, 103 patients were diagnosed with uterine leiomyosarcoma as primary sarcoma. As specimens of the most recent PM for one patient did not remain in Okayama University Hospital, this patient was excluded. Thus, the final cohort consisted of 102 patients.Patients were diagnosed with uterine leiomyosarcoma by histological examination of the primary lesion, and the presence of pulmonary metastasis was confirmed by histological examination of the surgical samples from PM. Patients who underwent PM for pulmonary metastases from uterine leiomyosarcoma met the following criteria: (a) the primary tumor was completely resected; (b) all metastatic diseases were completely resectable or controllable with local therapies; (c) the patients had a suitable performance status; (d) the planned procedure entailed acceptable anticipated complications; and (e) the respiratory function of the patients was sufficient to tolerate planned pulmonary resection.Clinical data for each patient were collected for the following variables: age at the first PM, histology; the date of the first PM; the maximum number of resected tumors among all the surgical procedures performed; the size of the largest lesion resected, frequency of pulmonary metastasectomy that each patient underwent as of the end of 2019; disease-free interval (DFI) from the date of the initial surgery for primary disease until the date of diagnosis of the first pulmonary metastasis; NLR immediately before the most recent PM; TILs and TLSs in the surgical specimens from the most recent PM. We evaluated TILs and TLSs by using the specimen of the biggest tumor in case of multiple lesions. The overall survival (OS) was calculated as the time interval from the first PM until death or the last recorded follow-up. All the patients were observed either until death or December 31, 2022.2O2 solution. After ultrapure water washing, nonspecific reaction was blocked using blocking serum . The sections were reacted with a rabbit monoclonal anti-CD3 antibody , a mouse monoclonal anti-CD8 antibody , a rabbit monoclonal anti-CD103 antibody , a mouse monoclonal anti-Foxp3 antibody , or a rabbit monoclonal anti-CD20 antibody  at 4 °C overnight. The sections were incubated with a secondary antibody  for 30 min at room temperature. After washing in phosphate-buffered saline, the sections were visualized using 3-3′-diamino-benzidine  for 40 s and counterstained with hematoxylin.We defined the clusters of CD20-positive B cells located inside the tumor, near the margin of the tumor, and in the stroma of the tumor as TLSs. Tumor specimens in paraffin-embedded tissue were cut into 4-µm-thick sections. After the sections were deparaffinized by xylene, they were hydrated by a series of decreasing concentrations of ethanol. The antigens were activated by citrate buffer . Endogenous peroxidase activity was blocked by incubation in a 3% HTumor sections stained with anti-CD3, anti-CD8, anti-CD103, and anti-Foxp3 antibodies were scanned at ×20 magnification. Five fields of view were randomly selected. We counted the number of positive cells and calculated the average number of CD3, CD8, CD103, and Foxp3-positive cells in five fields. Tumor sections stained with an anti-CD20 antibody were scanned at ×10 magnification to select three fields with the greatest area of intratumoral and peritumoral CD20-positive cells and the percentage area (%) of each field with CD20-positive cells was calculated by using ImageJ software . We defined the median as the cutoff value in all these five markers.24 Specifically, the software is a modified version of R commander (version 2.7-1), designed to add statistical functions frequently used in biostatistics.All statistical analyses were performed with EZR version 1.54 , a graphical user interface for R version 4.0.3 .p < 0.05 was considered statistically significant. In case of multigroup comparison, we performed Bonferroni’s multiple comparison test.Kaplan-Meier methods were used for calculating OS from the first PM. In terms of TILs and TLSs, we defined the median as the cutoff value. The cutoff value of NLR was determined by using a time-dependent receiver operating characteristic (ROC) curve analysis. A value of We reviewed the medical record of 102 patients with pulmonary metastases from uterine leiomyosarcoma who underwent PM. Table n = 51) and CD3-low group (n = 51); CD8-high group (n = 51) and CD8-low group (n = 51); CD103-high group (n = 52) and CD103-low group (n = 50); Foxp3-high group (n = 51) and Foxp3-low group (n = 51); and TLSs-high group (n = 51) and TLSs-low group (n = 51). The 5-year OS was significantly better in the patients with CD3-high group (38.1%) than in the patients with CD3-low group (15.0%) (p = 0.0181)    and NLR-low group (n = 40). The 5-year OS was significantly better in the patients with NLR-low group (39.3%) than in the patients with NLR-high group (17.7%) (p = 0.0179)  . We divided the patients into two groups according to the cutoff value: NLR-high group  , indicating that the status of tumor tissue and systemic inflammation were independent. Therefore, we assessed the impact of the combination of CD8-positive TILs or TLSs and NLR on the prognosis of patients undergoing PM for pulmonary metastases from uterine leiomyosarcoma. We divided the patients into four groups according to the combination of these factors, respectively. Regarding CD8-positive TILs and NLR, the OS of CD8-high/NLR-low group was significantly better than that of CD8-low/NLR-high group (p = 0.0078)   Fig. a. The 5-10) Fig. b. The 5-In this study, we assessed TME and systemic inflammation status of patients undergoing PM for pulmonary metastases from uterine leiomyosarcoma by means of tumor tissue and a peripheral blood count. Our results suggest that TILs, TLSs, and NLR are good predictors of the prognosis in patients undergoing PM for pulmonary metastases from uterine leiomyosarcoma. However, it is not sufficient to extract the group of the patients with better prognosis by using only one factor. Therefore, we introduced some combinations of biomarkers from tumor tissue and peripheral blood to predict survival in patients undergoing PM for pulmonary metastases from uterine leiomyosarcoma more accurately.25 All components play critical roles in the detection and elimination of tumor cells, thereby contributing to arresting tumor progression and proliferation. The activation of T cells requires the T-cell receptor complex, which includes the co-receptor CD3.26 The CD3-positive cells are likely reflecting immunocompetent T lymphocytes with antitumor activity; thus, CD3-positive TILs can be a prognostic factor. CD8-positive T cells play a pivotal role in the adaptive immune response to malignancy.14 Activated CD8-positive T cells are named cytotoxic T lymphocytes (CTL) as they can directly recognize and kill malignant and infected cells.27 The primary antitumor activity of CTL is to kill target cells via the exocytosis of cytotoxic granules containing perforin and granzymes as well as the production of cytokines such as interferon-γ and tumor necrosis factor.28 Thus, CD8-positive TILs also can be a prognostic factor.We explored whether TILs were correlated with the prognosis using some markers that distinguish the subtype of TILs. Although it was reported all the markers we used in this study could be prognostic factors in several malignancies respectively, only CD3 and CD8 were correlated with the prognosis in our study. The immune microenvironment is composed primarily of TILs, macrophages, and dendritic cells.29 acting as a predictor for the prognosis of various malignancies.30 There are many reports about the density of TLSs on the primary lesion, such as breast cancer, gastric cancer, and endometrial cancer.33 Although there are limited data on the presence and function of TLSs at metastatic sites, several reports describe the characteristics of TLSs in metastatic sites. TLSs in metastases seem to depend on both the primary tumor type and the organ in which the metastatic site is located. In the study of breast cancer that analyzed a large cohort of 355 metastases from four metastatic sites , TLSs were found in lung and liver metastases but not in brain and ovarian metastases.34 TLS densities in pulmonary metastases from different tumor types are highly variable and seem to mirror those of the primary sites, being at high densities in metastases from colon and prostate cancers, intermediate densities in metastases from thyroid, renal, liver, and breast cancers, and low densities in metastases from leiomyosarcoma and osteosarcoma.35 It may be significant to identify the group with better prognosis by extracting the cases with TLSs-high in the worse-prognosis disease, such as uterine leiomyosarcoma.In this study, we show that the presence of more TLSs can be a better prognostic factor. TLSs are crucial components of TME. They facilitate the recruitment of immune cells. For example, TLSs around a tumor play a crucial role in inducing antitumor, cytotoxic, T-cell proliferation,22 The impact of neutrophils and lymphocytes on the tumor microenvironment is increasingly recognized. Previous studies have shown that large numbers of neutrophils can inhibit the activation and cytolytic antitumor activity of lymphocytes and natural-killer cells.36 For tumor patients, the NLR reflects not only the systemic inflammation but also the immune state to tumors.We found low NLR immediately before the most recent PM could be a good prognostic factor in uterine leiomyosarcoma. The NLR is a systemic inflammation marker, which has been reported to be an independent prognostic factor for survival in several malignancies.37 there was no relationship between TILs or TLSs and NLR in this study. However, even in the studies that showed the correlation between TILs or TLSs and NLR, they did not suggest the reasonable mechanism, warranting further study. Instead, we consider that TME and the status of systemic inflammation are independent factors that affect the prognosis.Although there are several studies that demonstrated TILs or TLSs were correlated with the preoperative NLR,In this study, we combined TILs or TLSs and NLR to evaluate prognosis. Then, we were able to extract patients with better prognosis more accurately. Pulmonary metastases from uterine leiomyosarcoma often exist bilaterally, and they frequently recur in the lung after PM. Thus, several cases need repeat PM. In this cohort, 40% of all the patients underwent plural PM. These combined factors may be effective as not only prognostic factors but also indicators to decide surgical indication.39 Meanwhile, there is a study that the presence of TLSs predicted the efficacy of immune checkpoint inhibitor in solid tumors.40 If immune checkpoint inhibitors become a treatment option to uterine leiomyosarcoma, evaluating TLSs and TILs may be effective to predict responsiveness to the drugs.Although the role of immune checkpoint inhibitors in sarcoma treatment is unclear, some studies have demonstrated the efficacy of immune checkpoint inhibitors to sarcoma.35 Therefore, evaluating only TLSs of pulmonary metastases seems to be beneficial.To fully explore the role of TILs and TLSs to malignancies, it may be better to evaluate not only those of metastatic lesions but also those of primary lesions. However, there is a report that TLSs at metastatic sites have the same characteristics as in primary sites. Thus, regarding the prognostic impact of TLSs, those in metastatic sites parallel that of primary tumors.20 If we evaluate TLSs in detail as mentioned above, we may elucidate the function of TLSs on pulmonary metastases from uterine leiomyosarcoma. Although this is currently the biggest cohort of patients with pulmonary metastases from uterine leiomyosarcoma in which TILs and TLSs are assessed, it might still be underpowered. A multi-institutional study with a considerable sample size will be necessary to further clarify the prognostic value of TILs and TLSs in pulmonary metastases from uterine leiomyosarcoma.There are some limitations in this study. Regarding the evaluation of TLSs, we simply explored the CD20-positive cells, although there are several methods to evaluate the details of TLSs, such as CD21-positive cells and CD23-positive cells that classify the phenotype of TLSs, several T-cell markers contained in TLSs, and the chemokines associated with TLSs.We identified that the high CD3-positive TILs, the high CD8-positive TILs, the high density of TLSs, and the low NLR immediately before the most recent PM were better prognostic factors, respectively. Moreover, the combination of these TILs or TLSs and NLR resulted in clarifying the groups with better prognosis. Further investigations are warranted to clarify tumor microenvironment in pulmonary metastases from uterine leiomyosarcoma.Supplementary Fig. 1a Overall survival (OS) stratified by the status of CD103-positive tumor-infiltrating lymphocytes (TILs) (TIF 7699 KB)Supplementary Fig. 1b Overall survival (OS) stratified by the status of Foxp3-positive tumor-infiltrating lymphocytes (TILs) (TIF 7693 KB)Supplementary Fig. 2 Receiver operating characteristic (ROC) curve analysis to determine the cutoff value of neutrophil-to-lymphocyte ratio (NLR). AUC area under the curve (TIF 7605 KB)Supplementary Fig. 3a Linear-regression analysis for CD8-positive tumor-infiltrating lymphocytes (TILs) and neutrophil-to-lymphocyte ratio (NLR) (TIF 7595 KB)Supplementary Fig. 3b Linear-regression analysis for tertiary lymphoid structures (TLSs) and neutrophil-to-lymphocyte ratio (NLR) (TIF 7582 KB)Below is the link to the electronic supplementary material.


At the beginning of the COVID-19 pandemic, patients with primary and secondary immune disorders—including patients suffering from cancer—were generally regarded as a high-risk population in terms of COVID-19 disease severity and mortality. By now, scientific evidence indicates that there is substantial heterogeneity regarding the vulnerability towards COVID-19 in patients with immune disorders. In this review, we aimed to summarize the current knowledge about the effect of coexistent immune disorders on COVID-19 disease severity and vaccination response. In this context, we also regarded cancer as a secondary immune disorder. While patients with hematological malignancies displayed lower seroconversion rates after vaccination in some studies, a majority of cancer patients’ risk factors for severe COVID-19 disease were either inherent (such as metastatic or progressive disease) or comparable to the general population . A deeper understanding is needed to better define patient subgroups at a higher risk for severe COVID-19 disease courses. At the same time, immune disorders as functional disease models offer further insights into the role of specific immune cells and cytokines when orchestrating the immune response towards SARS-CoV-2 infection. Longitudinal serological studies are urgently needed to determine the extent and the duration of SARS-CoV-2 immunity in the general population, as well as immune-compromised and oncological patients. COVID-19 infection is a complex and heterogeneous disease, with the host response crucially determining its course: during the early phase of an infection, there is a substantial production of type 1 interferons, generated at the tissue level by infected as well as surrounding cells ,3. MoreoThe cytokine production is usually maintained in the first week, when the virus replicates and the most overt clinical symptoms usually appear; next, the antibody production phase takes place together with the expansion of the T-cell compartment. During a viral infection, the peak in T-cell expansion usually occurs about a week from the infection. Consequently, inflammatory mediators are key drivers of COVID-19-related morbidity . The knoIt is possible to draw some distinctions based on the severity of the disease. T-cell activation, DR expression and monocytes are critical; significant differences were found in T-cell activation and MHC DR expression, as well as in the effector memory T-cell population  . Many stFurthermore, conditions associated with poor outcomes in COVID-19 appear to be associated with general risk factors, such as type 2 diabetes, obesity and COPD; it is worth noting that these are traditional markers of poor outcomes in almost any severe disease. Aside from organ transplantation, nothing has been discovered to identify the immune system’s state as a major determinant . NonetheBecause ACE2 and TMPRSS2 variants and expression can be candidates for gender and country differences in COVID-19 severity, host genetics is also important in COVID-19 ,21. TherAlthough infection with SARS-CoV-2 was expected to activate the host’s immune response, data on this specific trait were initially scarce ,23. WhilA test designed to determine which pieces of SARS-CoV-2 are recognized by the immune system was supported by CD4+ and CD8+ epitopes predicted to play significant roles in SARS-CoV-2 . SpecifiT-cell and antibody responses in COVID-19 cases appear to be orchestrated by two main principles: in most humans, SARS-CoV-2 causes acute infections that resolve or cure. Both antibodies and T cells are important in modulating humoral responses, human vaccines and protecting against the virus . A betteOf note, T-cell reactivity to SARS-CoV-2 epitopes has also been observed in non-exposed individuals . In acutThe adaptive immune system has three branches in its arsenal against the virus, but there is no strong evidence of a causal negative association between adaptive immunity and disease severity. Conversely, age is a major COVID-19 risk factor, and adaptive immunity shortcomings seem to be part of the dysfunctional response when poorly coordinated T-cell responses and limited naïve T cells are in place .When considering the immune system ageing, we primarily consider T cells and thymic involution. The naive T-cell compartment contracts, thymus cells acquire senescent characteristics and some expanded oligoclonal T-cell populations are exhausted . These pListeria infection is usually insignificant in most young adults. However, after the age of 80, the risk of stroke and poor outcome increases significantly ,39: in vT cells have been shown to contribute to a better outcome in SARS-CoV-2 infections with lower viral loads  and playThe T-cell count has been discovered to be important in viral load control and infection eradication . HoweverMany studies have investigated the impact of HIV on COVID-19 outcomes and have suggested that the close monitoring of immune status and viral load, as well as adjustments to HIV treatment regimens, may be necessary for optimal management of COVID-19 in HIV-positive individuals. Additionally, early and aggressive treatment with antiviral therapy and corticosteroids has improved outcomes in COVID-19 patients, including those with HIV. COVID-19 vaccines are also recommended for HIV-positive individuals, although more research is needed to fully understand their safety and efficacy in this population. Finally, psychological and social support services are important considerations for HIV-positive individuals with COVID-19, as they may face unique challenges related to social isolation and stigma .The underlying trained innate immunity represents a significant breakthrough in COVID-19 prevention ,49. SARSCD4+ and CD8+ T cells exhibited similar behavior. The quality and duration of vaccination responses with various vaccine platforms have also been studied. Moderna, Pfizer, Johnson & Johnson, AstraZeneca and Novavax have all been tested for their ability to establish and maintain immune responses over time ,53. ThisWhen researchers examined factors contributing to COVID-19 mortality in European cancer patients, they discovered that onco-hematological pathologies, metastasis and additional comorbidities were risk factors for developing the severe disease . NonetheKuderer et al. defined factors associated with COVID-19 mortality in cancer patients, describing a not significant increase in risk for non-cytotoxic therapy . At the same time, they confirmed increased mortality for subjects with hemato-oncological conditions, the elderly, and patients with comorbid conditions ,65. LargRegarding humoral and cellular immunity, cancer patients are no exception to the general concept of vaccine responsiveness . PassiveThere is very little information available on the antibody responses against SARS-CoV-2 in cancer patients. The first prospective multicenter observational study evaluated the antibody response in cancer patients and oncology healthcare workers with confirmed or clinically suspected COVID-19 , demonstp < 0.001). Finally, lower seropositivity in cancer patients was associated with age and chemotherapy (p < 0.001) [A prospective, multicenter cohort study conducted in 2022 aimed to compare the spike IgG seropositivity rate in blood samples from 776 cancer patients and 715 non-cancer volunteers following inactive SARS-CoV-2 vaccination. The cancer patient group had a seropositivity rate of 85.2%, while the control group had a rate of 97.5%. Cancer patients not only had a significantly lower seropositivity rate but also lower antibody levels (p < 0.0001), this study still was the first to describe a positive immune response in this vulnerable patient subgroup [p < 0.001), and female sex , respectively) [A study from 2021 aimed to assess the SARS-CoV2 IgG seroprevalence in 74 older patients (aged ≥ 80 years) with cancer one month after receiving the second dose of the BNT162b2 vaccine. While median serum IgG levels in older cancer patients were lower compared to control , patient age  and cancer type. Therefore, administering a COVID-19 vaccine booster dose improved seroconversion and antibody levels. Patients with solid cancer consistently responded better to booster vaccines than patients with hematological cancer [Notably, the aim of Mai et al.‘s systematic review and meta-analysis was to determine the proportion of non-responders to COVID-19 primary vaccination in 849 patients with hematological cancer and 82 patients with solid cancer who seroconverted after a booster dose. Seroconversion occurred in 44% of patients with hematological malignancies, while a significantly higher seroconversion (80%) was observed among solid tumor patients. Higher antibody titers were found to be significantly associated with an increased duration between the second and third dose . However, there was no difference in neutralizing antibody production between COVID-19-positive and vaccinated patients who did not receive treatment (p = 0.14). As a result, it was suggested by the authors that vaccinated patients with MM on treatment who have not previously received a COVID-19 infection should be considered for booster vaccination [In particular, a recent study compared the development of neutralizing antibodies against SARS-CoV-2 in non-vaccinated patients with multiple myeloma (MM) and COVID-19 to patients who received two doses of the BNT162b2 vaccine, most likely due to immunoparesis ,78. Paticination . There is little information in the literature about the levels of autoantibodies in patients with paraneoplastic syndromes and whether these autoantibodies could interfere with the vaccine response in any fashion.Immunologic self-tolerance defects increase the risk of paraneoplastic autoimmune diseases and immune-mediated toxicity, which have been examined in patients with thymic epithelial tumors. Common COVID-19 vaccine adverse events among 54 participants in a 2021 US study included injection site pain, fatigue and headaches . Among tPrioritizing these populations who will benefit the most from SARS-CoV-2 vaccination and thus have a positive impact on the pandemic’s trajectory is critical. Patients with cancer, immunological disorders and close contacts should be prioritized. Customizing vaccination schedules could be one approach to developing more effective health policies based on evidence-based data. Alternative vaccine schedules are not insignificant, and they frequently affect vaccine efficacy. Similarly, adjusting the schedules to account for the risk of severe COVID-19 outcomes, as well as individuals’ ability to mount and sustain an immune response, is critical ,82.Researchers also investigated the potential contribution of systemic autoimmune disease (SAD) to COVID-19 disease severity. For this, they investigated the prognostic impact of SAD on COVID-19 mortality in a nationwide Spanish registry study performed before the introduction of SARS-CoV-2 vaccination. The presence of SAD in COVID-19 patients was generally associated with higher mortality. However, after adjusting for patient characteristics and comorbidities, SAD did not have a statistically significant effect on mortality . While cThe use of biologics and other immunosuppressive therapies has been a concern during the COVID-19 pandemic, as it was initially believed that these treatments could increase the risk of severe disease. However, studies have shown that the risk factors for COVID-19 hospitalization are similar in patients receiving biologics compared to the general population . TreatmeRecent studies have also shown that the IL-31/IL-33 axis plays an essential role in the immune response against SARS-CoV-2. In particular, the axis is involved in the regulation of cytokine production and immune cell activation . TherefoThe discovery of inborn errors in type I IFN immunity in patients with life-threatening COVID-19 paved the way for further research in this area . Inborn Based on this evidence, it is tempting to conclude that B cells can be harmful, especially considering additional evidence regarding acalabrutinib intake. Even though the study was small and uncontrolled, the authors assumed that acalabrutinib was extremely beneficial in their COVID-19 cohort . MoreoveMeyts et al. were the first to describe 94 patients with primary immunodeficiency  and coinfection with COVID-19. Specifically, COVID-19 was found in 20–25% of IEI patients, with 75–80% developing mild SARS-CoV-2 disease . IEIs, iCollectively, we may pave the way for representing two major causes of death, namely HLH in younger subjects and sepsis or renal failure in older subjects . BucciolHypersecretion of IFN-I, on the other hand, causes severe forms of Multisystem Inflammatory Syndrome in Children (MIS-C), a SOCS1-driven condition . AdditioOther IEIs, such as X-linked agammaglobulinemia or states with reduced or absent B lymphocytes, have a lower ability to eliminate the virus with a longer duration of infection . In thesFurthermore, data from the IPINet network’s 2021 and 2022 studies on a large population of Italian subjects with IEI revealed 74 cases of SARS-CoV-2 infection in 1161 patients with CVID, with an incidence comparable to that of the general adult population. The cumulative incidence is even lower in the pediatric population ,110. In The mortality rate was higher in Good syndrome and Del 22q11, both associated with a T-cell defect ,110. FouThe risk factors for increased severity of COVID-19 suggest that a more severe outcome correlates with the same risk factors seen in the general population, namely the male sex and associated comorbidities, such as chronic lung disease and chronic liver disease. On the contrary, rheumatological manifestations would not affect mortality. The same factors determine the duration of the infection (2 weeks vs. 2–3 months) . This maThe immunocompromised patient is an excellent candidate for an intense clinical investigation into the relationship between SARS-CoV-2 infection and host defenses. Three emerging paradigms in cancer treatment prioritization and frailty have a greater impact on the clinical outcome than cancer-related immune dysfunction. Of note, cancer-related immune dysfunction did not play a crucial role in COVID-19 disease severity in contrast to cancer-related frailty. SARS-CoV-2 infection was contracted by 20–25% of subjects with inborn errors of immunity, with 75–80% having a mild or asymptomatic clinical course. COVID-19 severity and mortality appear to be associated with comorbidities that manifest younger than the general population.Ultimately, the COVID-19 vaccines are safe for cancer patients, but they may be less effective than in healthy people, especially those with compromised immune systems.


Epstein–Barr virus (EBV) infection is ubiquitous and in sub-Saharan Africa, occurs early in life. In a population-based rural African cohort, we leveraged historical samples from the General Population Cohort (GPC) in Uganda to examine the epidemiology of infection with EBV over time, in the era of HIV.We used 9024 serum samples collected from the GPC in 1992, 2000, 2008, from 7576 participants across the age range (0–99 years of age) and tested for anti-EBV immunoglobulin G (IgG) antibodies to EAd, VCA, and EBNA-1 using a multiplex bead-based assay. The related gammaherpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV) seropositivity was also determined by detection of anti-KSHV IgG antibodies to K8.1 or ORF73 measured by recombinant protein enzyme-linked immunosorbent assay. Data on sex, age, and HIV serostatus were also collected. EBV seropositivity was modeled with age , sex, HIV serostatus, and KSHV serostatus using generalized linear mixed effects models to produce beta estimates.More than 93% of children were EBV seropositive by one year of age. EBV seropositivity was significantly associated with KSHV seropositivity. Anti-EBNA-1 antibody levels decreased with increasing age and were lower on average in people living with HIV. In general, anti-EAd antibody levels increased with age, were higher in males and KSHV seropositive persons, but decreased over calendar time. Anti-VCA antibody levels increased with age and with calendar time and were higher in KSHV seropositive persons but lower in males.This is the first study to identify factors associated with EBV antibodies across the entire life-course in rural sub-Saharan Africa. Consistent with other studies, EBV was near ubiquitous in the population by age one year. Patterns of antibodies show changes by age, sex and calendar time, but no association with HIV was evident, suggesting no relationship between EBV sero-epidemiology and the spread of HIV in the population over time in Uganda.The online version contains supplementary material available at 10.1186/s13027-023-00534-7. Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus and is the etiological agent for several cancers including nasopharyngeal carcinoma and Burkitt lymphoma. Similar to other herpesviruses, infection with EBV is lifelong with symptomatic disease only occurring in a small subset of infected individuals. Although EBV is ubiquitous worldwide, age of infection differs by region and in sub-Saharan Africa occurs early in life, with most children infected by two years of age in some regions , 2. IndiImmunosuppression impairs the ability of the immune system to control EBV infection. In many populations, the spread of HIV led to an increase in the incidence of EBV-associated cancers –26. WithThe GPC in Kalungu District, Uganda was established in 1989, by the UK Medical Research Council and the Uganda Virus Research Institute (UVRI) and includes residents from 25 adjacent villages. Data are collected during annual census rounds, the methods for which are described in more detail elsewhere . BrieflyWe identified serum samples collected during three GPC census rounds . Samples from these years were chosen because children were included in enrollment of those rounds. Subjects were selected at random, after stratification to ensure roughly equal sex and age distributions. After stratification, up to 3000 samples from each round were randomly selected with methods described elsewhere . Levels 3) and WHO clinical staging of HIV disease (stages 1–4) .EBV seropositivity was modeled with age, sex, KSHV serostatus, HIV serostatus, CD4 T cell count, and WHO clinical stage using generalized linear mixed effects models to account for within person correlations. Anti-EBV antibody levels were logWe tested 9077 samples collected from 7576 participants. The majority of samples came from different individuals in only one of the rounds (n = 6275), though samples from 1101 individuals were available from two rounds, and samples from 200 participants were available from all three census rounds. Overall KSHV seropositivity ranged from 91 to 94%. Participants did not differ in age, sex, or HIV serostatus by census year ; p < 0.001]. Among PLWH, there was no association between average CD4 T cell count or the highest WHO HIV disease stage in a census year and EBV seropositivity.We modeled risk factors for EBV seropositivity in participants over one year of age. EBV seropositivity was not associated with age or sex Table . After aTo identify factors associated with anti-EBV antibody levels we used linear mixed effects models restricted to EBV seropositive individuals over age one year  found no differences in anti-VCA, EAd, or EBNA-1 levels by HIV status, although EBV detection was significantly more frequent in blood and oral fluids of participants with HIV . In anotSeropositivity to KSHV, another gammaherpesvirus endemic to Uganda, was strongly associated with both EBV seropositivity as well as with higher average anti-EAd and anti-VCA antibody levels. In healthy Kenyan children, anti-EBNA-1 antibody levels were higher in KSHV seropositive children but there was no difference in anti-VCA antibody levels . And amoWhen we looked at changes in EBV serology over calendar time we found high overall EBV seropositivity in the 2000 census round compared to 1992, but no difference between EBV seropositivity in 1992 and 2008. We also identified significant increases in average anti-EBNA-1 and anti-VCA antibody levels but decreases in anti-EAd antibody levels over the three time points. The year 1992 was the peak of the HIV/AIDS epidemic in Uganda, with 18% of the general population infected; HIV prevalence decreased consistently until 2000 . The iniOur study had some limitations. We measured anti-EBV antibodies to three antigens that are important in the clinical diagnosis of EBV infection, EBNA-1, EAd, and VCA. However, more recent studies focused on the EBV vaccine target, gp350, or the immediate early protein, ZTA, could be more informative to the state of EBV infection and potential for vaccine efficacy in this population , 49. In We have identified early age of EBV infection in Uganda as well as several factors associated with changing anti-EBV antibody levels across two decades that included both pre- and post-ART timepoints. Our findings are an important contribution to understanding the epidemiology of EBV in sub-Saharan Africa. Future studies will be required to identify the significance of our findings and to apply them to potential intervention research.Additional file 1: Table S1. Risk Factors for log transformed anti-EBV antibody levels (MFI) in participants 1+ years of age (n=8495)1 using linear mixed effects regression2.


Up to 65% of patients with metastatic colorectal cancer experience disease recurrence following curative-intent metastasectomy. Certain oncology organizations recommend surveillance to detect recurrent disease at an early phase to allow for the greatest benefit from additional treatment. Surveillance programs may involve colonoscopy, physical examination, carcinoembryonic antigen testing, and computed tomography at regular intervals. Even though surveillance can be highly effective, there is little evidence for its use in metastatic colorectal cancer and no clear consensus on the best strategy. We used decision analysis and a population-based cohort to evaluate the cost-effectiveness of various surveillance strategies following curative-intent metastasectomy in patients with stage IV colorectal cancer. Our results show that surveillance with clinic visits and investigations every 12 months for 5 years would be cost-effective in a Canadian context. These findings demonstrate the utility of economic analysis for guiding the management of stage IV colorectal cancer after metastasectomy.Surveillance of stage IV colorectal cancer (CRC) after curative-intent metastasectomy can be effective for detecting asymptomatic recurrence. Guidelines for various forms of surveillance exist but are supported by limited evidence. We aimed to determine the most cost-effective strategy for surveillance following curative-intent metastasectomy of stage IV CRC. We performed a decision analysis to compare four active surveillance strategies involving clinic visits and investigations elicited from National Comprehensive Cancer Network (NCCN) recommendations. Markov model inputs included data from a population-based cohort and literature-derived costs, utilities, and probabilities. The primary outcomes were costs (2021 Canadian dollars) and quality-adjusted life years (QALYs) gained. Over a 10-year base-case time horizon, surveillance with follow-ups every 12 months for 5 years was most economically favourable at a willingness-to-pay threshold of CAD 50,000 per QALY. These patterns were generally robust in the sensitivity analysis. A more intensive surveillance strategy was only favourable with a much higher willingness-to-pay threshold of approximately CAD 425,000 per QALY, with follow-ups every 3 months for 2 years then every 12 months for 3 additional years. Our findings are consistent with NCCN guidelines and justify the need for additional research to determine the impact of surveillance on CRC outcomes. Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide . AlthougApproximately 10–20% of patients with de novo stage IV or recurrent metastatic CRC are eligible for curative-intent metastasectomy, a procedure associated with a potential for improved long-term outcomes and, sometimes, a cure ,10. The Intensive surveillance strategies after curative-intent resection of stage I-III CRC are advised by most major oncology organizations ,20,21,22To address these knowledge gaps, we performed a full-scale economic evaluation of potential surveillance strategies after curative-intent metastasectomy of stage IV CRC in a Canadian context. We constructed a decision analytic model to compare various modalities and intervals of surveillance based on NCCN guidelines . Costs, We performed a cost-utility analysis among patients who underwent curative-intent metastasectomy of stage IV CRC. We compared the following surveillance schedules for clinic visits, CEA testing, and CT with varying intensity: every 3 months for 2 years and then every 12 months for 3 additional years (“q3/q12”), every 6 months for 5 years (“q6”), every 6 months for 2 years and then every 12 months for 3 additional years (“q6/q12”), and every 12 months for 5 years (“q12”). We assumed that all patients under active surveillance also underwent colonoscopy at 1 year and 4 years , assuminWe built a Monte Carlo micro-simulation using a 1-month cycle length and 10-year time horizon, with 10,000 iterations. In the reference case, costs were calculated from a publicly funded healthcare payer perspective. We measured health outcomes in quality-adjusted life-years (QALYs). Outcomes included cost, QALYs, and cost per QALYs/LYs and were half-cycle corrected. We also conducted exploratory analyses using life-years (LYs) as an outcome. We discounted future costs and outcomes at 1.5% annually in accordance with Canadian Agency for Drugs and Technologies in Health (CADTH) guidelines . IncremeWe developed a Markov model using decision analysis software . All patients were initially assumed to have no evidence of disease following curative-intent metastasectomy for stage IV CRC, from which they could then develop recurrent disease. This recurrence may then be detected once symptoms develop or while undergoing surveillance during an asymptomatic phase. Upon detection, patients can be curatively treated, the likelihood of which is greater for asymptomatic disease, or undergo palliative treatment with either symptomatic or asymptomatic disease. We incorporated several general health states: (1) alive with no evidence of disease, (2) alive with undetected recurrent disease, (3) alive and receiving curative-intent treatment, (4) alive and undergoing palliative treatment for symptomatic disease, (5) alive and receiving palliative treatment for asymptomatic disease, and (6) deceased. Patients receiving curative-intent treatment were assumed to have undergone hepatic metastectomy and 6 months of peri-operative oxaliplatin-based chemotherapy. Palliative treatment consisted of FOLFOX, FOLFIRI, and bevacizumab. Molecular subtypes , more targeted treatment , and late-lines of treatment (TAS-102 and Regorafenib) were not accounted for, as they were not available during the study reference period. Each month, the patients could remain in the same health state, progress to a subsequent health state, or die .We modelled recurrence, repeat resection rates, and survival outcomes from the population-based outcomes of 257 patients who had curative-intent metastasectomy of stage IV CRC in British Columbia, Canada, and subsequently no evidence of disease . These pCosts, utilities, and probabilities are presented in We completed one-way and two-way sensitivity analyses to estimate the impact of plausible variations in time to symptomatic recurrence and the probability of repeat curative-intent resection. As the probability of repeat curative-intent resection was derived from observational data, we varied the rates by 25% in the opposite directions for symptomatic and asymptomatic recurrence to approximate a maximum and minimum difference between the observed rates. Using distributional assumptions of the input parameters , we alsoWe estimated the cost of surveillance for a patient after curative-intent metastasectomy of stage IV CRC to be CAD 19,890 for the q3/q12 strategy, CAD 14,191 for q6, CAD14,175 for q6/q12, and CAD 7707 for q12 . IncreasComparing the active surveillance strategies at willingness-to-pay thresholds of CAD 50,000–100,000 per QALY, the q12 strategy had the highest probability of being most the cost-effective . HoweverThe results of our base-case analysis were generally robust to uncertainty in the probability of repeat curative-intent resection . The q12Using a cohort of patients who have undergone curative-intent metastasectomy for stage IV CRC, we modelled and evaluated the cost-effectiveness of different surveillance strategies based on NCCN guidelines. We found that a surveillance scheme with clinic visits, CEA testing, and CT every 12 months for 5 years is likely favoured from a publicly funded healthcare payer perspective at the standard willingness-to-pay threshold of CAD 50,000/QALY . Only atActive surveillance following metastasectomy is the accepted clinical practice when resources are sufficient . Even thThis study focused on surveillance as a package, and although imaging contributed to upfront costs, the downstream costs of managing recurrence were the greatest driver of costs in our model. Further metastasectomy and varying systemic therapy regimens have demonstrated incremental cost-effectiveness in prior studies but when combined with surveillance in our model, their benefits appeared to attenuate ,50,51.The methodology used in this study was unique in its use of a population-based cohort to inform patient outcomes. Randomized studies have focused exclusively on the role of perioperative systemic treatments and metastatic resection, not formally on the role of surveillance afterwards ,53. The The differences in outcomes were largely driven by the differences in resectability for symptomatic and asymptomatic recurrence. Symptomatic disease was used as a surrogate for repeat resectability since increases in the size, number, and location of metastases are more likely to produce symptoms. Despite this assumption, our sensitivity analysis, which varied the difference in resectability between asymptomatic and symptomatic recurrence, led to a consistent ranking of the four strategies. True resectability is challenging to model, as it often involves interplay between the same disease burden characteristics, which can lead to symptoms, as well as surgical expertise to facilitate resection and any responses to perioperative systemic treatment . Our stuThe transition from asymptomatic to symptomatic recurrence in stage IV CRC was challenging to characterize, as it is not well described in the literature. We therefore performed a sensitivity analysis based on the only source identifiable for a possible range of possibilities . The oveBRAF mutation or sidedness of the primary tumor). Novel use of liquid biopsy for earlier stages of disease has also recently demonstrated benefits, though it is unclear what impact this may have on resected stage IV disease, but it may further refine risk stratification and surveillance. Given the era that the modelling data was constructed from, these details were not readily available for incorporation. Furthermore, many of these novel interventions have not been uniformly adopted in the real world. In addition, the existing risk-stratification Fong Clinical Risk Score was not utilized as it is only relevant for hepatic metastases, which represents a subset of our cases, and it reflects older approaches to metastatic resection where the focus was on the disease burden as opposed to the residual organ function required.This study is limited by various model assumptions. Modeling did not incorporate the impact of novel treatments , or the addition of biological agents  or more nuanced prognostic factors (e.g., the presence of a Surveillance can be an invaluable method for detecting recurrent disease in patients with stage IV CRC following curative-intent metastasectomy. In a Canadian context and based on NCCN guidelines, this cost-effectiveness analysis demonstrated that active surveillance with clinic visits, CEA testing, and CT every 12 months for 5 years were associated with the greatest cost-effectiveness when compared to alternative strategies with greater frequency of follow-up. Health economics considerations have an important role in guiding practice, but good clinical judgement is also required to optimize disease management in the individual patient. Further research is needed to assess adherence to surveillance guidelines and to confirm the impact of surveillance on patient outcomes in stage IV CRC.