Providing rehabilitation services in primary health care (PHC) is associated with numerous health, social, and economic benefits. Therefore, low and middle-income countries, such as Iran, should benefit from the advantages of integrating rehabilitation services into PHC. We conducted a qualitative study to determine policy solutions that could facilitate the integration of rehabilitation services into Iran’s PHC network.Semi-structured interviews were conducted with 38 participants, including health policymakers, rehabilitation managers, faculty members, and rehabilitation practitioners. Purposive and snowball sampling strategies were adopted to recruit participants. The WHO Health System building blocks framework analysis was applied to analyze the collected data.Participants’ perspectives and experiences outlined potential policy options including: (1) stewardship: increasing political support, strengthening the leadership of the rehabilitation sector, and promoting inter-sectoral collaborations; (2) service delivery: increasing the knowledge of healthcare professionals, using local volunteers, deploying mobile rehabilitation teams, using telerehabilitation, and improving referral pathways; (3) financing: increasing government funding, preparing a package of rehabilitation services, and using appropriate payment mechanisms; (4) human resources: expanding rehabilitation workforce, training rehabilitation assistants, and enhancing employment and social opportunities; (5) information systems: establishing a comprehensive information system and an effective surveillance system; and (6) technologies: facilitating access to a range of rehabilitation equipment and raw materials, especially for prosthetics and orthotics services.Based on the WHO six building blocks framework, this study identified several policy options for integrating rehabilitation services into the Iranian PHC Network. Some of the policy options include increasing political support, promoting inter-sectoral collaborations, increasing the skills and knowledge of healthcare workers, establishing effective referral pathways, strengthening team-working, and increasing government funding. In recent decades, demographic changes and improvements in acute healthcare have led to an increase in long-term conditions , 2. The Rehabilitation services seek to empower people living with disability to participate in their communities by improving functional ability and reducing environmental barriers , 7. RehaPHC services include diagnosing health conditions, recognizing functional problems, and referring patients for secondary and tertiary levels of care . Modern The concept of integration of health services as opposed to fragmented care is not new , and intIran’s Ministry of Health and Medical Education (MOHME) is the main provider and policymaker of health services. More than 90% of Iranians are covered by social insurance organizations for their healthcare, including the Iran Health Insurance Organization, the Social Security Organization, and the Armed Forces Health Insurance Organization . The MOHBehvarz During the interviews, the need to increase political support and strengthen the leadership in the field of rehabilitation in Iran was repeatedly mentioned. One of the participants said:“Various organizations are involved in financing and providing rehabilitation services. The welfare organization itself provides community-based rehabilitation services. However, if there is close co-operation between this institution and the Ministry of Health, these services can be integrated into primary healthcare.“[Faculty member 01]Participants emphasized the promotion of intersectoral collaborations as one of the potential solutions. Participants believed that due to the interdisciplinary nature of rehabilitation, several stakeholders are involved in providing these services, requiring inter-sectoral co-operation.“Interestingly, many health experts are still unaware that these [rehabilitation] services can be preventative and prevent many serious complications in the future. They always think that they should go to rehabilitation if treatment and therapy interventions fail!“[Physiotherapist 03]The level of awareness of policy- and decision-makers about the effects of rehabilitation services, especially their preventive effects, should be increased. Many relevant policymakers view these services as tertiary interventions and a strategy when other options have failed.“What do you expect from a policymaker with very little information about rehabilitation services? In my opinion, rehabilitation specialists should be involved in the care processes of care services to improve their rehabilitation position.“[Faculty member 04]Participation of rehabilitation professionals in the policy-making process can facilitate the strengthening of rehabilitation services, especially in PHC. One participant said:“Adherence to existing upstream laws and documents can be very effective in integrating rehabilitation services. Unfortunately, there is no good adherence to the existing laws.“[Health policymaker 03]Participants believed that national policy documents emphasized the provision of rehabilitation services in healthcare. They suggested that adhering to, and strengthening existing statutes could provide the necessary basis for strengthening rehabilitation services in PHC.“Recipients of rehabilitation services are usually disadvantaged and vulnerable groups and therefore do not have enough power to pressure policymakers. I think their needs can be better shown by holding rehabilitation campaigns and associations.“[Orthotist/Prosthetist 02]Individual service recipients, local community organizations and non-governmental organizations can campaign to improve services:“Scientific associations can provide a list of rehabilitation services that have preventive effects to consider in primary health care.“[Faculty member 05]Rehabilitation associations can also play a crucial role in demonstrating the effectiveness of rehabilitation interventions, and informing relevant policy makers:“We can teach some skills to current staff [Behvarzs] and ask them to provide basic musculoskeletal assessments and some training and exercises.“[Faculty member 02]Some participants proposed increasing the skills and knowledge of current healthcare workers to provide a range of rehabilitation services in PHC. A university professor believed that:“Many volunteers are interested in cooperating, especially in deprived and rural areas. They can be prepared to perform a set of tasks with short training courses.“[Rehabilitation expert 01]Given the current workload of health workers, suitably trained local volunteers could provide a range of rehabilitation services.“Rehabilitation professionals can serve in mobile clinics. For example, one may visit the covered areas every week and provide the necessary services.“[Health policymaker 05]Using mobile rehabilitation teams was mentioned as another solution. Participants believed that mobile rehabilitation teams could provide rehabilitation services, especially in rural and remote areas due to the lack of specialized personnel in non-urban settings.“We now have good Internet throughout the country that we can use to provide telerehabilitation services.“[Rehabilitation expert 04]Telerehabilitation could be used to facilitate the provision of rehabilitation services to remote areas. The development of the Internet across the country could greatly facilitate this approach.“Existence of an effective referral system is a prerequisite for proper integration of rehabilitation services in healthcare, especially primary health care.“[Health policymaker 10]Many participants described how rehabilitation services could be well integrated into PHC if there were effective referral pathways.“There are many professions involved in primary health care, and if there is no team-working, the desired goals will not be achieved!“[Health policymaker 07]As different professions are involved, team working was emphasized as one of the most important strategies for the effective integration of rehabilitation services.“Proper integration of these services in primary health care requires a clear understanding of the role of rehabilitation professions alongside other professions. Also, the knowledge of physicians and other healthcare workers should be enhanced about rehabilitation services to form a good team-working“[Faculty member 06]Nonetheless, clarity on the role of professions involved in PHC, including allied health professionals, and adequate knowledge of physicians and other healthcare workers about rehabilitation professions, can facilitate team working. In this regard, one of the participants said:“If we want to consider rehabilitation services in primary care, we need new financial resources. The government should allocate more resources to this area.“[Health policymaker 03]Integration of rehabilitation services into PHC requires an increase in government funding as a health policymaker said:“Some physiotherapy services can be provided to pregnant mothers during pregnancy. There are also rehabilitation services for infants and children that, if provided timely, can prevent many future problems.“[Physiotherapist 03]An interesting point raised was the need to provide a package of rehabilitation interventions that can be integrated into PHC, such as preventive interventions for pregnant mothers, infants, and children.“How you pay employees is very important to rehabilitation professionals and current primary health care staff. [Payment] Mechanisms should be used to motivate them to perform their tasks.“[Health policymaker 05]Many participants believed that having appropriate and competitive payment mechanisms for the PHC workforce could lead to more effective rehabilitation services provided by PHC staff.“If we want to provide these services well in primary health care, we need to train more rehabilitation professionals in physiotherapy, occupational therapy, and prosthetics/orthotics.“[Faculty member 02]The workforce is one of the most critical challenges addressed in this study. Several solutions were proposed, including increasing the training of the rehabilitation workforce.“Many rehabilitation graduates are not interested in working in primary health care, especially in deprived and remote areas. Rehabilitation assistants can be trained by taking two-year courses.“[Faculty member 04]A curriculum could be developed to train rehabilitation assistants who would be able to work in PHC.“Graduates will no longer be interested in working in the primary healthcare network until we improve the occupational and social status of the workforce in disadvantaged and remote areas.“[Physiotherapist 02]Nonetheless, even if the rehabilitation workforce were increased, there is still a lack of interest amongst rehabilitation professionals in engaging in PHC due to inadequate employment and social opportunities in disadvantaged areas. Therefore, job and social opportunities should be improved for deprived and less privileged areas.“How can we have an effective intervention if we do not have an information system of the needs and records of the patient?“[Faculty member 04]The need for a comprehensive information system was mentioned to facilitate patient follow-up and improve referral pathways in PHC.“There should be a comprehensive system that can well address the rehabilitation needs of the covered population.“[Health policymaker 07]An effective surveillance system that can prepare accurate and timely information on the rehabilitation needs of the population could provide the evidence base for rehabilitation services in PHC.“Some of the required equipment can be provided to employees in remote and rural areas, such as goniometers to measure skeletal deviations and range of motion... Also, some equipment is really expensive and it is better to be in the city or province“[Physiotherapist 03]Providing effective rehabilitation services requires equipment and materials. In this regard, some participants believed that basic equipment and materials could be provided at the rural level and more expensive and sophisticated equipment could be provided at city or provincial levels.Rehabilitation services have been a key but forgotten component of PHC in planning and providing health services. Although these services play an important role in all levels of care, their focus should be close to where people live and work to facilitate their utilization . At presThe first key finding of this study was to design an over-arching concept for integrating rehabilitation services into PHC with respect to legal frameworks, inter-sectoral co-operation, and participation of NGOs and scientific societies. Indeed, effective integration of rehabilitation within the health system depends on the quality of rehabilitation stewardship and governance , 32. AchProposed policy options for providing rehabilitation services at the first level of care include telerehabilitation, mobile rehabilitation teams, and using local capacity to provide rehabilitation services. Previous studies have shown that in health systems where rehabilitation in the primary care infrastructure is very poor, telerehabilitation is a potentially effective way to compensate for this deficiency . CollaboBased on the literature, inadequate financing is one of the most critical challenges of the rehabilitation sector in Iran . The useAn important issue addressed in this study is financing the integration of rehabilitation services into PHC, which means increasing government investment in this area and increasing the utilization of such services by the public. The collaboration of donors and NGOs can be very effective in financing rehabilitation services in PHC in countries like Iran, which face limited financial resources , 33. In Effective integration of rehabilitation services into PHC depends on a trained rehabilitation workforce. However, in addition to the small number of rehabilitation graduates in many countries, especially developing countries , many grThe national health information system often does not include rehabilitation-related information. This hinders rehabilitation policy at all levels and is one of the challenges of strengthening rehabilitation services globally . In IranThe approach to integrating rehabilitation into UHC in each country reflects the characteristics of that country’s health system, its values, the horizontal development of the social welfare system, and the availability of resources and infrastructure . TherefoDespite all the efforts made, the present study has some limitations. Rehabilitation is a broad field consisting of many different professions, of which this study focused on only three: physiotherapy, occupational therapy, and prosthetics/orthotics. In addition, service users were not included in this study, missing important viewpoints.This study has outlined policy options for integrating rehabilitation services into the Iranian PHC network based on the WHO six building blocks framework. In the governance dimension, the leadership of integration, legislation, and participatory decision-making were shown to be the main components of integration policy. Legislation with sufficient executive guarantees is required to put this issue on the policy agenda of the Iranian health system. Considering all policy stakeholders, especially the private sector as the leading actor and provider of secondary and tertiary care rehabilitation services in Iran, the Welfare Organization as the primary steward of disability in the country, and especially the people’s representatives as the users of services and main stakeholders of this policy, can facilitate the integration of rehabilitation services into PHC.


Desmoplastic small round cell tumor (DSRCT) is an aggressive, usually incurable sarcoma subtype that predominantly occurs in post-pubertal young males. Recent evidence suggests that the androgen receptor (AR) can promote tumor progression in DSRCTs. However, the mechanism of AR-induced oncogenic stimulation remains undetermined. Herein, we demonstrate that enzalutamide and AR-directed antisense oligonucleotides (AR-ASO) block 5α-dihydrotestosterone (DHT)-induced DSRCT cell proliferation and reduce xenograft tumor burden. Gene expression analysis and chromatin immunoprecipitation sequencing (ChIP-seq) were performed to elucidate how AR signaling regulates cellular epigenetic programs. Remarkably, ChIP-seq revealed novel DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including WT1  and FOXF1. Additionally, AR occupied enhancer sites that regulate the Wnt pathway, neural differentiation, and embryonic organ development, implicating AR in dysfunctional cell lineage commitment. Our findings have direct clinical implications given the widespread availability of FDA-approved androgen-targeted agents used for prostate cancer. Androgen receptor can promote tumour progression in desmoplastic small round cell tumour (DSRCT), an aggressive paediatric malignancy that predominantly affects young males. Here, the authors show that DSRCT is an AR-driven malignancy and sensitive to androgen deprivation therapy Given the inconspicuous tumor location, nearly all patients present in an advanced stage with abdominal pain or distention, nausea, constipation, and weight loss4. Given its rarity, with an age-adjusted incidence peak incidence of 0.3–0.74 cases per million5, it was not until 1989 that Gerald and Rosai first described DSRCT as a unique clinicopathologic disease6. Shortly thereafter, cytogenetic analyses demonstrated that DSRCT tumors harbor a pathognomonic t(p13:q12) chromosomal translocation that pairs the Ewing sarcoma (ES) gene (EWSR1) with the Wilms tumor suppressor gene (WT1)9. The resulting chimeric 59 kDa fusion protein (FP), in concert with the heterozygous functional loss of the WT1 tumor suppressor protein, promotes an oncogenic effect that reinforces tumor survival and growth10.DSRCT’s cell of origin is unknown, and these tumors universally exhibit a high-grade, poorly differentiated state characterized by cellular nests of unclear lineage, seeming to show epithelial, muscular, mesenchymal, and neural differentiation admixed with prominent desmoplastic stroma12. Given the striking observation that 90% of DSRCT cases occur in post-pubertal males (with an average age at diagnosis of 21.4 years), we investigated how AR contributes to tumorigenesis and survival14. A potential connection between the AR and DSRCT, first reported by Fine et al. in 2006, studied a series of twenty-seven advanced-stage DSRCT patients who had progressed through at least two chemotherapy regimens15. In that retrospective multi-center analysis, 37% of the samples were ≥2+ by immunohistochemistry, and surprisingly, three of six AR+ patients transiently benefited from second-generation combined androgen blockade (CAB) using Lupron and bicalutamide. Despite that promising signal of activity, AR targeting has not been pursued further in the clinic, and at the time of this writing, no additional studies were reported in the literature.The PI3K/AKT and androgen receptor (AR) signaling cascades are among the most frequently activated in cancer16. In addition, we present promising efficacy data using an experimental AR-targeted antisense oligonucleotide (ASO) that significantly delayed tumor growth by suppressing AR expression. Finally, we present chromatin immunoprecipitation sequencing (ChIP-seq) results that suggest a new mechanistic understanding of AR’s role in DSRCT tumorgenicity. Our research findings substantiate DSRCT as a second AR-driven malignancy and implicitly suggest a path toward clinical trials that center on AR-directed treatment options for this otherwise intractable pediatric cancer.In the present work, we use DSRCT xenografts and patient-derived tumor explants (PDXs) to extend the findings by Fine et al. to modern-day AR-targeted therapies, such as enzalutamide, that form the backbone of prostate cancer (PC) treatment19. Unsupervised double-hierarchical clustering correctly separated normal mesenteric tissues from DSRCT, which overexpressed Akt, Syk, PKC-α, and other proteins. Next, we identified proteins enriched in DSRCT compared to another malignancy, using ES as the closest molecular cousin that shares an N-terminus EWSR1 fusion partner. Interestingly, AR and Syk proteins were upregulated in most DSRCT specimens but nearly undetectable in ES  to identify proteins enriched in DSRCT. To determine tumor-specific proteins, we compared protein lysates from DSRCT nodules and paired adjacent normal-appearing mesenteric tissue from the same patients using a well-described RPPA platform enriched for known oncoproteins  from 60 cases treated at MD Anderson Cancer Center (MDACC) to determine the prevalence and cellular distribution of AR staining in patients treated at a single institution. Seventy-five percent of the cores available for analysis were positive for nuclear AR by immunohistochemistry Fig. , usually15.Given the male predominance of DSRCT, new AR protein expression data, and known abundance of AR-targeted therapies used for PC, we elected to pursue AR as a potential therapeutic target in DSRCT. The case report by Fine et al. hinted that DSRCT patients can respond briefly to a first-generation CAB, which heightened our enthusiasm to investigate how AR signaling contributes to DSRCT biology23, we performed a gene expression analysis comparing 22 DSRCT samples to 12 PC samples and a group of other diverse sarcoma subtypes, including 7 chondrosarcomas, 7 well-differentiated liposarcomas, 10 dedifferentiated liposarcoma, and 47 osteosarcoma samples, which served as negative controls. As expected, DSRCT demonstrated significant AR upregulation compared to these other sarcoma samples from chondrosarcoma, well-differentiated and dedifferentiated liposarcoma, and osteosarcoma (p < 0.01–p < 0.0001) but did not surpass the levels observed in PC  interaction pathway, known to initiate cell motility across the ECM barrier26.Compared to other sarcoma subtypes, most DSRCT samples clustered together based upon their expression of 89 genes associated with the androgen pathway, as defined by KEGG  have been implicated in PC tumor progression, an increased risk of biochemical relapse, and inferior overall survival outcomes31, we evaluated these AR activators within the same cohort of 11 DSRCT tumors at the proteomic level using a western blotting analysis. Among three alpha integrin  and beta integrin  subunits commonly observed in mesenchymal tissues, protein expression varied considerably and did not correlate with AR expression . The effects of AR-ASO and control ASOs were also assessed in NSG mice (5 mice/group) bearing a DSRCT PDX .Since only one DSRCT cell line exists, we extended our evaluation of the AR antagonists to the in vivo setting using the JN-DSRCT xenograft and available DSRCT PDXs. Immunocompromised NSG mice bearing JN-DSRCT xenograft tumors treated with enzalutamide or AR-ASO significantly reduced tumor burden and improved survival with the same efficacy, compared to placebo or control groups during the first two months of treatment Fig. . At 2 moPDX Fig. . As expe40. As a negative control, the corticosterone levels were unchanged by AR blockade. In addition, since the antineoplastic action of enzalutamide works by preventing ligand-AR binding, reducing AR shuttling to the nucleus, and impairing AR DNA binding affinity—instead of reducing AR levels  used in our preclinical experiments was well tolerated when administered to PC patients (NCT03300505). Therefore, one could theoretically investigate this AR-ASO drug candidate in DSRCT-specific phase 2 trials without delay. Given the limited nature of our preclinical studies, future studies with enzalutamide are also of interest.To gain a preliminary understanding of the short-term pharmacodynamic effects of AR suppression, a group of JN-DSRCT xenografts and DSRCT PDXs was collected 10 days into their AR-ASO treatment Fig.  were uprp < 1e−7) and heatmap . There, we identified WNT signaling and cell-adhesion as major drivers that are regulated at the chromatin level by AR-dependent active enhancer programs  in PC progressionams Fig. . The genDK6 Fig. . In addiInterestingly, in preclinical tumor samples, we also observed similar enhancer reprogramming. AR-ASO treatment of the DSRCT-xenograft significantly increased the active enhancer and promoter binding sites compared to control ASO treatment, whereas PDX samples showed a moderate increase , or pazopanib, which demonstrates preferential activity in DSRCT and other soft-tissue sarcomas49. Phase II studies testing neoantigen targeted monoclonal antibodies, for example, 8H9 in DSRCT, are also directed at unique sarcoma subtypes50.Ever since Ladanyi and Gerald discovered the EWSR1-WT1 chromosomal translocationAs three-quarters of all DSRCT patients typically succumb to their malignancy within 5-years, our RPPA study intended to define new molecular targets for DSRCT and expand our therapeutic arsenal of biologically targeted therapies that engage them Fig. . SurprisIn contrast to SYK, numerous FDA-approved and experimental AR antagonists were available for immediate preclinical evaluation, and potentially available to patients via compassionate access or early-phase clinical trials. Though our RPPA data and 9:1 male-to-female ratio hinted that DSRCT is an AR-driven malignancy, to prove this explicitly we proposed several criteria, akin to Koch’s postulates: (a) tumors must adequately express AR, (b) DHT must stimulate DSRCT cell proliferation, and (c) AR antagonists should curtail the tumor-promoting effects of androgen stimulation. The inclusion of mechanistic studies, including those directly tying AR to AREs, lends further credibility that DSRCT is a second AR-driven malignancy.51. As discussed briefly in the Introduction, Fine et al. evaluated protein expression of AR, c-Kit, EGFR, and other proteins by WB and IHC, scored using a 5-point scale that ranged between 0 (no staining) to 4+ (highly positive)15. Ten of 27 (37%) DSRCT patients in their case series scored 2+ or higher, but we highlight that 15 demonstrated no AR expression 52. Interestingly, among the pioneer factors that govern the lineage-specific binding of AR to specific genomic loci in PC55, and that control AR-mediated transcriptional regulation of prostate genes (such as PSA)56, FOXA1 was the most enriched MOTIF in JN-DSRCT cells  hexameric half-sites arranged as an inverted palindrome spaced 3-b.p. apart (IR3). Due to differences in local steroid metabolism, ligand abundance, chromatin accessibility, and cofactor occupancy, the DNA binding pattern of AR varies significantly in PC compared to other tissueslls Fig. . Shared lls Fig.  and SE . Shared dependence upon AR for tumor growth and survival provides an exciting opportunity to study AR signaling in a different cancer type and within a younger DSRCT-stricken patient population. Preclinical data using enzalutamide and AR-ASO raises the tantalizing possibility that AR-targeted drugs used for PC may also find utility to combat DSRCT.In compliance with all relevant ethical guidelines, patients provided written informed consent to collect and use their tumor specimens for research purposes using lab protocols LAB08-0151 or LAB04-0890, which are approved by MDACC’s Institutional Review Board. The charts and electronic medical records of patients with a confirmed diagnosis of DSRCT were included for analysis and archived at the MDACC biospecimen bank or the collaborator PIs laboratories. We identified 60 DSRCT patients treated at MDACC from 1990 to 2019 to generate a TMA. Also, we collected 16 DSRCT and 6 ES fresh frozen tumors, all of them were profiled by RPPA. Specialist pathologists used clinical information, immunohistochemistry, and cytogenic analysis for the EWSR1-WT1 or EWSR1-FLI1 fusions to confirm the DSRCT or ES diagnoses.n = 16) and ES (n = 6) specimens collected during a core-needle biopsy or surgical debulking procedures using clinical protocols approved by MDACC’s Institutional Review Board and specimens of normal-appearing mesenteric tissue adjacent to DSRCT obtained at the time of surgical debulking (n = 8) were used for the proteomic analysis (Supplementary Table 63. AR protein detection was performed using the CST antibody (#5153). Additional details about RPPA and WB analyses and normalized data are provided in the Supplementary Methods and Supplemental Data The available snap-frozen DSRCT n = 6 and ES 64. For calculation of gene expression, each gene’s raw count data were first obtained using HTSeq65, and are normalized by scaling the library size using calcNormFactors in the edgeR package66. Then, Voom transformation was applied to normalized counts and a linear model fit to the data for differential expression analysis using the Limma package67. Pathway analyses of differentially expressed genes between two sample clusters were performed using Gene Set Enrichment Analysis68. Fusion transcripts were detected from RNA-seq data using MapSplice69.Total RNA from primary tumor samples was extracted and libraries were made from cDNA using the NuGEN Ovation Ultralow Library System V2 . RNA sequencing reads of the samples were mapped to the hg19 reference genome using the STAR alignerA TMA was constructed from archival surgical pathology materials comprising 60 formalin-fixed, paraffin-embedded (FFPE) tissues from 60 DSRCT patients. Areas of the viable tumor were selected by pathologist review of whole slide H&E-stained sections. Selected areas were punched and transferred, in duplicate, to a recipient block using an ATA-100 Advanced Tissue Arrayer . All human specimens were utilized under an Institutional Review Board-approved research protocol (LAB04-0890) allowing for the retrospective sampling and analysis of existing archival materials collected during standard patient care. Immunohistochemical studies were performed using an autostainer  with anti‐AR  antibody. Additional details about TMA slide preparation and IHC analyses are provided in the Supplementary Methods.The JN-DSRCT, LNCaP, and TC71 cells were tested for their proliferation capacity in vitro using a colorimetric assay in 96-Well plates with WST1 reagent (Roche). The cells were seeded at 3000 cells/well in triplicates with 10% FBS DMEM (JN-DSRCT) or RPMI (TC71 and LNCaP) complete media. Additional details about WST1 cell proliferation assays are provided in the Supplementary Methods. JN-DSRCT and TC71 cell lines were supplied by JL. LNCaP and PC3 were obtained through MDACC’s cell line repository.JN-DSRCT cell line exhibiting a pathognomonic t translocation was generously provided from Dr M. Kikuchi’s laboratory . In addition, PC3, LnCaP, and TC71 cell lines are provided by the MDA cell lines core facility. All available cell lines in Dr Ludwig’s lab are registered within the MDA characterized cell line core (CCLC). Each cell line identity is validated twice per year in MDA CCLC using short-tandem repeat fingerprinting with an AmpFLSTR Identifier kit. Furthermore, according to the manufacturer’s protocol, all our cell lines are tested twice per year for mycoplasma contamination using the MycoAlert Detection Kit (Lonza Group Ltd.). In addition, cell lines are sent for third-party mycoplasma testing using a sensitive PCR testing approach any time a collection of cells are cryopreserved.Monolayer JN-DSRCT cell culture in 8 chamber slides was fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS). The primary JN-DSRCT xenograft and PDX tumors were harvested, fixed in 10% formalin, embedded in paraffin (FFPE), and then sliced in 5-μm sections before processing them for antigen retrieval using 0.1 M citrate buffer for 20 min and in a vegetable steamer. Altogether, monolayer and primary tumor slides were permeabilized and blocked with superblock buffer  for 1 h at room temperature. Slides were then incubated consecutively with primary antibodies to AR , (overnight at 4 °C) and Alexa Fluor 488-labeled Goat-anti-Rabbit  (for 1 h at room temperature). The nuclei were visualized using Hoechst , and the immunofluorescence was acquired after subtracting the background intensities using the Nikon A1-Rsi confocal microscope (Nikon). Fluorescent detection of proteins in the nuclei and cytosolic regions was quantified using the Imaris software (Bitplane) and its Cell module that use validated algorithms to define the segmentation by permitting the recognition of selected protein fluorescence in both nuclear and cytosolic regions.null mice 6 weeks in age  were subcutaneously injected with JN-DSRCT cells  or received PDX explants (2 mm) to generate DSRCT xenograft and PDX mouse models. The histologic and genetic analyses of DSRCT patients and PDX tumors are available in Supplementary Fig. 3, 5 mice per group received enzalutamide , or AR-ASOs , or control ASOs , or a placebo control (sterile vehicle buffer). Tumor volumes were measured using digital calipers at study initiation and 2–5 times per week after that for up to 85 days, or until their tumors reached 1500 mm3, whichever came first. Per institutional requirements, tumor size never exceeded 2 cm in maximal linear dimension. A Kaplan–Meier analysis was performed to assess drug efficacy. Statistical analyses between the control and treated groups or between different treated groups were performed with the log-rank (Mantel–Cox) test using GraphPad Prism 8.0.All experiments were conducted per protocols and conditions approved by the University of Texas MDACC  Institutional Animal Care and Use Committee (eACUF Protocols #00000712-RN03). Male NOD (SCID)-IL-2Rg40.Testosterone and corticosterone quantification was determined using Agilent’s Infinity II UHPLC in line with a 6495 triple quadrupole mass spectrometer and MassHunter workstation software (8.0.8.23.5). Briefly, DSRCT xenograft and PDX samples were homogenized using water containing internal standard  extracted with tert-butyl methyl ether (Sigma 34875), dried under nitrogen, and derivatized using hydroxylamine hydrochloride (Sigma 431362). The recovered ketoxime steroids were reconstituted in methanol/water (1:1 v/v) and injected into the Infinity II UHPLC. Ketoxime steroids were separated using a Chromolith reverse-phase column  and introduced into a JetStream source (Agilent) for triple quadrupole analysis. Data were analyzed and quantified using MassHunter software (Agilent)70 with optimized shearing conditions and minor modifications for JN-DSRCT cells. The antibodies used were: H3K27ac (Abcam ab4729) and AR (CST#5153). Briefly, 3 million cells per sample were cross-linked using 1% formaldehyde for 10 min at 37 °C. After quenching with 150 mM glycine for 5 min at 37 °C, cells were washed twice with ice-cold PBS and frozen at −80 °C for further processing. Later, cells were thawed on ice and lysed with ChIP harvest buffer  for 30 min on ice. Lysed cells were sonicated with Bioruptor (Diagenode) to obtain chromatin fragments. Antibody-dynabead mixtures were incubated for 1 h at 4 °C and cellular extracts were then incubated overnight with these mixtures. After overnight incubation, immune complexes were washed five times with RIPA buffer, twice with RIPA-500 (RIPA with 500 mM NaCl) and twice with LiCl wash buffer . For reverse-crosslinking and elution, immune complexes were incubated overnight at 65 °C in elution buffer . Eluted DNA was then treated with proteinase K (20 mg/ml) and RNase A and DNA clean-up was done using SPRI beads (Beck- man-Coulter). ChIP libraries were amplified and barcoded with use of the NEBNext® Ultra™ II DNA library preparation kit (New England Biolabs). After library amplification, DNA fragments were size-selected (200–500 bp) using AMPure XP beads (Beckman Coulter) and assessed using high sensitivity D1000 screen tape on the Bioanalyzer (Agilent Technologies). Libraries were multiplexed together and sequenced in HiSeq2000 (Illumina).Chromatin immunoprecipitation was performed as described earliert-tests with the GeneSpring GX software program version 12.6.1-GX (Agilent Technologies). For multiple comparison testing, proteins whose expression levels between specimen types were two-fold and significantly different (p ≤ 0.05) were subjected to unsupervised hierarchical clustering. Differentially expressed proteins identified by RPPA were confirmed by WB analysis. The RPPA data for the proteomic comparison of DSRCT patient tumors, normal mesenteric tissues, and ES patient tumors are available from the Gene Expression Omnibus repository under GSE108687 series.Raw log2 intensity values were normalized for global protein expression by median centering across 151 antibodies tested. Individual protein expressions in DSRCT specimens, ES specimens, and mesenteric normal tissue specimens were compared using two-sided unpaired 62. Lysis buffer  containing freshly added protease and phosphatase inhibitors (Roche Applied Sciences) was applied to lyse cellular washed pellets via cold incubation. Next, protein extraction from xenograft tumors was performed by homogenizing approximately 10 mg of frozen tissue in 500 μL of the lysis buffer using an electric tissue homogenizer (PRO Scientific). The homogenized tumors were incubated at +4 °C for 2 h to complete their dissociation and lysis. Altogether, the total lysed proteins from tumors were collected after centrifugation, quantified using BCA protein assay kit (Thermo Fisher Scientific), and stored at −80 °C until further analyses. WBs were performed as previously described by our group62. Protein detection was performed using a list of antibodies provided in the above supplementary table of antibodies used for WBs. The immune-reactive proteins were captured using horseradish peroxidase-conjugated secondary anti-rabbit IgG or anti-mouse IgG antibodies , amplified using the SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific), detected using the Chemi-Doc system (Bio-Rad), and quantified for their densitometry using the ImageJ Gel Analysis tool .The preparation of extracted protein from cells, DSRCT patient tumors, or xenograft animal tumors for western blotting validations was prepared as described previouslyFour‐micrometer unstained slides were prepared from this TMA. Immunohistochemical studies were performed using an autostainer  with anti‐AR  antibody. This primary antibody was detected using a Bond polymer refine detection kit according to the manufacturer protocol . Then, the slides were dehydrated in grade alcohols, cleared in xylene, and cover slipped. The TMA staining results were scored on intensity  as well as the percentage of tumoral labeling (positive (>50%), low positive (10–50%), focal (1–10%), and negative (0–1%)).The cells were cultivated for 24 h to let them adhere, before adding serially diluted concentrations of dihydrotestosterone , or AR-based drug treatments  for up to 48 or 72 h, respectively. WST1 was added, and cells were incubated for an additional 2 h. Cell proliferation was measured at 450 nm in a microplate reader . In both assays, the agonistic effect of DHT or cytotoxic effect of the drugs was expressed as a percentage of cell proliferation. IC50 values were calculated by the sigmoidal dose-response curve-fit using Prism GraphPad 8.0.Primary tumors from the DSRCT xenograft and PDX1 group of mice, untreated and treated with control-ASO or AR-ASO, were fixed in 10% formalin, embedded in paraffin, and then sliced in 5-μm sections. The EZ-retriever microwave-based pretreatment and antigen retrieval system  was used for dewaxing, rehydration, and antigen retrieval of these FFPE lung tissue sections. AR protein expression by IHC was evaluated on Leica Bond MAX Autostainer by using primary antibody, rabbit antibody anti-human AR . The primary antibody was detected according to the manufacturer's protocol . Then, the slides were dehydrated in grade alcohols, cleared in xylene, cover slipped, and imaged with Keyence Microscope  at ×20 resolution. The digital images were processed and quantified using the Visiopharm software version (2020.04) . An APP  was designed to quantify cell-based DAB staining in DSRCT preclinical samples using traditional thresholding methods. The algorithm was built around a three-step approach: (1) pre-processing: HDAB-DAB and HDAB-hematoxylin features were used to detect AR-positive cells (green masking) and negative cells (blue masking), respectively, by setting pixel values. A median filter of size 5 × 5 was used for the proper segmentation of cells. (2) Post processing: additional steps were designed to enhance the performance of the APP. Change by shape excludes the artifacts; merged cells were separated by separate labels step, and certain clear areas in the cells were filled with fill holes step to fully mask the cells. (3) Output variable: mean intensity of each cell and from each sample was extracted and exported into a spreadsheet. Finally, the data were plotted on a Scatter Plot using GraphPad Prism software, version 8 showing the AR Mean Intensity of each cell from all the analyzed samples, and the AR Average mean intensity from each preclinical group of treated mice.71, a snakemake72 based ChIP-seq pipeline. Briefly, raw reads were mapped by bowtie173 to hg19. Duplicated reads were removed, and only uniquely mapped reads were retained. RPKM normalized bigwigs were generated by deep tools74, and tracks were visualized with IGV75. Peaks were called using macs1.476 with a p value of 1e–9 for H3K27ac and 1e–7 for AR. Heatmaps were generated using R package EnrichedHeatmap. ChIP-seq peaks were annotated with the nearest genes using ChIPseeker77. SEs were identified using ROSE78 based on H3K27ac ChIP-seq data.ChIP-seq data were quality controlled and processed by pyflow-ChIP-seq79. The peaks were annotated with ChIPseeker R package77, using addFlankGeneInfo function for enhancers.To identify variable AR or enhancer domains enriched in specific DSRCT samples, enhancer peaks that overlap with 2.5 kb upstream and 2.5 kb downstream of any known TSSs were removed. The unique and shared peaks within multiple groups were identified by Intervene80 for pathway analysis, restricted to GO, KEGG, Hallmark, and WiKi gene sets. The Enrichplot package81 was used to generate dot plot and bar plot for gene sets enriched with a false discovery rate cut-off of <0.05.Differential AR binding sites and enhancers associated genes in each sample were imported into the ClusterProfiler82.To identify the motifs over-represented within AR-specific peak sets, we used the HOMER motif database and the coordinates of AR-specific peak setst-test p values are two-sided. To enhance reproducibility, no less than five animals were assigned per group. Three or more regions of interest were randomly assessed for each microscopy image, and representative images are shown.Bar and scatter dot plots are presented as mean values ± SD. Box plots indicate the median value and interquartile range; whiskers show minimum and maximum data points. All Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Mast cells (MCs) are innate immune system effector cells that play a pivotal role in innate immunity and modulating adaptive immunity, rendering them as key cells of allergic inflammation and thus of allergic diseases. MCs are typically located in body surfaces exposed to the external environment such as the nasal mucosa. Due to their location in the nasal mucosa, they are in the first line of defense against inhaled substances such as allergens. IgE-dependent activation of MCs in the nasal mucosa following exposure to allergens in a sensitized individual is a cardinal mechanism in the pathophysiology of AR. This review is a comprehensive summary of MCs’ involvement in the development of AR symptoms and how classical AR medications, as well as emerging AR therapies, modulate MCs and MC-derived mediators involved in the development of AR.Allergic rhinitis (AR) is a common rhinopathy that affects up to 30% of the adult population. It is defined as an inflammation of the nasal mucosa, develops in allergic individuals, and is detected mostly by a positive skin-prick test. AR is characterized by a triad of nasal congestion,  Allergic rhinitis (AR) is a common allergic inflammatory rhinopathy that affects up 20 to 30% of adults and up to 40% of children worldwide . Rhinitirhinorrhea, nasal congestion and blockage, sneezing, and/or itching of the nose. These symptoms may significantly impact the patients’ quality of life, often interfering with sleep and contributing to poor academic and work performance [AR is classically characterized by ongoing symptoms of formance .Diagnosis of AR is usually made clinically when an individual experiences one or more of the hallmark symptoms mentioned above in response to allergen exposure. AR is classified as seasonal when the symptoms are experienced seasonally  and perennial when the symptoms are experienced year-round. Another classification method is based on the length and recurrence of the symptoms. Intermittent AR (IAR) is defined as symptoms persisting for less than 4 days/week or less than 4 weeks, while persistent AR (PAR) is defined as symptoms lasting for more than 4 days/week for at least 4 weeks .To confirm the diagnosis, sensitization to allergens is assessed by an allergy skin-prick test or an in vitro antigen-specific IgE test. Clinical allergy is evidenced by active symptoms upon allergen exposure in a sensitized individual. A unique condition is local allergic rhinitis, which is characterized by local IgE production in the nasal tissues whilst systemic allergy testing is negative .Management of AR symptoms starts with allergen  avoidance. AR symptoms are mainly controlled via pharmacotherapeutic agents. These include antihistamines, intranasal corticosteroids, leukotriene receptor antagonists (LTRAs), cromolyn sodium, biologics, and immunotherapy .milieu of inflammatory cells and mediators contributes to the late and chronic phase response of allergic inflammation [As elaborated in detail later in this review, exposure to inhaled allergens in a sensitized individual starts the cascade leading to the AR symptoms via activation of MCs lining the nasal mucosa through crosslinking of antigens to a specific IgE antibody bound to the surface of MCs, resulting in the release of MC mediators. The latter contribute to the immediate allergic response and to further recruitment and infiltration of immune cells to the site of inflammation, including eosinophils, B cells, and T cells. The primary T-cell response in AR is mediated by T-helper (Th2) cells. Th2 cells secrete cytokines such as interlukin-4 (IL-4) and interlukin-13 (IL-13), which drive further IgE production. This ammation ,6.In addition to AR, MCs’ role is established in the pathogenesis of frequent comorbidities characterized by allergic inflammation such as atopic dermatitis (AD) and allergic asthma. To promote allergic inflammation, MCs interact with other inflammatory cells via soluble mediators and cell–cell contact. One of these interactions occurs through the allergic effector unit (AEU), where CD48 on MCs crosstalk with a 2B4 receptor on eosinophils, promoting an inflammatory outcome ,8,9.rhinorrhea, hyposmia, and facial pain for more than 12 weeks in combination with inflammatory signs confirmed by nasal endoscopy or by a computed tomography scan. Phenotypically, CRS is divided into two groups based on the presence or absence of nasal polyps: CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP) [A differential diagnosis of AR includes other types of sinonasal diseases because symptoms may overlap. In fact, more than one type of rhinitis may coexist at the same time . An exam(CRSsNP) ,10,11. EAnother condition associated with CRSwNP is aspirin-induced respiratory disease (AERD), which is defined as a triad of adult onset asthma, CRSwNP, and anaphylactic response to COX-1 inhibitors . Activation of MCs by classical (IgE-mediated) and nonclassical pathways is crucial in the pathophysiology of AERD due to the secretion of a diverse array of inflammatory mediators that activate different Type 2 inflammatory pathways involved in AERD pathogenesis .As demonstrated above, MCs are essential players in the development of allergic diseases, including AR. The allergic reaction consists of an early phase mediated by the release of inflammatory mediators from preformed granules of MCs and a late phase characterized by the influx of inflammatory cells, which are mostly modulated by MCs and their secreted mediators ,11.MCs are located in the major body surfaces exposed to the external environment. This includes the skin, where connective tissue MCs are primarily found; the epithelium of the gastrointestinal tract; and the respiratory tract, where mainly mucosal MCs are found. The content of the preformed granules divides MCs into two phenotypic subtypes: mucosal MCs producing only tryptase and connective tissue MCs producing tryptase, chymase, and carboxypeptidase .Due to their strategic positioning in the exposed body linings and the stored granules laden with preformed mediators, MCs may respond to invading stimuli more rapidly than other tissue-resident immune cells ,15, thusFcεRI is a high-affinity IgE receptor found on the surface of MCs and basophils. Allergen crosslinking of the IgE-bound FcεRI receptors results in the release of preformed and newly synthesized mediators in a phasic fashion. A few seconds after activation, MCs degranulate and release the stored content of the preformed granules, which contain histamine, heparin, proteases, and TNFα .In addition, arachidonic acid is freed from membrane phospholipids and de novo synthesized arachidonic acid–lipid metabolites—mainly prostaglandin D2, leukotriene LTC4/D4/E4, and LTB4—are also promptly released as part of the immediate MC response. ,18 LaterMCs promote the trafficking of immune cells to the site of infection or inflammation, making them an essential initiator of the inflammatory response. MCs recruit circulating eosinophils through direct secretion of eosinophil-attracting chemokines  and indirectly via secretion of histamine, inducing eotaxin secretion by endothelial cells .MCs and eosinophils are the key effector cells in allergy and communicate with each other in a bidirectional manner in what is defined as the “Allergic Effector Unit”. This crosstalk is mediated by physical cell–cell contact through cell surface receptors/ligands and by released mediators, including specific granular factors, chemokines, cytokines, and their respective ligands. In the physical cell–cell crosstalk, CD48 on MCs and its high-affinity ligand, the 2B4 receptor on eosinophils, form a pivotal couple, initiating and maintaining this proinflammatory crosstalk .Through complex cellular processes, MCs can influence the induction, amplitude, and function of the adaptive immune response. Via histamine secretion, MCs increase vessel permeability, aiding in the recruitment of adaptive cells to the site of inflammation .Dendritic cells (DCs) are antigen-presenting cells (APCs) identified by their high expression of major histocompatibility complex (MHC) class II molecules (MHC-II). In the tissue, they detect homeostatic imbalances and process antigens for presentation to T lymphocytes, linking the innate and the adaptive immune responses with one another . MCs sec+ T lymphocytes (Th0s) in the draining lymph nodes. Th0s are activated and transformed into T-helper 2 CD4+ lymphocytes (Th2s) under interleukin-4 (IL-4) cytokine stimulation. Th2 cells, together with ILC2, release IL-4, IL-5, and IL-13 to initiate the adaptive immune response. IL-4 and IL-13 then stimulate specific B-cell lymphocytes to differentiate into antibody-producing plasma cells [In the nasal mucosa, APCs are located in paracellular and intercellular channels adjacent to the basal epithelial cells. APCs process inhaled allergen-derived proteins deposited in the nasal mucosa and present them to naïve CD4ma cells ; IL-4 anma cells ,22.Additionally, exposure to allergens induces structural cells in the nasal mucosa  to release cytokine alarmins including thymic stromal lymphopoietin (TSLP), IL-25, and IL-33, which in turn promote the downstream production of proinflammatory Type 2 inflammatory cytokines by effector immune cells ,23,24.mepolizumab) and anti-IL5R antibodies  that interfere with the IL-5 pathway [As mentioned earlier, eosinophils engage MCs in a physical and soluble crosstalk via the AEU. MCs and eosinophils produce mediators that enhance the activation status of both cells . Humaniz pathway  may thus pathway .tezepelumab) in asthma and atopic dermatitis [TSLP, which is secreted by structural cells in the nasal mucosa, binds the TSLP receptor (TSLPR) expressed by MCs, inducing their release of various cytokines and chemokines  . Due to rmatitis .rhinorrhea. Activation of H1 and H2 receptors of nasal blood vessels by histamine contributes to increased vascular permeability and vasodilation, leading to symptomatic nasal congestion and enhanced leukocyte recruitment to the inflamed nasal mucosa [Histamine, one of the preformed mediators in MCs, is known to be a major factor of the acute allergic response. Its release from MCs initiates the immediate (or early) phase response, typically occurring within minutes after allergen exposure and lasting for 1–2 h after exposure. Histamine binds to four types of G-coupled receptors. In the nasal mucosa, histamine induces activation of H1 receptors on sensory nerves of the afferent trigeminal system, which in turn transmit signals to the central nervous system (CNS), causing symptoms of itching and motor reflexes; i.e., sneezing. Via activation of sensory and parasympathetic nerves, histamine stimulates mucous glands to secrete a watery discharge, which manifests clinically as l mucosa ,28,29.rhinorrhea in AR [In addition, MCs release growth factors  and vascular endothelial growth factor (VEGF)), which are angiogenic and increase vasodilation and vascular permeability in the nasal walls, leading to enhanced inflammatory cell infiltration, local edema, and swelling of the nasal mucosa, which contribute to the clinical symptoms of nasal congestion and watery ea in AR . During the early and late phase responses, MCs release cytokines and chemokines to attract additional inflammatory cell types to the nasal mucosa, including neutrophils and eosinophils, group 2 innate lymphoid cells (ILC2), and Th2 cells. An influx of these cells in the nasal mucosa characterizes the late-phase response, which typically occurs within 5 h following the initial allergen exposure and lasts up to 24 h. The late-phase response is complex due to the secretion of various cytokines and chemokines from an array of migratory cells that interact together to sustain the inflammation and prolong its symptoms through the release of additional cytokines  Figure .Cysteinyl leukotrienes and PGD2 released from MCs act by increasing the vascular permeability in the nasal mucosa ,31,32. IResolution of inflammation can be expected after the two phases of allergy (early and late phases). However, due to the continuous exposure to the allergen(s) and/or the severity of the disease or atopy, allergic inflammation may evolve into a chronic phase when the allergic reaction fails to resolve . During CD48 is the high-affinity ligand for 2B4, an activating receptor expressed notably on eosinophils. The interaction between CD48 and 2B4 on MCs and eosinophils is an important mechanism by which MCs and eosinophils affect and communicate with one another within the AEU.Our group demonstrated in a recent immunohistochemical study that MC infiltration in nasal polyps (NPs) obtained from CRSwNP patients was higher in allergic patients in comparison to nonallergic patients. In addition, membranal CD48 (mCD48) expression on eosinophils infiltrating nonallergic asthmatic NPs was highly elevated in comparison to allergic asthmatic polyps, allergic nonasthmatic polyps, and nonallergic nonasthmatic polyps. Similarly, mCD48 and the expression of its high-affinity ligand m2B4 on eosinophils from enzymatically digested NPs were significantly higher in nonallergic asthmatics in comparison to allergic asthmatics, possibly indicating that allergy attenuates mCD48 expression on eosinophils .Recent findings demonstrated that sCD48 (the soluble form of CD48) levels were significantly higher in an intermittent allergic rhinitis group (IAR) of patients in the pollen season compared to their levels (in the same group of patients) not during pollen season. These results suggested that sCD48 may be a biomarker to the exacerbation phase in patients with IAR and that CD48 could be plausibly implicated in the pathogenesis of IAR .A recent study aimed to compare the immune gene expression profiles in the nasal mucosa and peripheral blood samples between adults with AR and healthy controls. The results showed a significant upregulation of allergy-related genes in the nasal mucosa samples obtained from the AR group in comparison to the control group. Notably, chemokines genes, including CCL17, CCL26, and TPSAB1 (which encodes tryptase) were upregulated. Counts of differentially expressed genes (DEGs) in the nasal mucosa samples positively correlated with eosinophil and dust-mite-specific immunoglobulin E (IgE) counts in the blood. The study concluded that distinct gene expression profiles in the blood and nasal mucosa samples were observed between AR patients and healthy controls. The results also provided evidence for the close interaction between the local site and systemic immunity. The genes identified in this study contributed to the current knowledge on AR pathophysiology and may serve as biomarkers to evaluate the effectiveness of treatment regimens or as targets for therapeutic development .The allergic reaction in AR is characterized by an interplay between inflammatory cells infiltrating the nasal mucosa and released mediators that provokes the main symptoms of AR. Available therapeutic strategies in allergic diseases including AR, asthma, and atopic dermatitis are aimed largely at targeting MCs to suppress the allergic process .Once a diagnosis of AR has been established, the standard of care includes a treatment plan that considers the severity of the disease; the presence of concomitant allergic diseases; and most importantly, a shared decision-making process that focuses on the patient’s preferences. The standard treatment algorithm for AR begins with allergen avoidance, including limiting exposure to relevant allergens, maintaining a humidity of less than 40% at home to prevent dust mites and mold, and use of high-efficiency particulate air (HEPA) filters to remove allergens from the inhaled air. If the symptoms persist despite avoidance strategies, pharmacologic options are discussed with the patient. The first-line approach is to use symptomatic treatment starting with antihistamines (AHs) and intranasal glucocorticosteroids (GCSs). For more severe disease, topical anti-inflammatory therapy is considered, and lastly, allergen-specific immunotherapy (AIT) may be employed, thus providing a bonus benefit due to its disease-modifying effects. Still, none of these modalities is optimal .Antihistamines are considered the first line of treatment for symptomatic AR and elicit their effects by blocking the histamine receptors expressed on nasal mucosa cells, thus preventing their activation by histamine secreted by MCs. Currently available oral antihistamines for the treatment of AR include first-generation antihistamines  and second-generation antihistamines . Both first- and second-generation antihistamines are effective at controlling symptoms of AR [ms of AR .Intranasal antihistamines  are also available and are more efficacious and rapid in their symptomatic relief than oral antihistamines . Anti-H3Intranasal GCSs  may also be used as a first-line symptomatic monotherapy, as an adjunct to intranasal antihistamine, or in combination with oral antihistamines in patients with mild, moderate, or severe symptoms. Studies have shown that intranasal GCSs are superior to antihistamines in effectively reducing nasal inflammation and improving mucosal pathology. Oral and injectable GCSs may cause significant systemic adverse effects; therefore, even though they have been shown to be effective in alleviating AR symptoms, their use is not recommended and routine use should be avoided [ avoided ,39.Concerning the mechanism of action of GCSs in AR, they are primarily used due to their ability to suppress various stages of the allergic inflammatory reaction as a result of modifications in gene transcription . Their mLeukotriene receptor antagonists (LTRAs) such as montelukast and zafirlukast block leukotriene receptors on immune cells, thus blocking activation of the latter by leukotrienes secreted from activated MCs and other inflammatory cells such as eosinophils. LTRAs were found to be beneficial in patients with AR and resulted in significant improvements in daytime nasal symptoms and the quality of life [rhinorrhea by blocking leukotriene-activated inflammation in the nasal lavage fluids and airways [ of life . Moreove airways . In termCromolyn sodium (CS) is an MC stabilizer that prevents the subsequent release of inflammatory mediators including histamine and leukotrienes. It is an FDA-approved medication for adjunctive treatment of allergic rhinitis that is used in the form of nasal inhalation and is capable of effectively reduce sneezing, rhinorrhea, and nasal pruritus [pruritus . Recent pruritus .Omalizumab is a human monoclonal antibody designed to target and block IgE. It is capable of reducing free (and hence bound) IgE, downregulating high-affinity IgE receptors, and limiting MC degranulation, thus minimizing the release of mediators throughout the allergic inflammatory cascade. A meta-analysis that assessed the efficacy and safety of omalizumab in inadequately controlled AR in randomized controlled trials (RCTs) found that it reduced the Daily Nasal Symptom Severity Score (DNSSS), improved the Daily Ocular Symptom Severity Score (DOSSS), improved the Rhinoconjunctivitis Quality of Life Questionnaire, and reduced the mean daily consumption of antihistamines. In addition, in a recent study, long-term therapy with omalizumab was demonstrated to be effective and safe in treating severe persistent AR and concomitant asthma. These results supported the efficacy and safety of omalizumab in the management of patients suffering from inadequately controlled AR in conventional treatment [reatment ,45. Omalreatment . Howeverreatment .Dupilumab is a fully humanized monoclonal antibody that exerts its effects by targeting the interleukin IL-4Rα subunit of the IL-4 and IL-13 receptors, thus inhibiting the signaling of IL-4 and IL-13 , a primary molecule in MCs’ preformed granules. Cytokines such as IL-33, IL-25, TSLP, and eotaxin should be further investigated as well due to their ability to promote continuous recruitment of eosinophils and T-helper cells to the inflamed tissue, which maintains the chronic inflammatory phase. Finally, further investigation of these ligands and the role of cytokines in AR should be carried out, as they may be potential novel targets for directed AR pharmacologic therapy or biomarkers for the evaluation of AR treatment regimens.


Spinal stretch reflex (SSR) hyperexcitability reflected by the H-reflex has been reported in more strongly affected extremities after stroke. The H-reflex in the lower extremities is modulated by body position normally and alternatively modulated post-stroke.This study aimed to preliminarily explore how upper extremity (UE) H-reflexes are modulated by body position after stroke, which remains unknown.Three patients after stroke with hemiparesis/hemiplegia were included. Bilateral flexor carpi radialis (FCR) H-reflexes were examined in the supine position while standing. Other clinical evaluations include the modified Ashworth scale (MAS) and postural stability measurement.The three cases herein showed that (1) SSR excitability was higher in more strongly affected UEs than less-affected UEs, (2) down-modulation of SSR excitability occurred in less-affected UEs in static standing compared with the supine position, but modulation of SSR excitability in more-affected UEs varied, and (3) bilateral UE SSR excitability in case 3 was down-modulated the most. Moreover, case 3 showed no difference in muscle tone of the more affected UE between supine and standing positions, and case 3 showed the best postural stability.Spinal stretch reflex hyperexcitability in strongly affected UEs could commonly occur in different phases of recovery after stroke. Down-modulation of SSR excitability could occur in less-affected UEs in the standing position compared with the supine position, while modulation of SSR excitability might be altered in strongly affected UEs and vary in different phases of recovery. There could be some correlation between postural control and UE SSR hyperexcitability. The H-reflex may help to offer a new perspective on rehabilitation evaluation and interventions to promote UE motor control after stroke. Upper extremity (UE) movement dysfunction is the most common and stable symptom in stroke survivors , often pIt is widely accepted that the increase in flexion synergy patterns is mediated by an exaggerated spinal stretch reflex (SSR). The H-reflex has been usually employed as a measure of SSR excitability to investigate spinal neuronal pathway health in patients with neurological disorders. It is evoked by low-intensity electrical stimulation of the Ia afferent, resulting in the monosynaptic excitation of motor neurons. Changes in H-reflex amplitude are usually used to assess different types of inhibition acting on Ia afferent terminals, including homosynaptic depression, presynaptic inhibition, reciprocal Ia inhibition, Ib inhibition, and recurrent inhibition .It has been reported that SSR hyperexcitability is reflected by the H-reflex in more strongly affected upper and lower extremities after stroke , 6. SeveThe objective of this study was to preliminarily explore how UE H-reflexes were modulated by different body positions after stroke. The study presented three cases in different phases of stroke recovery. The muscle chosen for the H-reflex examination was the flexor carpi radialis (FCR), which has been commonly used. Furthermore, measurements of the modified Ashworth scale (MAS) in two different body positions as well as postural stability in static standing were also assessed. We hope that this study can provide new perspectives for evaluation and intervention in the rehabilitation of strongly affected UEs after stroke.The patients enrolled in this study had been participating actively in rehabilitation before the study commenced, and they had a great interest in learning about their motor recovery in their UE. Thus, they agreed to be enrolled in the study and were well informed regarding the study's purpose and their rights as participants.A 38-year-old right-hand-dominant male patient suddenly presented with slurred speech, a drooping mouth, and weakness in his left extremities. The patient was diagnosed with ischemia in the right parietal lobe and received thrombolytic therapy. Two hours later, he had a severe headache and was nauseated. A new CT scan showed a hemorrhage in his right basal ganglia. Craniotomy was suggested but refused by the patient. He was then given conservative treatment. After 18 days in stable condition, he was referred to rehabilitation to receive physical, occupational, speech, and acupuncture therapy. He was admitted to our study 59 days after stroke onset, in the rehabilitation phase of recovery. The patient's relevant past medical history included hypertension and long-term use of valsartan (80 mg qd) to manage blood pressure.via the left lateral fissure under general anesthesia, and an intracranial pressure probe was placed. After 38 days in stable condition, the patient was referred to rehabilitation for physical, occupational, speech, and acupuncture therapy. He was admitted to our study 221 days after stroke onset, in the chronic phase of recovery.A 38-year-old right-hand-dominant male patient underwent cerebral hemorrhage in his left basal ganglia as well as radiographic coronal area and had a seizure. In the emergency room, the intracerebral hematoma was removed Six and a half months after stroke onset, to reduce spasticity in his more affected UE, he received multipoint Botox injections in the right pectoralis major, biceps brachii, brachioradialis, teres pronator, and triceps surae.A 71-year-old right-hand-dominant female patient presented with weakness in her left extremities and dizziness. She was diagnosed with ischemia in her right basal ganglia and lateral paraventricular region. There were also multiple older ischemic foci and lumens in the bilateral basal ganglia, lateral ventricle, and the centrum semiovale. The patient then received active symptomatic treatment. One day later, her motor dysfunction had gotten worse and she suffered from slurred speech and occasional choking. Ganglioside and other active symptomatic treatments were further prescribed. After 28 days in stable condition, she was referred to rehabilitation for physical, occupational, speech, and acupuncture therapy. The patient was admitted to our study 50 days after stroke onset, in the sub-acute phase of recovery.The case data were retrieved from the Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine. We included three cases in total. The present study followed the international recommendations of the CARE checklist for drafting reports of case studies . All parThe FCR H-reflexes recruitment was recorded using electrodiagnostic equipment . The signals were amplified and digitized using a three-channel amplifier . Data were collected at a sampling rate of 48 kHz and band-pass filtered . Active shielding and grounding of the cables were used to minimize the power line (50 Hz) interference. Before FCR H-reflex examination, the skin of the patient's forearm was cleaned with 75% isopropyl alcohol solution, and FCRs in bilateral UEs were located by finding the fullest FCR muscle belly, at about one-third of the distance between the medial epicondyle and the radial styloid . SurfaceWith the intensity of the electrical stimulation current increasing gradually starting from 0.0 mA, the H-reflex trace began to appear in the EMG traces as the excitation threshold of the Ia afferents was reached. When increasing stimulus intensity, the M-wave appeared and was followed by the H-reflex as the excitation threshold of α motor neurons was reached. The peak-to-peak amplitude of the H-reflex increased until a maximum value (Hmax) was reached and subsequently decreased. As more α motor neurons were recruited, the M-wave increased until its maximum value (Mmax) was reached. Hmax and Mmax were recorded, and the Hmax/Mmax ratio was calculated, which was considered to represent the proportion of the motor neurons recruited by a monosynaptic reflex in the motor neuron pool and reflect the excitability of motor neurons and inhibition that mediate the Ia afferent volley . In ordeThe FCR H-reflex was examined in bilateral UEs in both the supine and standing positions. First, the patients were instructed to lie in the supine position on the examination bench, keeping their extremities extended and relaxed, with both palms facing up, the legs slightly spread, and the head and trunk in a centered neutral position. The patients were instructed to relax on the couch for 2 min before the FCR H-reflex examination. After the FCR H-reflex examination in the supine position, the patients were instructed to change the standing position without support. In standing position, the patients were asked to stand still with their head and trunk centered and neutral, their feet hip-width apart with their toes facing forward, and UEs hanging naturally on both sides of the torso. The examination in the standing position was started after a 2-min relaxation period. The examination is illustrated in The MAS was assessed in the elbow and wrist flexors of the more strongly affected UEs in both the supine and static standing positions. The proximal part of the UE was stabilized, and the distal part was moved passively by the evaluator through its available range of motion from a position of maximal flexion to maximal extension over a period of about 1 s by counting “one thousand and one”. Two repetitions were performed, and the evaluator took an average score of the two.The resistance of muscle tone was scored on the following 6-point scale: 0, no increased resistance; 1, slightly increased resistance ; 1+, slightly increased resistance ; 2, clear resistance throughout most of the range of motion; 3, strong resistance with which passive movement is difficult; and 4, rigid flexion or extension.The patients were instructed to stand with their head and trunk upright, eyes looking forward, both UEs hanging naturally, and their feet hip-width apart standing on force plates . The patients were instructed to stand still on the force plates without any assistance for 5 s. The evaluation is illustrated in The force plates recorded their center of pressure (COP) trajectories over the 5 s of standing. To evaluate the postural stability, mean sway amplitude  was automatically calculated in the system and was returned as a percentage, referring to the rate of MSA, with a lower percentage indicating better postural stability.Flexor carpi radialis (FCR) H-reflex data collected in the three cases are presented in Table 1Clinical evaluation of cases.Despite the body position, the FCR Hmax/Mmax ratios of the more strongly affected UEs were higher than those of the less-affected UEs. Between the supine and standing positions, the FCR Hmax/Mmax ratios of the less-affected UE decreased by 12.50% in case 1, 77.27% in case 2, and 76.92% in case 3. However, the FCR Hmax/Mmax ratios of the more strongly affected UE increased by 5.12% in case 1, decreased by 3.77% in case 2, and decreased by 34.38% in case 3.Modified Ashworth scale (MAS) data in the three cases are presented in The postural stability was assessed in both mediolateral and anteroposterior axes . No joinFlexor carpi radialis (FCR) H-reflex measurements showed that the Hmax/Mmax ratios of the more-affected UEs were higher than those of the less-affected UEs in both the supine and standing positions in all three cases, which is consistent with a previous study in patients with chronic stroke , indicatPrevious research has shown obvious down-modulation of lower extremity (LE) SSR excitability in the sitting and standing positions than in the prone position in healthy subjects . In patiSpasticity can be divided into its neurological and biomechanical components . The neuThe UEs play an important role not only in motor control but also in balance and postural control. A previous study confirmed that increased flexor muscle tone or flexion synergy patterns when standing have been regarded as a compensation strategy for postural instability . LookingIn addition to the fact that the RST has a strong role in modulating SSR excitability, it also plays a key role in both postural control and motor control involving proximal and distal UE muscles . It has The present study had some advantages and limitations. This study was the first to measure FCR H-reflexes in UEs in patients after stroke in different body positions, and the first to preliminarily explore the possible relationships between postural control and UE SSR excitability after stroke. However, this study included only three cases. Thus, the results were drawn from limited sample size and obtained after data collection at a single time point. Further studies using larger sample sizes and long-term follow-up should be conducted to obtain more comprehensive results. Variables  and factors  should be also controlled in further study. Some devices or methods are also required to ensure the consistency of body positions among the participants. Additionally, more functional situations  could be considered. Moreover, more evaluation methods such as brain functional imaging and evoked potentials could be applied to get more holistic results and to further explore the possible mechanisms that underlie postural control and motor control deficits after stroke.Spinal stretch reflex (SSR) hyperexcitability in strongly affected UEs could commonly occur in different phases of recovery after stroke. Down-modulation of SSR excitability could occur in less-affected UEs in the standing position compared with the supine position, while modulation of SSR excitability might be altered in strongly affected UEs and vary in different phases of recovery. There could be some correlation between postural control and UE SSR hyperexcitability. We further hypothesized that changes in RST output might underly SSR hyperexcitability in different phases of stroke recovery and altered SSR modulation in different body positions. We hope that the H-reflex can help to offer a new perspective for evaluation and intervention in UE rehabilitation after stroke.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Yueyang Hospital, affiliated with Shanghai University of Traditional Chinese Medicine. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.J-GX and C-LS designed the study. M-XZ and X-YH interpreted the results. J-YM and J-JW analyzed the data and wrote the manuscript. All authors revised and approved the final version of the manuscript.This study was supported by the National Key R&D Program of China (Grant Nos. 2018YFC2001600 and 2018YFC2001604), the National Natural Science Foundation of China , the Shanghai Rising-Star Program (Grant No. 19QA1409000), and the Shanghai University of Traditional Chinese Medicine (Grant No. 2021yyjq07).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Clinical Gastroenterology and Hepatology that demonstrated an increased risk of graft failure and death in liver transplant (LT) recipients with acute rejection (AR) in the post-2000 era.1 However, an important challenge raised was the recipients’ hepatitis C virus (HCV) status given the frequent diagnostic uncertainty in distinguishing AR from recurrent HCV. This was relevant as that study population originated from an era in which HCV constituted a high percentage of LT recipients with active viremia given the lack of effective direct-acting oral antiviral (DAA) therapies. Nevertheless, the association between AR and inferior recipient outcomes persisted despite stratification by HCV status. Our objective was to re-evaluate the association between AR and post-LT outcomes in the DAA era, in which sustained virological responses are nearly 100%, and thus report data on the impact of AR that is more applicable to current practice.In 2017, we published a study in A retrospective cohort of adult LT recipients was identified using the United Network for Organ Sharing (UNOS) database. Pre-DAA LTs included those performed between 1/1/2010 and 12/31/2013, while the post-DAA period spanned 1/1/2014–12/31/2019. While more recent years of data were available, a substantial increase in missing data with regards to AR was noted beginning 1/1/2020, likely reflecting delays in data reporting and the COVID pandemic. Multiorgan and prior organ transplants were excluded. The last follow-up date for the cohort was 7/1/2021.AR in the first year post LT was evaluated as a binary exposure. Among patients alive with their native allograft at 1 year post LT, 1-year AR was unable to be determined due to missing data in 18.5% . All other exposures were measured at LT including sex, age, race/ethnicity, HCV vs non-HCV liver disease, Model for End-stage Liver Disease score, ascites and encephalopathy severity, patient location, dialysis status, history of hepatocellular carcinoma, receipt of living donor LT, receipt of allograft from a donor after circulatory death, and donor age. Primary outcomes of interest included patient and graft survival.1 Cox proportional hazards models evaluated the association of AR with patient and graft survival among patients surviving at least 1 year with their initial allograft, adjusting for the aforementioned covariates. All analyses were performed using STATA v17 .Using a similar approach to the original manuscript,P < .001; P < .001) and 9.8% vs 13.1% post-DAA (P < .001). During the post-DAA era, the etiologies of liver cirrhosis that had the highest rates of 1-year AR era were autoimmune hepatitis (19.3%), primary sclerosing cholangitis (18.3%), primary biliary cholangitis (16.6%), and alcohol-related liver disease (12.8%) (P < .001 comparing 1-year AR by diagnosis). Changes in 1-year AR over time were less pronounced in HCV than in non-HCV recipients  and HCV  were independent predictors of mortality. This association was also observed with respect to graft survival  for 1-year AR and  for HCV. Stratifying by DAA era, HCV was associated with patient survival pre-DAA , but not post-DAA . HCV remained a predictor of graft survival pre-DAA and post-DAA, though to a lesser degree .Among all patients surviving the first year with their initial liver allograft (89.9%), 1-year AR . This temporal phenomenon was not observed among non-HCV patients, in whom the risk of mortality associated with 1-year AR remained stable   . Similarctively) .1 that AR continues to be a predictor for graft failure and patient death in the non-HCV patient cohort, independent of DAA era. This has also been substantiated in a study by Tanaka et al.2 As in our prior study,1 this is contrary to data prior to 2000 which suggested that AR had no effect on outcomes.4 Ultimately, AR continues to be a significant adverse event, mainly because treatment with high-dose immunosuppression contributes to complications , likely more so than the impact of AR on the graft itself. These findings may indicate that closer monitoring is required for post-LT patients at higher risk for AR, such as those transplanted for autoimmune liver disease.In this study, we corroborate our prior studyStrengths of our study include a large patient population with good generalizability. Limitations of the study include the retrospective nature and missing data in the UNOS database. For instance, we assume that post-DAA patients were cured of HCV, but this cannot be confirmed from this database. Another limitation is that rejection in the UNOS database is based upon provider clinical documentation and entered by transplant coordinators rather than biopsy-proven rejection, making it a less accurate of a measurement. There is also no information on the immunosuppression regimen and compliance of the patients in this dataset. Our power also may be limited since the sample size of HCV recipients was much smaller than non-HCV recipients in the post-DAA era.In conclusion, as liver failure and need for LT due to HCV is rapidly declining with the introduction of DAA therapy, it is important to focus on the clinically relevant, current population of non-HCV LT recipients. Using updated recent era data, AR continues to be deleterious in the LT population, and avoidance and/or early detection and treatment of AR will likely improve LT outcomes.1


The androgen receptor (AR) is a steroid hormone receptor widely detected in breast cancer. Evidence suggests that the AR might be a tumor suppressor in estrogen receptor alpha-positive (ERα+ve) breast cancer but a tumor promoter in estrogen receptor alpha-negative (ERα-ve) breast cancer. Modulating AR activity could be a potential strategy for treating breast cancer. For ERα+ve breast cancer, activation of the AR had been demonstrated to suppress the disease. In contrast, for ERα-ve breast cancer, blocking the AR could confer better prognosis to patients. These studies support the feasibility of utilizing AR modulators as anti-cancer drugs for different subtypes of breast cancer patients. Nevertheless, several issues still need to be addressed, such as the lack of standardization in the determination of AR positivity and the presence of AR splice variants. In future, the inclusion of the AR status in the breast cancer report at the time of diagnosis might help improve disease classification and treatment decision, thereby providing additional treatment strategies for breast cancer. AR gene is located at locus Xq11–12 on the X-chromosome. It harbors eight exons encoding a protein of 110 kDa molecular weight. The AR protein is composed of three main functional domains: the N-terminal domain (NTD) (encoded by exon 1), the DNA binding domain (DBD) (encoded by exon 2–3) and the C-terminal ligand binding domain (LBD) (encoded by exon 5–8), together with a hinge region (HR) (encoded by exon 4) [The androgen receptor (AR), also known as NR3C4, is one of the sex steroid hormone receptors widely expressed in males and females. The receptor plays a vital role in multiple physiological processes, such as the development of the reproductive system . The AR  exon 4) ,7,8. The exon 4) . LikewisAccording to GLOBOCAN 2020, the latest global cancer statistics reported by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO), female breast cancer was estimated to be the most prevalent cancer worldwide, with approximately 2.26 million newly diagnosed cases  and 0.68 million death cases in 2020 . Breast SEC14L2, EAF2 and ZBTB16, to repress tumor growth directly [CCND1, or may enhance other tumor suppressors, including FOXO1 and FOXO3a [In ERα+ve breast cancer, ERα-mediated signaling is the driving factor for cancer development. The AR, by interfering with the function of ERα, may have a tumor suppressor function. The importance of cross-talk between AR and ERα has been supported by mechanistic studies which suggest that AR and ERα can interact with each other through the AR’s N-terminal domain (NTD) and ERα’s ligand-binding domain (LBD). This protein–protein interaction can inhibit the activity of both proteins . Studiesdirectly . Furtherd FOXO3a ,21. Due d FOXO3a ,23, and d FOXO3a ,25,26. TWNT7B. Activation of the WNT/β-catenin pathway would result. In addition, the AR could further form a complex with β-catenin. The complex could promote the transcription of HER3, which is a critical factor for the oncogenic activity of HER2 [In ERα-ve breast cancer patients, including HER2+ve and TNBC, the AR shows oncogenic effects. In HER2+ve breast cancer, it has been reported that an active AR could induce the expression of HER2, which subsequently activates the MAPK pathway. The activated MAPK pathway induces the AR’s expression, resulting in a positive-feedback loop . Moreove of HER2 . In TNBC of HER2 . These a of HER2 ,31,32,33The possibility of modulating AR activity for breast cancer treatment can be traced back to 1939, when the first record of breast cancer patients who might benefit from receiving testosterone propionate to activate the AR was published . SubsequIn a retrospective study, 508 postmenopausal and ovariectomized women who had received testosterone implants in addition to conventional hormone therapy were followed up for a mean duration of 5.8 years. The incidence of breast cancer was recorded, and the results showed that using testosterone might reduce the occurrence of conventional-hormone-therapy-induced breast cancer . A prospThe combination of testosterone and anastrozole was also applied to treat menopausal symptoms in 72 breast cancer survivors. The treatment was effective in relieving the symptoms without elevating estradiol; no tumor relapse was found for up to 8 years . CombiniAnother study provided more direct evidence to support that androgens as a single drug could be an effective anti-cancer agent for treating ERα+ve breast cancer patients. The study recruited 53 ERα+ve/PR+ve metastatic breast cancer patients whose tumors no longer responded to anti-hormonal therapies. These patients were treated with testosterone propionate. Approximately 60% of the subjects showed disease regression or stabilization, representing a positive result . An indeAs proof of principle, a phase II trial (NCT01616758) on evaluating the efficacy and safety of enobosarm, a synthetic and selective AR activating agent, in ERα+ve metastatic breast cancer demonstrated that around 50% (8/16) of the patients reached the best response with a median duration of 4.5 months. The drug was well tolerated . Due to More recently, a pre-clinical study also demonstrated that activation of the AR by either DHT or enobosarm in ERα+ve endocrine-resistant breast cancer patient-derived xenografts (PDXs) significantly suppressed the estrogen-induced tumor growth. By contrast, inhibition of the AR activity via the AR antagonist enzalutamide enhanced the ability of estrogen to stimulate tumor growth . These sMany AR suppressive agents are available for treating prostate cancer. These agents significantly contributed to improving patients’ treatment outcomes. Due to the importance of the AR in ERα-ve breast cancer, studies on evaluating the efficacy of anti-androgens, alone or combined with other anti-breast cancer drugs, have been documented. A phase II clinical trial (NCT02091960) was conducted to evaluate the therapeutic value of the AR antagonist enzalutamide. In the trial, enzalutamide was used on advanced ERα-ve/AR+ve/HER2+ve breast cancer patients. In all, 24% of the patients exhibited a clinical benefit at 24 weeks . A case Another phase II study (NCT00468715) demonstrated bicalutamide in AR+ve/ERα-ve/PgR-ve metastatic breast cancer. The results showed a clinical benefit rate of 19% at 6 months and a median progression-free survival of 12 weeks, comparable to other chemotherapies studies in TNBC patients . SimilarAn ongoing phase II trial (NCT03383679) combined an androgen-receptor antagonist darolutamide with a chemo-drug capecitabine in advanced AR+ve/TNBC patients recently reported the first-stage result of the study that darolutamide is well tolerated and 26.3% of the patients present a clinical benefit at 16 weeks. The project is now moving to its second stage . AnotherOther than AR inhibitory drugs, agents targeting androgen synthesis also showed promising potential in clinical application for TNBC patient management. Abiraterone acetate is a CYP17A1-specific inhibitor that suppresses the production of androgens . CombineA novel antiandrogenic drug seviteronel which functions by a unique dual mechanism of action that lowers the biosynthesis of androgen via inhibiting CYP17 lyase, as well as blocking AR activation via antagonizing AR, has attracted great attention since the drug is more effective than abiraterone acetate in suppressing AR activity . An openIn pre-clinical studies, blocking AR signaling in ERα-ve breast cancer models has been shown to suppress cell growth. Inhibition of AR activity by shRNA or enzalutamide could interfere with cell proliferation in AR+ve/ERα-ve/HER2+ve breast cancer cell lines and xenograft models; when combined with trastuzumab, it could further enhance the suppressive capacity. Enzalutamide could reduce the expression of the proliferation marker Ki-67 and induce the expression of active caspase-3 in the xenograft model, demonstrating an anti-proliferative property after AR suppression in HER2+ve breast cancer .A combination of enzalutamide and trastuzumab or mTOR inhibitor everolimus in AR+ve/ERα-ve/HER2+ve and AR+ve/TNBC could synergistically inhibit cell proliferation . AdditioBlocking the AR could significantly suppress proliferation and increase apoptosis in both in vitro and in vivo AR+ve/ERα-ve models, which might result from the inhibition of Wnt/β-catenin and EGFR signaling pathways ,65. ERα-Unlike other prognostic and predictive factors such as ERα that have been standardized for detection in breast cancer, the AR detection protocol to define positivity in patients has not been standardized. The cut-off scores for pathological examination by immunohistochemistry (IHC) of AR positivity varied in different studies. Moreover, the antibodies against the AR used for IHC staining in different studies have been diverse . Other sAlternative splicing of the AR can give rise to variants, some with truncation of the LBD. These variants may be innately and constitutively active even in the absence of ligands. Based on current studies, at least 18 AR-Vs have been identified , many ofFortunately, several strategies for targeting AR-Vs have been established  for the AURKA, BIRC5, CCNB1, MKI67 and UBE2C) than those with a value less than 2 [The AR tends to be an ERα suppressor in breast cancer. Activating the AR properly in AR+ve/ERα+ve patients to counteract the activity of ERα should be a prioritized option for controlling disease progression. However, without the influence of ERα, such as in ERα-ve breast cancers, the AR can also promote disease progression. It has been reported that breast tumors with an AR-to-ERα (AR/ERα) ratio greater than or equal to 2 have higher expression of proliferation signatures  and a higher risk of tumor aggression in breast cancer clinical studies ,101. AltThe different roles of AR in ERα+ve and ERα-ve breast cancers have been discussed separately. As a heterogeneous cancer, the different intrinsic molecular profiles of breast cancer provide the basis for breast cancer molecular subtyping, which largely determines treatment strategy for each patient. Although the AR is expressed in a considerable proportion of breast cancer, due to its undetermined role, the expression status of AR has not been included in the current molecular subtyping. However, novel molecular subtypes of TNBC involving the AR have identified a subtype called the luminal androgen receptor (LAR) . InteresSince the AR plays a critical role in multiple normal biological processes, it is not surprising that treating breast cancer patients with AR modulators, either agonist or antagonist, will alter the activity of the AR. This may have adverse effects. AR agonists have been reported to induce acne, hirsutism and alteration in lipid profile , while ACurrently, many conventional AR-targeting drugs that modulate the activity of AR signaling can be applied in cancer therapy. Patients who benefit from these drugs have largely had their survival time prolonged, with improved disease treatment outcomes, regardless of their standard treatment for prostate cancer or their clinical trial treatment for breast cancer. In addition, more novel drugs produced by advanced techniques are in development. The proteolysis-targeting chimaera (PROTAC) molecule was designed for utilizing the ubiquitin–proteasome system (UPS) to degrade specific proteins by its bound E3 ubiquitin ligase. The model was proposed by Sakamoto et al. in 2001 , and subTargeting AR by N-terminal inhibitors is another possible strategy. Some constitutively activated AR-Vs may lack the LBD but retain an intact NTD. Inhibitors targeting the NTD of the AR may inhibit not only the function of the entire length of the AR, but also the function of AR-Vs. One of the AR NTD inhibitors are the EPI compounds. In vitro and in vivo studies demonstrated that these compounds could suppress the growth of different prostate cancer models ,87,88. CThe roles of the AR should be clarified in different subtypes of breast cancer. Modulating the activity of the AR will be a promising strategy for treating breast cancer. However, more studies are needed to address the existing issues in the hope of translating these strategies into the clinical setting.