Early-stage and castration-sensitive prostate cancer (PCa) growth is solely mediated by androgen signaling pathways. AR signaling inhibitors (ARSIs) have significantly improved clinical outcomes among men with PCa. In the metastatic castration-resistant PCa, there is presence of both androgen-dependent and androgen-independent cells driving the tumor growth. Despite the use of ARSIs, disease progression ultimately occurs in all patients with PCa and is due to genetic alterations in ARs, resulting in the outgrowth of androgen-independent cells. The possible mechanisms include development of AR splice variants of which AR-V7 is more common, AR point mutations, and AR overexpression. In addition, restoration of downstream signaling through alternate pathways can also lead to androgen-independent growth of PCa. Therapeutic strategies to overcome these resistance mechanisms and establish predictive biomarkers are still in clinical trials. This review article details the current evidence on clinically relevant driver mechanisms, relevant biomarkers, and treatment modalities to overcome resistance.Understanding of the molecular mechanisms of prostate cancer has led to development of therapeutic strategies targeting androgen receptor (AR). These androgen-receptor signaling inhibitors (ARSI) include androgen synthesis inhibitor-abiraterone and androgen receptor antagonists-enzalutamide, apalutamide, and darolutamide. Although these medications provide significant improvement in survival among men with prostate cancer, drug resistance develops in nearly all patients with time. This could be through androgen-dependent or androgen-independent mechanisms. Even weaker signals and non-canonical steroid ligands can activate AR in the presence of truncated AR-splice variants, AR overexpression, or activating mutations in AR. AR splice variant, AR-V7 is the most studied among these and is not targeted by available ARSIs. Non-androgen receptor dependent resistance mechanisms are mediated by activation of an alternative signaling pathway when AR is inhibited. DNA repair pathway, PI3K/AKT/mTOR pathway, BRAF-MAPK and Wnt signaling pathway and activation by glucocorticoid receptors can restore downstream signaling in prostate cancer by alternative proteins. Multiple clinical trials are underway exploring therapeutic strategies to overcome these resistance mechanisms. Prostate cancer (PCa) is the second most common malignancy among men . Based oAndrogen Receptor (AR) plays a key role in the pathogenesis of PCa. AR is a ligand-activated nuclear transcription factor that belongs to the steroid hormone receptor family. As testosterone (produced by Leydig cells in the testes) or 5-alpha-dihydrotestosterone (DHT)  binds to AR, the receptor dimerizes and translocates into the nucleus to bind to the androgen response element (ARE) . The AR Abiraterone acetate is an androgen synthesis inhibitor derived from pregnenolone and is an irreversible inhibitor of 17, 20-lyase and 17-alpha hydroxylase which are products of the CYP17 gene. The drug inhibits the production of androgen in the testes, adrenal glands, and tumor cells . The ageThe drug is currently approved by the US Food and Drug Administration (FDA) for use in newly diagnosed metastatic castration-sensitive PCa (CSPC) and metastatic CRPC. Clinical trials that led to FDA approval of individual drugs are shown in Androgen receptor antagonists (ARA) block the androgen binding site of AR and inhibit the nuclear translocation of AR and subsequent association of AR with nuclear DNA. This results in attenuation of coactivator mobilization leading to cellular apoptosis and decreased prostate tumor volume. The first-generation ARAs including bicalutamide, nilutamide, and flutamide do not completely block AR activity. Enzalutamide, apalutamide, and darolutamide are the currently utilized second-generation ARAs and have no agonistic activity on AR compared to first-generation ARAs. Enzalutamide and apalutamide have a similar mechanism of action and side effect profile . DarolutEnzalutamide is currently approved in de novo mCSPC, non-metastatic CRPC, mCRPC in chemo naïve patients, and mCRPC which progressed after chemotherapy. In addition to overall survival benefit, the medication also improved health-related quality of life and benefited patients above 75 years of age with mCRPC ,22. ApalSince abiraterone and apalutamide act by different mechanisms, clinical trials are evaluating whether the combination provides superior outcomes. Preliminary reports from abiraterone plus apalutamide combination trial showed improved PSA response while results from abiraterone plus enzalutamide trial showed no improvement in OS and increased side effects ,25.n = 55) gave mPSA-P of only 3.9 months and a PSA decline ≥50% was seen in 44% of the patients [n = 33) had an mPSA-P of 2.8 months and a PSA decline ≥50% in 67% of patients. These studies emphasize the development of AR-mediated cross-resistance which limits the clinical benefit for subsequent use of alternate ARSI.In the phase IV Safety Study of Continued Enzalutamide Treatment In Prostate Cancer Patients (PLATO) trial that evaluated 251 patients who were on abiraterone after progression on enzalutamide, the median time to PSA progression (mPSA-P) was 2.8 months, median radiologic progression free survival (rPFS) was 5.7 months, and only 4 patients had PSA decline ≥50% ,27. Thispatients . The cohDespite the efficacy of ARSI, secondary resistance to these agents develops in nearly all the patients due to molecular changes from selective pressure on AR. The various secondary resistance mechanisms are discussed below with an emphasis on mechanisms that have potential therapeutic implications and are compiled in De novo absence of efficacy with ARSI occurs in approximately 5–10% of mCRPC patients and progression ensues in nearly all the patients who initially respond to these drugs. These failures can be partly attributed to AR variants ,56,57. TAndrogen receptor isoform splice variant 7 (AR-V7) is the most common variant detected and is not targeted by available ARSIs . AR-V7 iDetecting these variants previously required serial biopsies but the use of widely available and validated liquid biopsy has made testing more accessible. Treating physicians should consider testing for AR-V7 in patients who experience disease progression after ARSI. This approach can guide further treatment with an alternative ARSI versus chemotherapy. The presence of CTC AR-V7 is not associated with primary resistance to taxane chemotherapy . TaxanesAlthough apalutamide and darolutamide target full-length AR with no effect on AR-V7 activity, resistance is seen in AR-V7 expressing enzalutamide- and abiraterone-resistant models. This effect could be mediated by concurrent AKR1C3 enzyme activation in these models which converts weak androgens to the more potent products: testosterone and DHT. AKR1C3 also stabilizes AR-V7 and full-length AR (AR-FL), which results in increased c-MYC expression that in turn activates AR target genes ,65. KnocTAS3681 is an oral AR antagonist with full length-AR and AR-V7 downregulatory activity and was shown to have antitumor efficacy in enzalutamide-resistant models. The open-label phase I trials among 56 patients refractory to abiraterone (14.2%), enzalutamide (46.4%), or both (39.3%) showed PSA response lasting up to 16.3 months with a manageable safety profile . The expAR-V567es is another splice variant identified in xenografts after prolonged ADT exposure and increased in enzalutamide-resistant PCa cells ,38. CTC Point mutations in the hinge region or the LBD, which result in reduced ligand specificity and increased trans-activation, are commonly found in mCRPC . CTC DNASome mutations convert AR antagonists into potent agonists. CTC DNA studies in mCRPC patients resistant to apalutamide or enzalutamide, as well as animal studies, have demonstrated that the F877L mutation converts enzalutamide and apalutamide into agonists ,47. F877TRC253 is a high-affinity competitive binder of wild-type (WT) and mutant AR with proven efficacy in F877L mutant mice models. The drug is currently being studied in a phase I trial and is enrolling for the dose-expansion phase including patients with an F877L mutation ,51.PROTACs are protein degrading agents, of which, ARV-110 is currently being evaluated in a phase I/II trial in mCRPC. It degrades the AR in PCa cell lines and animal models with high potency leading to lower expression of PSA ,53. ThisApproximately 80% of patients with CRPC have marked increase in AR mRNA and protein expression ,75,76,77p = 0.02) [p = 0.020), and a similar pattern was seen with OS . Another study with 66 patients who received radioligand therapies  showed that 47% had AR amplification or resistant mutations. These patients were less likely to experience a PSA decline ≥30% compared to wild type; it was also associated with inferior OS  [Although in most cases (92%), AR amplification in CTC DNA corresponds to amplification in matched solid biopsy samples, exceptions do occur. A 10-fold AR amplification was detected on CTC DNA analysis in a patient with bone and lymph node metastasis while the same was not detected in a corresponding lymph node biopsy. It was postulated that the tumor clones with high AR amplification were localized to sites of bony metastasis . AR ampl = 0.02) . AR ampl = 0.02) . Mechani = 0.02) ,85,86,87 = 0.02) . Patient= 0.043) .AR overexpression can also occur without gene amplification by stabilization of the mRNA or protein or by increasing transcription rates ,90,91. TThe phase II TRANSFORMER trial compared the efficacy of bipolar androgen therapy (BAT) by cycling polar extremes of near-castrate and supraphysiologic testosterone levels with enzalutamide in asymptomatic men with CRPC after progression on abiraterone. Differences in PFS and OS were not statistically significant with either approach. However, patients who underwent BAT followed by enzalutamide had better PFS2 compared to enzalutamide followed by BAT . The OS was also superior in the BAT followed by enzalutamide group compared to the enzalutamide alone group . In this study, 38% of the entire study population overexpressed AR and 9% had AR-V7 in CTCs. These aberrations conferred numerically shorter PFS and OS on BAT and enzalutamide therapies; however, the study was not powered to determine if these aberrations could be used as treatment selection biomarkers . It is pBromodomain and extra-terminal chromatin readers (BET) inhibitors including miverbresib and ZEN-3694 regulate AR gene transcription to decrease AR expression and AR-V7 signaling, which results in tumor suppression . Single Resistance to AR-targeted agents in PCa can also be mediated by activation of alternate signaling pathways that are induced by peptide growth factors, PI3K/AKT/mTOR pathway, glucocorticoid receptor (GR) pathway, and through restoration of downstream signaling by alternative proteins.DNA repair pathway: Patients with tumor cells harboring pathogenic or likely pathogenic variants of BRCA1 or BRCA2 benefit from PARP inhibitors such as olaparib or rucaparib. Although data is limited, these agents can also be used with other homologous recombinant repair (HRR) gene defects such as ATM, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCL, PALB2, RAD51B, RAD51C, RAD51D, or RAD54L . PARP-men = 521, median 19.1 vs. 14.2 months, HR 0.65, 95%CI 0.45–0.95) or PIK3CA/AKT1/PTEN-alterations by next generation sequencing  [PI3K/AKT/mTOR pathway: The PI3K/AKT/mTOR pathway is commonly altered in PCa and signaling can be activated by enzalutamide through stabilization of AKT phosphatase ,106,107.44–0.88) . Another44–0.88) ,112. Oth44–0.88) ,114,115.44–0.88) ,117. Sam44–0.88) . GSK263644–0.88) .BRAF-MAPK pathway: Alternative signaling through the MAPK pathway was identified as a potential growth pathway in 2 patients with enzalutamide resistant PCa harboring a BRAF-K601E mutation ,121. PhaWnt signaling pathway: PCa cells can also gain the ability to synthesize and secrete specific ligands and receptors that help sustain survival through the wnt-beta-catenin pathway independent of androgen signaling . A studyp = 0.395) [Glucocorticoid receptor (GR) activation: Enzalutamide resistance has been attributed in some cases to increased GR expression, which can drive transcription of AR-related genes ,132. GR = 0.395) ,136. Ano= 0.395) . In pati= 0.395) . The lowNeuroendocrine differentiation: Treatment-related neuroendocrine differentiation is quite prevalent in mCRPC. Studies showed that 16.9% of patients who had disease progression after ADT demonstrated small cell histology on biopsies of metastases . DevelopPD-1/PDL-1 immunoinhibitory pathway: Higher expression of PDL-1 in patients with enzalutamide-resistant PCa prompted the phase III IMbassador250 trial that combined enzalutamide with the PDL-1 blocker atezolizumab in mCRPC after progression on abiraterone or chemotherapy . HoweverSignificant advantages have been made in the management of CRPC with second-generation androgen receptor antagonists and androgen synthesis inhibitors. However, the benefits are often short-lived due to the rapid development of resistance to these drugs. Extensive studies on the resistance mechanisms have opened the way to new drug developments which are aimed at reducing the emergence of resistant clones as well as targeting them. These drugs are still in the pipeline with clinical utility being evaluated in numerous clinical trials. Although preclinical data have been promising, many agents were not clinically beneficial. This result is possible because of the interaction of multiple crosstalk pathways and genetic aberrations occurring concurrently, which makes targeted monotherapies less effective. Further understanding of the nuances of resistance mechanisms and wider utilization of clinical trials can help in development of these agents.


Aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, has been implicated in the onset and development of the ocular complications of diabetes, including cataracts and retinopathy. Despite decades of research conducted to address possible mechanisms, questions still persist in understanding if or how AR contributes to imbalances leading to diabetic eye disease. To address these questions, we created a strain of transgenic mice engineered for the overexpression of human AR (AR-Tg). In the course of monitoring these animals for age-related retinal phenotypes, we observed signs of Müller cell gliosis characterized by strong immunostaining for glial fibrillary acidic protein. In addition, we observed increased staining for Iba1, consistent with an increase in the number of retinal microglia, a marker of retinal inflammation. Compared to age-matched nontransgenic controls, AR-Tg mice showed an age-dependent loss of Brn3a-positive retinal ganglion cells and an associated decrease in PERG amplitude. Both RGC-related phenotypes were rescued in animals treated with Sorbinil in drinking water. These results support the hypothesis that increased levels of AR may be a risk factor for structural and functional changes known to accompany retinopathy in humans.  Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase that has been extensively studied as the first and rate-limiting enzyme of the polyol pathway . The elep < 0.001; We previously reported on a series of mutant mouse strains designed for the over-expression of human aldose reductase (AKR1B1) in the lens epithelium and the outer cortical fiber cells to support mechanistic studies of diabetic cataract formation ,7. ConsiGiven the profound effect of Sorbinil on the prevention of RNFL complex thickening in AR-Tg mice, we next sought to verify expression of human AR in the affected tissue layer. Immunofluorescence staining for human AR in cryosections of retina taken from WT and AR-Tg mice showed strong immunostaining in AR-Tg mice. The staining appeared to not be limited to a particular laminar region, but rather showed patterns that resembled columns that radiated from the outer to inner retinal layers . Müller glia (MG) are known to extend from the inner to outer retina and form foot plates at the interface of the inner retinal compartment and the retinal inner limiting membrane. To ascertain if MG populate the area of thickened cells adjacent to the inner limiting membrane observed in our AR-Tg model, we carried out immunostaining for cellular retinaldehyde binding protein (CRALBP), a protein abundantly expressed in the MG and retinal pigment epithelium . As showAxons extending from retinal ganglion cells (RGC) organize into bundles that course along the RNFL and leave the eye through the optic nerve to connect to the brain. Given the phenotype of an apparent activation of Müller glia to a proinflammatory and gliotic state, which is known to induce apoptosis of RGCs, we next determined whether such changes to Müller glia may result in a loss of RGCs in our AR-Tg model ,11. The The loss of RGCs revealed in immunostaining studies was also reflected in a loss of ganglion cell function, measured by pattern electroretinography (PERG). PERG is a noninvasive method used to measure the light-evoked electrical activity of RGCs. We observed through longitudinal studies that AR-Tg mice experienced an age-dependent loss of RGC function over a period of 35 weeks. A potential role for AR in this reduction in PERG amplitude was demonstrated by a substantial delay, relative to untreated AR-Tg mice, in the loss of PERG signal activity when mice were treated with Sorbinil. AR-Tg mice showed a substantial loss of PERG amplitude compared to wild-type controls, beginning at 10 weeks of age . In contp < 0.001). Similarly, cells staining positive for Iba-1, a marker for retinal microglia, were significantly elevated in comparison to the wild type (p < 0.001). The treatment of AR-Tg mice with Sorbinil largely prevented the upregulation of both of these markers of inflammation.Elevated expression of AR has been associated with a shift toward a proinflammatory phenotype characterized by increased levels of activated retinal microglia . Since aAldose reductase, the first and rate-limiting enzyme of the polyol pathway of glucose metabolism, has been implicated in the pathogenesis of diabetic complications in the eye, including retinopathy and cataracts . AccelerSeveral lines of evidence lead us to suspect that the activation of AR expression in Müller glia is responsible for the phenotypes we observe in our AR-Tg mouse line. The thickness of the RNFL complex in AR-Tg mice maintained on Sorbinil was essentially indistinguishable from WT, whereas AR-Tg mice not treated with the AR inhibitor showed marked thickening. Previous studies have shown that RNFL thickness is influenced by contributions from Müller cells , and MülDiabetic retinopathy has for many years been considered a microvascular disease. However, current thinking is shifting toward a realization that the modulating influence of neuronal processes on the retinal inflammation and apoptosis of retinal ganglion cells may play an important role in the effects of diabetes in the retina . While oThe research in this study was conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, as well as an animal protocol approved by the University of Colorado Medical Center Animal Care and Use Committee (IACUC). C57BL/6J (WT control) mice were purchased from the Jackson Laboratory , and transgenic AR-Tg mice (homozygous for the AKR1B1 gene) were generated by our lab on a C57BL/6 background, as reported previously . BrieflyEnucleated eyes used for the histological and immunohistochemical (IHC) staining were fixed using a modified Davidson’s fixative  for approximately 18 h, transferred to 10% neutral buffered formalin  for a further 4 h, and then embedded in paraffin. Sagittal eye sections (3.5 µm) were cut and stained with Mayer’s hematoxylin and eosin Y  or used for IHC analysis. Bright-field color images were obtained using a Nikon Eclipse 80i microscope  equipped with a Nikon D5-Fi1 color camera, a Nikon CFI 4x or 20x/Plan Fluor objective lens, and the NIS-Elements software package.The thickness of the RNFL complex was determined using the length measurement tool contained within the Nikon NIS-Elements software package version 5.20.02. For each image, at least six evenly spaced measurements were taken (while avoiding areas with blood vessels), representing the distance from the base of the RGC layer to the retinal inner limiting membrane. Measurements were taken over a distance of approximately 100–400 µm extending in either direction from the center of the optic nerve. Enucleated eyes used for immunofluorescence staining were fixed for 2 h in 4% paraformaldehyde, infused at 4 °C with an increasing concentration series (10–30%) of sucrose solutions in phosphate buffered saline (PBS), and then flash frozen in OCT . Sagittal eye sections (10 µm) were first washed in PBS to remove excess OCT and then blocked for 1 h at room temperature in PBS containing 10% bovine serum albumin (Sigma-Aldrich) and 10% normal goat serum . Tissue sections were then incubated with the primary antibody in blocking solution overnight at 4 °C and washed three times in PBS containing 0.1% Tween-20 . For detection of unconjugated primary antibodies, tissue sections were incubated for 40–60 min at room temperature in the dark with the appropriate secondary antibody (Alexa488 or Alexa594) diluted in blocking solution followed by three PBST washes. All sections were then mounted under coverslips using Fluoro-Gel II  containing DAPI as a nuclear stain. Immunofluorescent images were collected using a laser scanning confocal microscope  equipped with a Nikon 20x/0.75  software package. Each set of experimental images was captured using similar microscope configurations, including laser intensity and brightness. At least three replicates for each set of experiments were analyzed.The following mouse monoclonal antibodies were used at the given dilutions: anti- glial fibrillary acidic protein  diluted 1:150, anti-ionized calcium-binding adapter molecule 1  diluted 1:150, monoclonal anti-Brn3a conjugated with Alexa488  diluted 1:75, monoclonal anti-AR conjugated with Alexa594  diluted 1:100, and anti-CRALBP antibody  diluted 1:250. Secondary antibodies (diluted 1:500) included Alexa 488 goat anti-mouse IgG and Alexa594 goat anti-mouse IgG . Immunofluorescence analysis: The analysis of immunofluorescent images was performed using the Nikon NIS-Elements analysis software package. The percent of Brn3a-positive cells within the nerve fiber layer was determined using the “Object Count” feature. A 400 micron long region of nerve fiber layer was first isolated using the “polygon” region of interest (ROI) tool. Objects in the DAPI channel  and Alexa488 channel (Brn3a-positive nuclei) were identified by first eliminating background fluorescence  using the “Thresholding” tool, setting “Smooth” option to “1x”, “Separate” option to “ON”, and restricting the size to objects greater than 2 microns. The areas of positive staining were obtained using the “Field Measurement” feature. A 400 micron by 200 micron region of the retina was first isolated using the rectangular ROI tool. After eliminating the background fluorescence  using the “Thresholding” tool, field measurements were performed to obtain the area of positive fluorescent signal. At least three replicates per each set of experiments were analyzed.PERG recording was performed as previously described using the JORVEC instrument  ,28. Micet-tests or one-way ANOVAs, followed by Tukey’s Multiple Comparison Test, in statistical software by GraphPad Prism 7.0 . * p value < 0.05 was considered statistically significant.All data shown are means ± SEM. Quantifications of immunofluorescent-positive cell staining were analyzed using


Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with ill-defined therapeutic targets. Androgen receptor (AR) and tumour-infiltrating lymphocytes (TILs) had a prognostic and predictive value in TNBC. The relationship between AR, TILs and clinical behaviour is still not fully understood.Thirty-six TNBC patients were evaluated for AR (positive if ≥1% expression), CD3, CD4, CD8 and CD20 by immunohistochemistry. Stromal TILs were quantified following TILs Working Group recommendations. Lymphocyte-predominant breast cancer (LPBC) was defined as stromal TILs ≥ 50%, whereas lymphocyte-deficient breast cancer (LDBC) was defined as <50%.p = 0.1) and median CD20 was 20% and 7.5% (p = 0.008) in AR− and AR+, respectively. Mean CD3 was 80.7% and 93.3% (p = 0.007) and CD8 was 75% and 80.8% (p= 0.41) in AR− and AR+, respectively. All patients who were ≥60 years old expressed CD20. LDBC was found to be significantly higher in N+ versus N− patients (p = 0.03) with median TILs of 20% versus 50% in N+ versus N−, respectively (p = 0.03). LDBC was associated with higher risk of lymph node (LN) involvement .The mean age was 52.5 years and 27.8% were ≥60 years. Seven patients (21.2%) were AR+. All AR+ cases were postmenopausal (≥50 years old). LPBC was 32.2% of the whole cohort. Median TILs were 37.5% and 10% (AR expression was evident in older age (≥50 years). Median CD20 was higher in AR− TNBC, while mean CD3 was higher in AR+ tumours. LDBC was associated with higher risk of LN involvement. Larger studies are needed to focus on the clinical impact of the relation between AR and TILs in TNBC. TNBC) is a challenging heterogeneous disease with distinct molecular subtypes that does not have receptors for oestrogen, progesterone hormones and the human epidermal growth factor receptor 2 (HER2) protein. TNBC was grouped into six molecular subtypes: basal-like (BL) 1, BL2, mesenchymal (M), mesenchymal stem-like (MSL), immunomodulatory (IM) and luminal androgen receptor (LAR) [et al [Triple negative breast cancer . Moreover, as the immunotherapy landscape continues to evolve, there is interest in whether the immune system could be playing a more substantial role in TNBC specifically. The association between TNBC subtypes and the impact of TILs is still not fully understood. However, accumulating evidence from several studies indicates that intra-tumoural levels of TILs in TNBC are: a) predictive for response to neo-adjuvant chemotherapy and b) prognostic in patients treated with adjuvant chemotherapy, being correlated with improved overall survival (OS) and disease free survival (DFS) .Besides the immune cell markers, the androgen receptor (AR), which controls the transcription of different genes including the immune response genes, has been recognised as a valuable biomarker in TNBC . The AR In this cross-sectional, pilot study, patients’ records were reviewed retrospectively to select patients with TNBC. TNBC was deﬁned based on the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations (2010) , as tumoPathological evaluation was performed by a dedicated pathologist (TH), who was blinded for the clinical data. Haematoxylin and eosin-stained sections were revised for the negativity of ER, PR and Her2 and assessed for the histologic type and grade of the tumour. Then, the sections were examined to quantify the stromal TILs according to the 2014 TILs International Working Group , where iFormalin-fixed, paraffin-embedded tissue specimens were available for the evaluation of both of AR and TILs in 28 patients, AR alone in 5 patients and TILs alone in 3 patients. The AR expression (Code 200M-18) was evaluated by immunohistochemistry (IHC), and considered positive if ≥1% nuclear staining of the tumour cells . Also, it-test of significance and Mann–Whitney (z) test were used to compare two means and non-parametric data, respectively. Analysis of Variance (ANOVA) test and Kruskal–Wallis test were used to compare more than two means and multiple-group comparisons in non-parametric data, respectively. Chi-square (x2) test was used in order to compare proportions between qualitative parameters. As multivariate analysis is not suitable for small sets of data, estimates are represented according to univariate analysis. Spearman’s correlation coefficient (r) test was used to assess the degree of association between two sets of variables. The p-value was considered significant if ≤0.05.Recorded data were analysed using the Statistical Package for Social Sciences, version 20.0 . Quantitative data were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR). Qualitative data were expressed as frequency and percentage. Independent-samples Thirty-six TNBC patients with available enough tumour material were identified for analysis. The patients’ characteristics are shown in Supplementary Material, p = 0.47). LNs were involved in 77% and 85.7% in AR− and AR+, respectively, (p = 0.61). No statistical difference was found in median OS between AR− and AR+ groups . The clinico-pathological parameters according to AR expression are shown in AR was tested in 33 patients and it was expressed in 21.2% (7 patients). All AR+ cases (100%) were postmenopausal (≥50 years old). Although patients with AR+ tumours were older than those who were AR− (mean age: 55 versus 51.6 years), there was no statistically significant difference in age between the two groups (p = 0.10). Median CD20 was significantly higher in AR− versus AR+ , as depicted in p = 0.007), as depicted in The majority of AR+ tumours (85.7%) was LDBC subtype, with median percentage of TILs was 37.5% and 10% in AR− and AR+ tumours, respectively, (p = 0.03), as depicted in p = 0.03) as illustrated in p = 0.04).In the 31 patients, where TILs were evaluated, the median TILs were 30% (range = 1%–70%), while LDBC and LPBC were 67.7% and 32.3%, respectively. CD20 and CD4 were negative in 9.6% and 54.8%, respectively. p = 0.45). Moreover, all patients who were ≥60 years old expressed B-cell marker (100%)  = 0.591, p ˂ 0.001), while significant inverse correlations were present between CD3 and CD20 , CD8 and CD20  and CD8 and CD4 , as illustrated in On the other side, when analysing the relationship between the age and TILs , in univariate analysis [p = 0.63), OS , distant-DFS  or recurrence-free survival , regardless of the confounding factors and heterogeneity that existed among included studies. Our study results had matched the latter meta-analysis results, where no statistical difference in median OS  or relapse/progression rate  was found between AR− and AR+ groups.Available evidence about the prognostic value of AR in TNBC is controversial. Some reports suggested that AR-positivity was associated with good outcomes , 13, wheanalysis  demonstrImportantly, the presence of more frequent special histological subtypes with poor prognosis as medullary carcinoma, ILC and adenoid cystic carcinoma in the AR− versus the AR+ group (42% versus 28%) in our cohort, may have an impact on survival as pointed to by other studies .et al [et al [Compared to other subtypes, TNBC was shown to exhibit higher levels of TILs . There iet al  reportedl [et al  describeet al [et al [p = 0.45).Little is known about the association between TNBC clinico-pathologic features and lymphocytic predominance. A pooled analysis of nine large studies by Loi et al  demonstrl [et al  reportedp = 0.04). This is in agreement with Loi et al [Interestingly, we found that patients with LN involvement were significantly more likely to be LDBC, where a total TILs expression < 50% (LDBC) was associated with higher risk of LN involvement  compared to non-LAR, although this did not reach statistical significance , similarp = 0.008). Whereas, mean CD3 was significantly lower in AR− versus AR+ . On the other side, previous publications reported that CD8+ were more frequent in AR+ than AR− tumours [Studies about the immune cells subsets composition of TILs according to AR expression are very scarce. In our study, median CD20 was significantly higher in AR− tumours compared to those with AR+ . Intra-tumoural and adjacent stromal CD8+ T-cell infiltration have been found to be significantly associated with ER negativity and basal phenotype , 33. Infp = 0.008).The role of tumour-infiltrating B cells (CD20) as components of TILs in BC subtypes is still unclear. A positive correlation between higher numbers of total CD20+ B cells and ER and PR negativity, and basal phenotype has been reported . In our et al [in situ expression of 36 proteins in 41 patients with TNBC, suggested that all patients with B cells had also CD4 T cells and CD8 T cells [Using a digital pathology computational workflow to quantify the spatial patterns of five immune markers  in TNBC, Mi et al  demonstr T cells . In contImmune cellular subpopulations in BC representing the innate immunity  and adaptive immunity (CD3+ cells (CD8+ or CD4+) and CD20+ cells) , worth tAs no prior studies evaluating the exact relation between AR and TILs in this unique disease entity ‘TNBC’, we tended to represent all the statistical analyses and correlations which we evaluated, keeping with the aim of this exploratory study which may help future research. This study had mainly described the expression patterns of AR and TILs in TNBC. Moreover, the correlation between AR and the total and different TILs subpopulations was illustrated. TILs were evaluated by one pathologist who was blinded to the clinical characteristics and according to the International Working Group. However, our findings should be interpreted carefully. The limitations of our study include: i) the retrospective nature, ii) the small sample size  and iii) the survival data was not mature due to the short follow-up duration (median: 39 months).This study highlighted the probable relationship between the AR and total and differential TILs expression in TNBC; and the clinico-pathological characteristics as well. Understanding the immune micro-environment in a subset of tumours with poor prognosis and less identified therapeutic targets like TNBC, may pave the way for the advent of immunotherapy in specific group of patients. Moreover, lower TILs density may identify a subpopulation of TNBC who warrants more radical regional LNs management. The prognostic relevance and the potential predictive impact of AR and TILs in TNBCs merit further evaluation in larger scale studies.All authors declared no conflicts of interest.Conception and design: HE, JB, TH, AMA, MK, DZA, MO Ahmed, MO AlorabiAcquisition and interpretation of data: all authorsDrafting the manuscript and revising it critically for important intellectual content: all authors.None.


Novel coronavirus disease 2019 (COVID-19) has been the subject of a numerous research projects over the past year. In cases with a severe disease course or threatening long-term impairment due to disease, the German health care system offers insured persons the possibility of medical rehabilitation. In contrast to what was observed and expected at the beginning of the pandemic, COVID-19 patients with varying degrees of disease severity are represented in rehabilitation. To date, there is no common consensus on the content and aftercare of rehabilitation nor is there any knowledge about the short- and long-term effects of such a rehabilitation programme. In addition, these aspects were not considered with regard to the varying severity of the course of the disease. The present research project investigates this question.N = 350 rehabilitants after COVID-19 and a comparison group (CG) with N = 230 rehabilitants suffering from an obstructive respiratory disease. The participants will be recruited at five German rehabilitation facilities and undergo medical rehabilitation. This prospective, exploratory, multicentre, mixed-methods study will be evaluated as follows: (A) The quantitative portion includes questionnaires at different points in time  and contains standardised measurement instruments. For example, participation limitations, quality of life, health status, fatigue, psychomental limitations and disorders, performance in different areas of life and ability to work are measured. (B) Qualitative interviews are held at different times , and an expert workshop is conducted. Topics are rehabilitation content, satisfaction and aftercare as well as different outcomes on subjective health and participation impairments.The study sample will consist of Studies on other indications have already shown that pneumological rehabilitation has positive effects. Thus, it is expected that an improvement in different dimensions will be observed at the end of rehabilitation in both groups. With regard to the different severities of COVID-19, this study evaluates the long-term developments. Subsequently, the authors will elaborate concrete recommendations for medical rehabilitation after different courses of disease with regard to existing pneumological rehabilitation concepts.www.drks.de.Identifier: DRKS00023642; Registered: 01.12.2020.Date and version identifier: 08.04.2021; version 4.German Register of Clinical Trials,  COVID-19 is caused by the SARS-CoV-2 virus and is characterised by an unspecific, varying and diverse course of the disease, ranging from the lack of symptoms to severe pneumonia with lung failure and death . HoweverThere are currently no uniform definitions of the long-term effects of COVID-19 (“long COVID”) , 6, reduDue to suspected long-term consequences of COVID-19, the literature provides recommendations for physiotherapeutic treatment and early rehabilitation , 11 and Case reports in social networks and on public television show that the long-term effects of COVID-19 will have to be scientifically investigated in the future. These reports also demonstrate that persistent symptoms can also diverge from a linear course and vary in intensity and impairment over time. As a result, on the one hand, persons with a severe course of the disease can be found in the context of follow-up rehabilitation. On the other hand, patients with mild or moderate disease progression are impaired by long-lasting symptoms and can improve their occupational and social participation disorder through rehabilitation.Given the novelty of the disease, there is no uniform approach to medical rehabilitation after COVID-19. Neither the rehabilitation content nor outcomes are known. In addition, the aftercare that is offered, recommended and received is also unknown. This observational study will provide recommendations for action, include a comparison group and consider the severity of COVID-19.The aim of our study is to describe the health status after COVID-19 in the context of medical rehabilitation and its medium- and long-term effects in different dimensions. For this purpose, the study examines the burden of disease and changes in health status under medical rehabilitation and after six and twelve months. Based on the experience of studies on pneumological rehabilitation, we expect positive effects on the general state of health and psychomental disorders at the end of rehabilitation. At follow-up, we expect that in addition to the outcomes already mentioned, rehabilitation success is represented in the dimensions of participation, quality of life, performance in various areas of life, ability to work, and subjective prognosis of employment.Including rehabilitants from different clinics, it is possible to describe different therapeutic approaches, groups of patients and courses of disease. Statements are made about the extent to which existing rehabilitation approaches meet the needs of COVID-19 rehabilitants, the extent to which new rehabilitation therapy concepts have to be developed and how these are constituted. To date, it is unclear which requirements rehabilitants have with regard to aftercare, which recommendations rehabilitation clinics make and to what extent rehabilitants implement them.The analyses also considered the severity of the disease course and compared the study results with a reference group with obstructive airway disease . Finally, the study will provide concrete recommendations for rehabilitation after COVID-19.As an observational study, there is no intervention in the strict sense. The rehabilitants receive medical rehabilitation directly following hospitalisation or with greater time after SARS-CoV-2 infection. The duration of rehabilitation is 21 days with the possibility of extension. Given that the study participants are recruited from different clinics, the rehabilitation programmes differ in terms of therapy concept and structure.At the MEDIAN Klinik Heiligendamm, the therapeutic elements of rehabilitation include general strengthening, mobilisation and normalisation of respiratory functions. This is intended to improve respiratory distress, respiratory muscle strength, pulmonary resilience, psychomental impairments and/or stress-induced mental disorders of the disease. The main components of the therapy include respiratory gymnastics with respiratory muscle training as well as dissolving phlegm. If required, group discussions (1–2 h per week) are offered to exchange experiences of coronavirus disease, relaxation training and supportive psychological counselling to cope with trauma, fear or depression.The Reha-Zenrtum Schömberg treats patients in the pneumology department after severe respiratory insufficiency caused by a respiratory disease  and after pneumonia. With this experience, the rehabilitation centre also receives COVID-19 patients for rehabilitation. The therapy contents include physical and balneotherapy, physiotherapy and exercise therapy, occupational therapy and psychological support.Ostseeklinik Schönberg-Holm offers medical rehabilitation for various respiratory diseases. These illnesses range from bronchial asthma and COPD to the novel disease COVID-19. This facility also pursues a multimodal and holistic treatment approach during pneumological rehabilitation.The comparison group is recruited in the MEDIAN Klinik Flechtingen and MEDIAN Klinik Flachsheide Bad Salzuflen as well as in the Ostseeklinik Schönberg-Holm and Reha-Zentrum Schömberg. The CG consists of COPD and asthma bronchial patients. These patients receive rehabilitation and aftercare according to the standards of the German Pension Insurance. The rehabilitants receive treatments comparable to those after COVID-19. Depending on their needs, the rehabilitation includes physical training , respiratory physiotherapy, respiratory therapy, education, psychological interventions, nutritional therapy, counselling by social services and inhalation.The particular therapeutic approaches are documented and described within the study.Rehabilitants after varying degrees of COVID-19 who are able to work will be recruited to the study. The rehabilitants may no longer remain in isolation. Participants are between 18 and 65 years old and provide written consent. Participants in the control group have a diagnosis of obstructive airway disease, such as bronchial asthma or COPD. This group will be similar based on age, sex and ability to work.Individuals who do not provide consent and who do not have sufficient knowledge of German are excluded. Furthermore, rehabilitation may not be paid by German Social Accident Insurance.This is a prospective, explorative, longitudinal multicentre observational study without any specific intervention. Different methodological components  are included. The aim of the study is to obtain an overview of the problems associated with the new disease COVID-19 to derive recommendations for future clinical practice.The Institute for Social Medicine and Epidemiology at the University of Lübeck leads the study. Consecutive recruitment will occur in five different rehabilitation clinics in Germany . Each clinic designates a study representative who recruits suitable rehabilitants during their first days in rehabilitation. If possible, the study representatives personally contacted the study participants and provides them with the information material and the declaration of consent.0), at the end of rehabilitation (t1), six (t2) and twelve months after rehabilitation (t3). A questionnaire with standardised and validated instruments will be completed by all study participants at each measurement point.To measure the long-term burden of COVID-19 and the effects of rehabilitation, the study includes four measurement points: at the beginning , the IMET  measures disease , 19. Thehealth-related quality of life (HRQoL) at the beginning of rehabilitation and at catamnesis. The VR-12 is based on the SF-36 and the Veterans Health Study. It covers eight health domains, and a total score is provided [The VR-12 measures provided . We use provided .depression, anxiety and stress are assessed with the DASS. The instrument is mainly used in patients with pain, but it is also used for patients without pain-specific symptoms. The short version in Germany comprises 21 items and 7 items for the respective scales of depression, anxiety and stress. The individual scales of the DASS have sufficient to good reliability and have also been classified as valid [Core symptoms of as valid .generalised anxiety disorder as well as its symptom severity is measured using the GAD-7 [The presence of a he GAD-7 . For thiDepressiveness is measured with the PHQ-9 [he PHQ-9 , which ihe PHQ-9 .effects of chronic obstructive pulmonary disease. The CAT is a reliable instrument designed to measure the impact of COPD on health status and daily life [The COPD Assessment Test (CAT)  assessesily life . A semanwork ability. In numerous studies, this score provided acceptable values in terms of validity and reliability [The Work Ability Score (WAS) of the Work Ability Index (WAI) measures the iability . The higsubjective prognosis of employment. To divide the study participants into two risk groups, the respondents assess their occupational risk with three items from which an overall score is formed. The validity and reliability of the SPE scale was positively evaluated in a large sample [The German version of the SPE scale is used to predict the e sample .occupational changes are measured, including job retention interventions and types of job loss [Using individual items, job loss .Fatigue is measured with the EORTC-FA12, which has been validated in large international and multicentre studies [ studies . With th studies , a high  studies .COVID-19-associated life events. Questions are asked about the death of a relative due to COVID-19, COVID-19-associated physical and/or social deprivation, loss of autonomy and loss of economic livelihood. Participants are also asked if they infected someone else, how they feel about it and if they feel that their general practitioner takes their symptoms seriously.Single items without standardisation capture Performance in different areas of life is assessed with three scales from 0 to 10 for the areas of work, everyday life and leisure. The individual scales are valid instruments for determining performance limitations. A low sum score indicates a high level of performance limitations [itations , 33.physical activity, questions are asked in conjunction with Menski’s Federal Health Surveys [To assess the extent of  Surveys .The moderating variables include comorbidity , risk faThe rehabilitants receive the questionnaires at four measurement points. Some of the outcomes that are not likely to change during rehabilitation are only measured at the beginning of rehabilitation and at catamnesis. These are parameters that probably change only insignificantly during rehabilitation  or outcomes addressing everyday life and the domestic environment. Table Telephone interviews and an expert workshop occur during the study. The guidelines for the telephone interview were pre-tested with COVID-19 rehabilitants. Rehabilitants provided feedback on the comprehensibility of the questions and the length of the interview.Regarding telephone interviews, nine rehabilitants after COVID-19 were interviewed at three points in time: after medical rehabilitation, six and twelve months after rehabilitation. In the comparison group, eight interviews were held six months after rehabilitation. The interview participants varied in terms of sex, age and educational background. In the semi-structured interviews, the participants were asked about the course of their disease, the contents and goals of their rehabilitation, their satisfaction, their current disease burden, their social and occupational participation and their rehabilitation aftercare.The expert workshop involves clinical staff, funding agencies and scientists. The topics of the workshop include the rehabilitation procedure, general conditions of rehabilitation after COVID-19, and existing pneumological therapy concepts in rehabilitation. Finally, recommendations for rehabilitation after COVID-19 are formulated.Both the interviews and the expert workshop follow guidelines. The guidelines and the results of the qualitative research are discussed by the project team and at the brain trust “Arbeitsgruppe qualitative Methoden” (AQUAM). The interviews last approximately 30 min, while the expert workshop is correspondingly longer. With the agreement of all participants, the interviews and the workshop are recorded and transcribed word by word. The evaluation of data is based on qualitative content analysis.Study participants are uniquely organised and documented in all clinics using an automated documentation template. All study representatives are briefed on the template before recruitment.The included study participants receive an identification number (ID) to pseudonymise their personal data. The first number is used to classify whether the participant receives rehabilitation after COVID-19 or belongs to the comparison group. The second number of the ID refers to the rehabilitation clinic where the participant is recruited. The subsequent numbering corresponds to the number of recruited participants. Personal data are only documented in a separate Excel file; appropriate linkage occurs using the ID. The data will not be sent to the ISE until the personal contents are deleted. This collection includes only standardised documentation in the clinics. The data from the questionnaire are collected and kept separately from the ISE.The data for the quantitative part of the study (A) are generated by standardised written questionnaires. The answers to the questionnaires are entered into SPSS 22.0 by a student assistant under the guidance of the research assistant. The entered data are assessed for random duplicate entries (5 − 10 %) by checking their validity and plausibility.To determine an appropriate sample size, it is assumed that the indicators used to characterise the sample should be described with sufficient precision. Sufficient precision should be applied to continuous indicators if the range of the 95 % confidence interval around a sample mean does not exceed a pre-specified value. For categorical variables, it is analogously assumed that the range of the 95 % confidence interval should not exceed a pre-specified value by a sample proportion. In the case of a continuous variable, the limits mentioned are set at +/- 5 points for a scale with a value range of 0 to 100. In the case of proportions, the range of the 95 % confidence interval should not exceed +/- 10 % points in the range of percentages from 10 to 40 %. The required width of the 95 % confidence interval is achieved with a continuous variable from a sample size of N = 100. In the case of a binary variable in the range between 10 and 40 %, the width of the 95 % confidence interval will be less than 20 % points for sample sizes of 100 or more. To compensate for smaller deviations in the required number of cases, a 20 % larger sample is taken compared to this net number of cases, i.e., N = 120 cases. We also expect a dropout rate of 20 % , 38. ThuIn the course of the study, no predefined hypotheses are confirmed. Rather, the rehabilitation groups are precisely characterised. Comparative analyses are used to determine the relevant differences between the subgroups and the comparison group. Accordingly, we mainly provide descriptive evaluations on the burden of disease as well as on health-related changes during and after rehabilitation.For the interval-scaled variables, variance analyses with measurement repetition are calculated to evaluate the pre-post differences of the outcomes at the various measurement times. Depending on the data constellation, the t-test for dependent or independent samples is used as a parametric significance test. Non-parametric tests are performed for ordinally scaled data. Subgroup analyses will be planned for sex, age and education as well as risk factors and comorbidities. To classify the findings in COVID-19 patients, the stress manifestations and the measured changes are compared with those in the CG.In addition to the analysis of the health-related changes in relation to a comparison group, the treatment concepts from different rehabilitation facilities are also examined. The type and extent of the therapy, characteristics of the included rehabilitants, perceived benefits of the rehabilitation and subjectively experienced health improvements are recorded. All calculations are performed with SPSS 22.0.The interviews and focus group are analysed using qualitative content analysis –41. We uThe novelty of the disease and the increasing use of rehabilitation services by COVID-19 patients enhances the call for research. At present, COVID-19 rehabilitation does not follow uniform guidelines. Therefore, it is necessary to review the different therapeutic approaches and to formulate recommendations for pneumological rehabilitation after COVID-19. Furthermore, the rehabilitation needs of COVID-19 patients and their rehabilitation capacity will be assessed. The concrete short-, medium- and long-term effects of COVID-19 medical rehabilitation have not yet been evaluated.When assessing rehabilitation, it is also essential to analyse rehabilitation aftercare. To date, there is no evidence on the needs at the end of rehabilitation, nor is there a uniform approach to rehabilitation aftercare. This study clarifies which aftercare recommendations are given to these rehabilitants, to what extent they are realised and how the needs of the rehabilitants are met.It now appears that COVID-19 survivors of any disease severity seek rehabilitation. Therefore, the study results are also differentiated against the different degrees of severity and additionally contrasted with a comparison group. Finally, recommendations for rehabilitation and rehabilitation aftercare need to be developed for COVID-19 patients. During the telephone interviews and written survey, the study participants are asked about their aftercare recommendations, the use of aftercare programmes and their wishes or needs for the time after rehabilitation. By classifying the results on the basis of a comparison group, conclusions are drawn for further projects on suitable aftercare strategies in the novel indication COVID-19. Further study could aim at maintaining long-term rehabilitation effects and maintaining or restoring employability. In addition, it may be possible that different recommendations are made for the varying severity of the disease course; therefore, further consideration is necessary.As this is an observational study, the baseline situation and the changes in the mentioned outcomes are described. These results are reported in relation to the comparison group, and statements on the extent to which there are similarities and differences are provided. The rehabilitation contents, needs and ability; aftercare recommendations and their implementation are also described descriptively for different degrees of disease severity.Although the rehabilitants do not receive an intervention in the strict sense, they all undergo rehabilitation. Based on previous studies on pneumological rehabilitation as well as from the current experience of rehabilitation physicians, we expect positive changes in outcomes during rehabilitation. For example, we expect a significant reduction in the burden of disease and psychological impairments at the end of rehabilitation. The extent to which the effects on outcomes change over all four measurement points will be described in this study.The results of the study will show whether rehabilitants have different needs in terms of rehabilitation, its contents and aftercare due to their individual course of illness, their previous illnesses or other personal prerequisites. With regard to aftercare, it is assumed that individual needs range from their own initiative and classic aftercare programmes to more intensive aftercare.In recent months, it has become apparent that the need for rehabilitation services for COVID-19 survivors is increasing. To offer a treatment that meets the needs of rehabilitants, appropriate research is necessary. At the end of the present study, practical recommendations for rehabilitation after COVID-19 will be developed. A common consensus in the rehabilitation procedure after COVID-19 can guarantee a universal standard in German rehabilitation.The observational study is conducted in five German rehabilitation clinics. However, it is possible to transfer the results and provide recommendations to other pneumological rehabilitation clinics.To ensure equal treatment, all characteristics of the participants  and the disease  are documented and compared between groups. If differences arise between the two groups, they are comparably integrated in the analysis . Based on our present knowledge, we assume that structural changes in the context of this project are very unlikely.


Prevotella and Escherichia were associated with AR. Furthermore, significant statistical interactions were observed between retinol and Prevotella. The age- and sex-adjusted odds of AR were 25-fold lower in subjects with high retinol intake and high Prevotella abundance compared to subjects with low retinol intake and low Prevotella abundance. Our data provide insights into complex interplay between dietary nutrients, gut microbiome, and the development of AR.Several studies suggest the involvement of dietary habits and gut microbiome in allergic diseases. However, little is known about the nutritional and gut microbial factors associated with the risk of allergic rhinitis (AR). We recruited 186 participants with symptoms of AR and 106 control subjects without symptoms of AR at the Hitachi Health Care Center, Japan. The habitual consumption of 42 selected nutrients were examined using the brief-type self-administered diet history questionnaire. Faecal samples were collected and subjected to amplicon sequencing of the 16S ribosomal RNA gene hypervariable regions. Association analysis revealed that four nutrients  were negatively associated with AR. Among 40 genera examined, relative abundance of  The classical symptoms of AR are nasal itching, sneezing, rhinorrhoea, and nasal congestion, and ocular symptoms are also frequent3. Over 500 million people worldwide are suffering from AR1. The burden of disease related to AR is substantial due to reduced patients’ quality of life (QoL), lower performance at work and school, poorer sleep quality and cognitive function, and increased irritability and fatigue4. Moreover, AR is a risk factor for asthma3. Despite recognition of AR as a global health problem, its aetiological risk factors have yet to be fully understood1.Allergic rhinitis (AR) is a symptomatic disorder of the nose caused by immunoglobulin E (IgE)-mediated reactions against inhaled allergens5. Diet is an important source of nutrients that may modulate the risk of allergic diseases6. For instance, systemic oxidative stress enhances inflammatory responses relevant to allergy; and therefore, antioxidants may play a key role in the prevention of allergic diseases7. Furthermore, epidemiological studies have suggested that fruits and vegetables that contain several antioxidants protect against allergic diseases8. Therefore, researchers have intensively investigated the nutrients exhibiting a protective (or adverse) effect on the development of allergic diseases, such as asthma8. However, little is known about the relationship between nutrients and the risk of AR.The prevalence of allergic diseases, including AR and asthma, has increased in many countries, and changes in dietary habits may be one of the factors responsible for this trend9. The gut microbiota produces bioactive metabolites from dietary materials10. These metabolites influence host immune responses through the interplay with regulatory T (Treg) and dendritic cells. Therefore, the composition of the intestinal microbiota potentially links with the development of allergic diseases11.Recent studies have suggested that gut microbiota may be involved in the pathogenesis of allergic diseases. The gut microbiota is mainly composed of bacteria, and its composition is highly dynamic and influenced by dietary habits of their hostIn the present cross-sectional study, we investigated the relationship between daily nutritional intake, gut microbiome composition, and self-reported AR symptoms in middle-aged workers. Our aim was three-fold: (i) to suggest which nutrients are associated with AR; (ii) to search for microbes whose abundances are associated with AR; and (iii) to examine whether nutrients and microbes have a combinatorial effect on AR. Various dietary components are metabolised by gut microbiome enzymes; and therefore, the effect of dietary nutrients on the odds of AR may be modulated by intestinal microbiota.14. They have continuously undergone annual comprehensive health examinations, including cardiovascular and cancer screening. Therefore, we could select visitors based on the results of their previous health examinations. Previous visitors without a history of serious illness  and unhealthy risk factors  were invited to participate in this study by e-mail. The number of enrolled volunteers was 301, and they were asked at visit reservation whether they had symptoms of seasonal or perennial AR. They re-visited the HHC Center for their annual health check-up from September 2018 to February 2019. The study participants were asked to complete a questionnaire on their dietary habits and symptoms of AR. In addition, stool samples were collected. Nine subjects were excluded due to withdrawal of consent (n = 2) or their inconsistent report of AR symptoms in the questionnaire compared to that at visit reservation (n = 7). Finally, 186 participants with symptoms of AR and 106 control subjects without symptoms of AR were included in the study. This study was reviewed and approved in advance by the Hitachi Hospital Group Ethics Committee , the Institutional Review Board of the Hitachi Ltd. (Approved No. 220-1 and 238-1), and the Research Ethics Committee of RIKEN (Approved No. H30-5). All participants provided written informed consent. This study was conducted according to the principles of the Declaration of Helsinki.The study participants were recruited at the Hitachi Health Care (HHC) Center, where annual health examinations of employees and their spouses from 35 affiliated companies  are performed. Most visitors at the HHC Center were employed by electronics-related companies and performed generally representative (not industry-specific) jobs, including general affairs, design, research and development, sales, and manufacturing. The characteristics of the visitors were described previously16. The BDHQ does not consider the portion size. Instead, standard portion sizes were defined according to recipe books for Japanese diets16. Based on the standard tables of food composition in Japan revised in 201017, crude estimates for dietary intake of total energy and 42 selected nutrients were calculated. Energy adjustment was performed using the density method18. Adjusted values for energy-providing nutrients were calculated by dividing the amount of energy obtained from each nutrient by the total energy intake. Adjusted values for non-energy-providing nutrients were defined as the amount of weight of each nutrient per 10 MJ of dietary energy intake.Nutritional consumption data for the study participants were obtained using the brief-type self-administered diet history questionnaire (BDHQ). The questionnaire asked about the intake frequency of 58 selected food and beverage items, which are commonly consumed in Japan, in the preceding monthg for 10 min at 4 °C. The pellets were washed with 35 mL PBS once, further washed with TE20 buffer , and subjected to DNA extraction. Bacterial DNA was isolated and purified from the faecal samples according to enzymatic lysis methods described previously19.Fresh faecal samples were collected in plastic containers containing glass beads (Tomy Seiko) and RNAlater Reagent (Life Technologies Japan). The samples were transported at 4 °C to the laboratory. In the laboratory, the faecal samples (~ 0.2 g) were suspended in 15 mL phosphate-buffered saline (PBS) buffer and filtered with a 100-μm-mesh nylon filter (Corning) to remove human and eukaryotic cells and debris from the faecal sample. The debris on the filter was washed twice with PBS. The bacteria-enriched pellet was obtained by centrifugation of the filtrate at 9000×19. PCR was performed using 50 μL of 1 × Ex Taq PCR buffer composed of 10 mM Tris·HCl (pH 8.3), 50 mM KCl, and 1.5 mM MgCl2 in the presence of 250 μM dNTPs, 1 unit Ex Taq polymerase (Takara Bio), forward and reverse primers (0.2 μM), and ~ 20 ng of template DNA. PCR was performed in a 9700 PCR System (Life Technologies Japan), and the following cycling conditions were used: initial denaturation at 96 °C for 2 min, followed by 20 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min, with a final extension at 72 °C. PCR amplicons were purified using AMPure XP magnetic purification beads (Beckman Coulter) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Japan). An equal amount of each PCR amplicon was mixed and subjected to multiplexed amplicon sequencing with MiSeq (2 × 300 paired-end run), according to the manufacturer’s instructions.The 16S V1–V2 region was amplified using polymerase chain reaction (PCR) with barcoded 27Fmod (5′-agrgtttgatymtggctcag-3′) and the reverse primer 338R (5′-tgctgcctcccgtaggagt-3′)http://microbiome.osu.edu/), and a reference genome sequence database obtained from the NCBI FTP site . From the filter-passed reads, 10,000 high-quality reads per sample were randomly chosen. The total reads  were then sorted by frequency of redundant sequences and grouped into operational taxonomic units (OTUs) using UCLUST with a sequence identity threshold of 97%. The representative sequences of the generated OTUs were subjected to a homology search against the databases mentioned above using the GLSEARCH program for taxonomic assignments. For assignment at the phylum, genus, and species levels, sequence similarity thresholds of 70%, 94%, and 97%, respectively, were applied.Two paired-end reads were merged using the fastq-join program based on overlapping sequences. Reads with an average quality value of < 25 and inexact matches to both universal primers were filtered out. The reads lacking both forward and reverse primer sequences were removed. Filter-passed reads were used after trimming off both primer sequences. Reads having BLAST match lengths < 90% with the representative sequence in the 16S databases (described below) were considered as chimeras and removed. Finally, filter-passed reads were used for further analysis. The 16S database was constructed from three publicly available databases: Ribosomal Database Project (RDP) , CORE  using R software (v4.1.1). The genus-level relative abundance was represented as a percentage. Genera with a mean relative abundance of ≥ 0.2% were considered for statistical analyses.Three measures of alpha-diversity 21. In the logistic regression analysis, nutritional and microbial variables were classified into four categories based on quartiles (Q1–Q4). The same definition of variable categories was also used in multivariate analyses that incorporated age, sex, multiple nutrients, and/or multiple microbial genera. Statistical interaction was tested by adding a multiplicative interaction term in an age- and sex-adjusted logistic regression model and by using the chi-squared test for the comparison with and without the interaction term. Two-sided P < 0.05 was considered statistically significant, and two-sided P < 0.1 was regarded as marginally significant in all analyses. All statistical analyses were performed using the R software 3.6.1 .Differences in clinical characteristics between the AR and control groups were assessed using the Wilcoxon rank sum test for continuous variables or Fisher’s exact test for discrete variables. Similarly, potential associations of nutritional and microbial variables with AR were tested using the Wilcoxon rank sum test. For nutrients and microbial genera associated with AR, age- and sex-adjusted logistic regression analysis was performed. Analysis of the receiver operating characteristic (ROC) curve and area under the curve (AUC) was performed with ‘pROC’ R packageP = 0.006 for systolic blood pressure; P = 0.003 for diastolic blood pressure; and P = 0.035 for triglyceride). No significant differences between the groups were observed in body mass index, HbA1c, serum total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol.The number of subjects who reported the symptoms of AR was 186, and the number of control subjects was 106 .The total dietary energy was similar between the AR and control groups  Table . Among tP for trend = 0.0004). Q4 had a significantly lower odds of AR than Q1 (P = 0.0001), whereas Q2 and Q3 did not. Similar dose-dependent trends were observed in the association of vitamin A and cryptoxanthin with AR  suggested that a high level of retinol intake (Q4), a high level of cryptoxanthin (Q4), and intermediate levels of copper intake (Q2 and Q3) were independently associated with decreased odds of AR , possibly because the intake levels of retinol and vitamin A were highly correlated . We observed no statistical interaction between the four nutrients and sex .The energy-adjusted intake levels of the four nutrients were not statistically different between males and females  and between males and females . In total, 40 genera with mean relative abundance exceeding 0.2% were included. Among them, the relative abundances of three genera  were significantly different between the AR and control groups .Four measures of microbial alpha-diversity were not significantly different between the AR and control groups Table . The micPrevotella was either zero or near zero in a large proportion of subjects  in their gut microbial community, whereas the relative abundance of Prevotella widely ranged in other subjects (relative abundance reached as high as 55.6%). Compared to the first quartile (Q1) of relative abundance of Prevotella, the age- and sex-adjusted odds ratios for Q2, Q3, and Q4 were 0.67, 0.83, and 0.35, respectively , but not in Q2 and Q3. A significant dose-dependent trend was observed between relative abundance of Prevotella and the age- and sex-adjusted odds of AR (P for trend = 0.017).The relative abundance of cts Fig. a: 35.5% Bifidobacterium seemed to be higher in the AR group than in controls . Compared to Q1 of relative abundance of Bifidobacterium, neither of Q2, Q3, nor Q4 showed significant difference in the age- and sex-adjusted odds of AR , whereas Q2 and Q3 did not.The AR group showed a higher relative abundance of ols Fig. e. A cleaPrevotella and Escherichia  and a high relative abundance of Escherichia (Q4) were independently associated with decreased and increased odds of AR, respectively  in combination to distinguish the AR and control groups was only modest . We observed no statistical difference in the relative abundance of the two genera between males and females  and no statistical interaction between the two genera and sex .The ability of the microbial variables  and between vitamin A and Prevotella   indicated that a high level of retinol intake (Q4), intermediate levels of copper intake (Q2 and Q3), a high relative abundance of Prevotella (Q4), and a high relative abundance of Escherichia (Q4) were independently associated with AR , whereas vitamin A was not statistically associated with the AR  and between vitamin A and Prevotella (P for interaction = 0.041), but not for the other 6 combinations , compared to the reference group with Q1–Q3 of retinol intake and low relative abundance of Prevotella (Q1–Q3)  compared to the reference. In the Q4 retinol intake group with high abundance level of Prevotella (Q4), a combinatorial protective effect was observed (P for interaction = 0.048); the age- and sex-adjusted odds ratio for this group was 0.04, which was 6.5-times smaller than the expectation based on a simple multiplication that assumes no statistical interaction (0.04 < 0.64 × 0.40 The age- and sex-adjusted odds ratio of AR was 0.64 in the low or intermediate retinol intake group Q1–Q3) with a high relative abundance of  with a hPrevotella  find nutrients associated with AR; (ii) discover gut microbes associated with AR; and (iii) examine the combinatorial effect between nutrients and gut microbes on AR. Towards these ends, we recruited 186 subjects with AR symptoms and 106 controls and examined their dietary nutritional intake and composition of the gut microbiome. An association analysis suggested that retinol, vitamin A, cryptoxanthin, and copper were associated with the age- and sex-adjusted odds of AR. Furthermore, the relative abundances of 16, we defined dietary intake of vitamin A  as the sum of retinol, β-carotene/12, α-carotene/24, and cryptoxanthin/24. β-Carotene, α-carotene, and cryptoxanthin are common provitamin A carotenoids, which can be converted into retinol by enzymes in the intestinal mucosa22. As expected from this definition, dietary intake levels of vitamin A and retinol were highly correlated in our study population (R = 0.96). Vitamin A is essential for multiple functions in the human body, including embryonal development, good vision, epithelial differentiation, and maintenance of immune function23, particularly in the differentiation of naïve T cells. Without retinoic acid, a metabolite of vitamin A, transforming growth factor beta (TGF-β) promotes the differentiation of naïve T cells into T helper 17 (Th17) cells, which are involved in inflammation, autoimmunity, and allergic disorders25. In the presence of retinoic acid, TGF-β helps intestinal dendritic cells to mediate the differentiation of naïve T cells into Treg cells, which suppress autoimmune responses26. Natural Treg cells develop in the thymus, whereas Treg cells that develop in non-thymus tissues are called induced Treg (iTreg) cells27. iTreg cells play a crucial role in the maintenance of intestinal homeostasis, including tolerance to commensal bacteria28. In addition, vitamin A is important for gut mucosal turnover and barrier function29, intestinal IgA secretion30, and T cell homing to the intestine28. Taken together, this finding that vitamin A and retinol are associated with a decreased odds of AR may be related to the role of vitamin A in modulating intestinal immune responses.Principal dietary sources of vitamin A were retinol from animal foods and provitamin A carotenoids present in fruits, vegetables, egg yolk, and butter. As in a previous study8. From this perspective, vitamin A and carotenoids are the most effective antioxidants at low oxygen tensions that are typical of human tissues31. Among the carotenoids examined in this study, only cryptoxanthin was associated with AR, whereas α-carotene and β-carotene were not. As all three of these carotenoids have antioxidant activity33, it remains unclear whether the association of retinol, vitamin A, and cryptoxanthin with AR is related to their antioxidant activity.Dietary antioxidants may protect against allergic diseases34. As such, it is involved in iron metabolism, antioxidant activity, neuropeptide synthesis, and immune function35. Copper is essential for humans but toxic at high levels; and therefore, both copper deficiency and excess can produce adverse health effects36. Copper deficiency influences the oxidant defence system, resulting in increased oxidative damage to lipids, DNA, and proteins37. Similarly, the toxic effects of copper at high concentrations are related to the generation of oxygen free radicals, and excess copper enhances lipid peroxidation and DNA damage38. In this study, we observed a reverse J-shaped relationship between copper and AR. This reverse J-shaped curve may be related to the anti- and pro-oxidant activities of copper at appropriate and high concentrations, respectively.Copper, an essential trace metal, is a cofactor of many redox enzymes.39, compositions of the human gut microbiome are classified into three clusters, which are named enterotypes. The first enterotype was characterised by Bacteroides (B-type), whereas the second and third enterotypes were dominated by Prevotella (P-type) and Ruminococcus (R-type), respectively. Later studies reduced the number of enterotype clusters into two, because the B- and P-type enterotypes were consistently identified, but the identification of R-type was dependent on clustering and modelling methods42. Note that the concept of enterotypes has been challenged by some researchers because the B- and P-type enterotypes were not clustered separately in large-scale datasets, but instead samples were distributed in a gradient across the B- and P-type groups43. Long-term dietary patterns are associated with enterotypes40. Individuals who habitually consume a Western diet high in protein and animal fat tended to have the B-type enterotype40. In contrast, vegetarian, or Mediterranean diets rich in fruits and vegetables were positively associated with P-type enterotype44. Additionally, plant-rich diets abundant in carbohydrates and fibres were associated with the P-type enterotype42. Based on these studies, it can be inferred that the relative abundance of Prevotella may reflect adherence to healthy dietary patterns. We observed a negative association between Prevotella and AR, and the association was still significant even after adjusted for AR-associated nutrients. Thus, it is unlikely that the association between Prevotella and AR is confounded by dietary patterns.The human gut harbours hundreds of microbial species. According to Arumugam et al46. For example, a study of Chinese nomads showed that the prevalence of the P-type enterotype gradually decreased with the degree of urbanisation47. Given this gradient of Prevotella along with urbanisation, our finding of the association between Prevotella and AR reminds us of the microflora hypothesis of allergic diseases, which states that an unhealthy microbiota composition attributable to urbanisation or westernisation contributes to the development of allergies48. Several studies support this hypothesis. First, since the 1950s, industrialisation and urbanisation have accelerated around the world, and simultaneously, the prevalence of allergic diseases has increased in urban areas49. Second, exposures to animals, early day-care attendance, and increased number of siblings were associated with a decreased risk of allergen sensitisation50, indicating that early exposures to multiple types of microorganisms may facilitate development of the immune system49. Similarly, having older siblings was also associated with a decreased risk of AR in children51. Third, reduced exposure to microorganisms in early life is responsible for a shift in the balance between type 1 and type 2 helper T (Th1/Th2) cells towards the overactive Th2 arm, which stimulates IgE-mediated allergic responses52. Based on the microflora hypothesis, our data indicate that the abundance of Prevotella in adults may serve as a surrogate marker for early exposure to particular microorganisms that are essential for immune system development. Alternatively, Prevotella itself may be one of the essential microorganisms for the development of immune system.Several observational studies found that the P-type enterotype was frequently observed in rural or isolated populations, whereas the B-type enterotype was abundant in industrialised countriesEscherichia species and allergic disorders. The intestinal relative abundance of Escherichia was higher in children with asthma or rhinitis than in controls53. The positive association between Escherichia and asthma was replicated in another study54. In infants, a high abundance of E. coli was associated with an increased risk of atopic eczema55. To our knowledge, little is known about the molecular mechanisms underlying the association between Escherichia and allergies. We expanded previous findings in children by providing evidence of a positive association between Escherichia and AR in adults.Several studies have reported the relationship between 56 reported that bacterial diversity in the early intestinal flora 1 and 12 months after birth was inversely associated with the risk of developing allergic sensitization, peripheral blood eosinophils, and AR during the period until 6 years of age. Meanwhile, gut microbial diversity at 7 years of age was not associated with the presence of AR symptoms57. A recent study compared gut microbial diversity between 33 AR and 31 non-AR adults (mean age was 32 years old in both groups) and showed that the alpha-diversity in AR group was significantly higher than that of the non-AR group58. Our data showed that there was no statistical difference in gut microbial diversity between AR and control groups. Further researches, including meta-analyses and systematic reviews, are required to conclude whether the alpha-diversity of adult gut microbiome is associated with the risk of AR.Bisgaard et al.Prevotella as well as between vitamin A and Prevotella, suggesting that a combination of high dietary intake of retinol and carotenoids with high abundance of Prevotella may have a protective effect on the development of AR. Retinoic acid, which is derived from retinol or carotenoids, modulates the intestinal immune system59. In humans, retinoic acid is irreversibly synthesised from retinal by a host enzyme, aldehyde dehydrogenase60, which is in turn reversibly produced from retinol by alcohol dehydrogenases or retinol dehydrogenase60, or from carotenoids by β-carotene-15, 15’-oxygenase 161. Interestingly, some gut microorganisms encode enzymes that are potentially involved in retinal biosynthesis62. For example, Prevotella marshii DSM 16,973 harbours a gene belonging to the brp/blh family63, which encodes an enzyme that produces retinal from β-carotene64. The observed statistical interaction of retinol and vitamin A with Prevotella may indicate a complex interplay of host and bacterial genes in the metabolism of retinol and carotenoids.We observed a statistical interaction between retinol and 65. Under the presence of nondifferential misclassification, the observed association is usually underestimated66. Accordingly, it is unlikely that our findings are an artefact caused by the self-reported definition of AR. Third, ~ 90% of the subjects were male. Although we observed no evidence of sex-specific effects of nutritional and microbial factors on the odds of AR, further research is warranted to reveal whether our findings are consistent with female populations.The present study has several limitations. First, we searched for nutritional and gut microbial factors associated with AR and incorporated 42 nutrients and 40 genera in association analyses. We did not apply multiple testing correction. Therefore, our findings should be carefully interpreted. Although we found multiple candidate factors associated with AR, further studies are needed to confirm the associations. Second, our definition of AR was based on self-reported symptoms. A clinical diagnosis based on interviews, rhinoscopy, skin tests, and allergen-specific IgE tests is required for a more accurate definition. Although AR and control groups could be misclassified due to self-reporting, epidemiological studies based on self-reported AR symptoms have assumed that the misclassification was most likely nondifferential Prevotella and Escherichia) were associated with the age- and sex-adjusted odds of AR. In addition, a combinatorial protective effect of retinol and Prevotella was observed, and the age- and sex-adjusted odds of AR was 25-fold lower in subjects with a high level of dietary retinol intake and a high abundance of Prevotella compared to those with low retinol intake and a low abundance of Prevotella. Our results provide insight into the complex interplay between dietary nutrients, gut microbiome, intestinal immune systems, and the development of AR.In conclusion, we suggest that four nutrients  and two gut microbial genera (Supplementary Information.


Androgen receptor (AR) signaling inhibitors represent the standard treatment in metastatic castration resistance prostate cancer (mCRPC) patients. However, some patients display a primary resistance, and several studies investigated the role of the AR as a predictive biomarker of response to treatment. This study is aimed to evaluate the role of AR in liquid biopsy to predict clinical outcome to AR signaling inhibitors in mCRPC patients.Six milliliters of plasma samples were collected before first-line treatment with abiraterone or enzalutamide. Circulating free DNA (cfDNA) and exosome-RNA were isolated for analysis of AR gain and AR splice variant 7 (AR-V7), respectively, by digital droplet PCR.n = 40) or enzalutamide (n = 44) as first-line therapy. Twelve patients (14.3%) presented AR gain and 30 (35.7%) AR-V7+ at baseline. Median progression-free survival (PFS) and overall survival (OS) were significantly longer in AR-V7− vs AR-V7+ patients . Patients carrying the AR gain had a median PFS of 4.8 vs 24.3 months for AR normal patients (p < 0.0001). Median OS was significantly longer in AR normal vs patients with AR gain . A significant correlation between AR-V7 and AR gain was observed . The AR gain/AR-V7 combined analysis confirmed a strong predictive effect for biomarkers combination vs patients without any AR aberration .Eighty-four mCRPC patients received abiraterone (The present study demonstrates that cfDNA and exosome-RNA are both a reliable source of AR variants and their combined detection in liquid biopsy predicts resistance to AR signaling inhibitors. AR gain and/or point mutations [AR gene gain are associated with shorter progression-free (PFS) and overall survival (OS) with abiraterone or enzalutamide in mCRPC, even if a direct comparison of these two biomarkers was not provided to date [AR gain and AR-V7 in circulating free DNA and in RNA of plasma-derived exosomes, respectively, on clinical outcome in  chemotherapy-naive mCRPC patients, treated with first-line abiraterone or enzalutamide, in order to identify a biomarker strategy for the early identification of resistance to treatment.Prostate cancer is the most frequent cancer in men and is rated as the second cause of cancer deaths in the Western world . The sigutations . Liquid utations , 11 thanutations , and itsutations . A pleth to date , 14–16. AR gain in circulating free DNA (cfDNA) and AR-V7 in exosomes-derived RNA to predict treatment outcome with first-line abiraterone or enzalutamide. Blood samples for plasma analysis were obtained from eligible patients at baseline and treatment strategy was at the physician’s choice without awareness of AR status. Laboratory investigators were blinded to the clinical information on patients’ outcome.This retrospective study evaluated the ability of Patients with mCRPC starting first-line treatment with abiraterone or enzalutamide were enrolled. All patients should have a histologically confirmed diagnosis of prostate adenocarcinoma without neuroendocrine differentiation, ongoing treatment with LHRH analog and no prior treatment with docetaxel. Eligibility criteria included also an Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0–2, adequate cardiac, renal, hepatic, and bone marrow function. Serum prostate-specific antigen (PSA) was assessed within 1 week from starting treatment and monthly thereafter. In addition, biochemical parameters, such as: serum LDH, alkaline phosphatase (ALP), complete blood count were also measured at therapy baseline. Radiographic disease was assessed with computed tomography (CT) and bone scan at the time of screening and every 12 weeks. Progressive disease (PD) was assessed according to Prostate Cancer Clinical Trials Working Group 3 (PCWG3) guidelines . Laboratg for 10 min at 4 °C within 2 h after drawing. Plasma was divided into 2 aliquots of 2 ml and stored at −80 °C until analysis. Circulating free DNA was extracted with the QIAamp Circulating Nucleic Acid Kit  and the total DNA was quantified using the Quant-iT high sensitivity PicoGreen double-stranded DNA Assay Kit (Invitrogen) or by spectrophotometric evaluation . A multiplex digital droplet PCR  assay was performed to assess plasma AR gain, as previously described [AR gain was defined as copy number amplification when over the 2.01 threshold [Peripheral blood samples were collected from each patient within 30 days before starting abiraterone or enzalutamide treatment. Overall, 10 ml of blood were collected, transferred in ethylene-diamine-tetra-acetic acid (EDTA) tubes and centrifuged at 1900 × hreshold .AR gain values, the Spearman’s rho coefficient was calculated. The relationship between AR-V7 status and PSA decline >50% as well as between AR gain and PSA decline >50% have been evaluated by Chi-square test. Statistical significance was defined by a p value <0.05. All statistical analyses were performed with MedCalc Statistical Software version 14.8.1 . Categorical variables, such as ECOG PS, tumor stage and Gleason score at diagnosis, type of hormonal treatments, presence of bone, lymph node, and visceral metastases, were described by absolute and relative frequencies, whereas quantitative factors as time from diagnosis to start hormonal therapy and baseline total PSA level by median and range.The Kolmogorov–Smirnov test was performed to evaluate the normality of the quantitative data distributions. Kaplan–Meier method was used to evaluate time-to-event outcomes, including clinical and radiographic PFS and OS, survival differences were compared by the log-rank test. To evaluate the correlation between AR-V7 and Neutrophils-to-lymphocytes ratio (NLR) >3 was used to define unfavorable prognosis together with the number of disease sites, considering with a good prognosis patients with one non-visceral metastatic site and with worse prognosis patients with at least two non-visceral metastatic sites  or one visceral site .n = 16) were previously described in Conteduca et al. [n = 77, 84.3%) and showed no visceral metastases . Table AR gain was detected in 14.3% patients and 36% were AR-V7+ at baseline, 9 patients had both AR gain and AR-V7. AR gain positive patients had a median PFS of 4.8 vs 24.3 months in AR normal patients  was also found. In the univariate and multivariate model, AR-V7 and AR gain, resulted significantly associated with shorter PFS and , and their association was maintained at the multivariate analysis  from circulating free DNA and RNA using next-generation sequencing and PCR, respectively. This study provided new insights into how AR aberrations were also associated with a worse outcome in earlier stages of prostate tumor, even at a lower frequency (up to 20%) than that observed in CRPC. Alternative acquired mechanisms of resistance may be AR-dependent or independent [AR gain and AR-V7+ using cfDNA and exosomes was independent of clinical prognostic factors such as NLR or site score, supporting the hypothesis that AR status is an independent predictive biomarker of resistance to abiraterone and enzalutamide. Considering the OS, in addition to AR gain and AR-V7+ also the NRL showed to be an independent predictive factor, suggesting that, being NLR the link between innate (neutrophil) and adaptive (lymphocyte) immune response, NLR may represent the dynamic change of host in the inflammatory and immune response, and treatment effect [A recent sub-analysis of the TITAN trial  in mCRPCependent , and incependent , alternaependent , 28, gluependent  and addiependent . The negt effect .The small sample size of the cohort and its retrospective design represent the major limitations of the present study. Other AR aberrations such as mutations  and somap < 0.0001).We found that the presence of AR-V7 or/and AR gain at baseline were related with more aggressive cancers and suggest these should be considered when deciding on treatment. In conclusion, the present study demonstrates that the AR gain/AR-V7 combined analysis has a strong value as prognostic and predictive biomarker, since both OS and PFS were significantly shorter compared to patients without any AR aberration  already demonstrated to be affordable and that can be widely implemented in clinical laboratories , 20, 36.Supplementary figures legendSupplemental Material 1Supplemental Material 2