AR) copy numbers (CN) and mutations in plasma circulating cell-free DNA (cfDNA) and evaluated the AR statuses of patients with CRPC. AR amplification was detectable in VCaP cell line (AR amplified) genomic DNA (gDNA) diluted to 1.0% by digital PCR (dPCR). AR mutation were detectable in LNCaP cell line (AR T878A mutated) gDNA diluted to 0.1% and 1.0% by dPCR and target sequencing, respectively. Next, we analyzed AR status in cfDNA from 102 patients. AR amplification and mutations were detected in 47 and 25 patients, respectively. As a biomarker, AR aberrations in pretreatment cfDNA were associated with poor response to abiraterone, but not enzalutamide. In serial cfDNA analysis from 41 patients, most AR aberrations at baseline diminished with effective treatments, whereas in some patients with disease progression, AR amplification or mutations emerged. The analysis of AR in cfDNA is feasible and informative procedure for treating patients with CRPC. cfDNA may become a useful biomarker for precision medicine in CRPC.The therapeutic landscape of castration-resistant prostate cancer (CRPC) has rapidly expanded. There is a need to develop noninvasive biomarkers to guide treatment. We established a highly sensitive method for analyzing androgen receptor gene ( Potent AR axis-targeted agents (ARATs) such as abiraterone and enzalutamide prolong survival of patients with CRPC; however, nearly a third of the patients show primary resistance to ARATs4. Taxanes such as docetaxel and cabazitaxel are also known to prolong survival of patients with CRPC. In addition, multiple AR targeting and non-AR targeting agents are currently in development. Given the wide selection of treatments for CRPC, it is important to optimize the treatment sequence and promote precision medicine based on molecular and genetic biomarkers.Prostate cancer (PCa) is the second most frequently diagnosed cancer among males worldwide. PCa is driven by androgen receptor (AR) signaling, with the standard treatment for advanced disease being androgen deprivation therapy. However, most patients eventually gain resistance and develop castration-resistant prostate cancer (CRPC). The primary mechanism underlying CRPC is reactivation of the AR pathway. This includes 7. However, it is difficult to routinely obtain tissue in PCa, which predominantly metastasizes to bone. Even if successfully collected, the sample may contain low tumor volume and not amenable to genetic studies. Furthermore, CRPC is a highly heterogeneous disease, and biopsy of a single site may not reflect the molecular characteristics of all tumor sites. As an alternative to tissue biopsy, there is a growing interest in liquid biopsies such as circulating tumor cell (CTC) and circulating cell-free DNA (cfDNA). In PCa, several groups have recently shown the association of AR amplification and mutations in plasma cfDNA with poor responses to abiraterone and enzalutamide in patients with CRPC12. However, predictive values of these aberrations remain controversial13. Moreover, there is still a technical challenge to analyze highly fragmented (150–200 bp) and diluted (nanograms per 1 ml plasma) cfDNA and reliably detect those shed from tumor cells  which generally comprises less than 10% of cfDNA. Herein, we established a robust method to analyze the AR copy number (CN) and mutations in plasma cfDNA by combined use of digital PCR (dPCR) and multiplex PCR based target sequencing. We analyzed the AR status of Japanese patients with CRPC to evaluate the utility of cfDNA as a novel biomarker.In the past 5 years, several large studies have identified recurrent somatic mutations, copy number alterations, and chromosomal rearrangements from CRPC metastatic sitesAR CN analysis by dPCR was first tested using VCaP (AR amplified) and LNCaP  cell lines. AR CN in VCaP genomic DNA (gDNA) and LNCaP gDNA were 25.02 copies/µl and 0.94 copies/µl, respectively . These results confirmed the establishment of a successful assay for CN analysis in cfDNA  gDNA and cfDNA from mice implanted with LNCaP, but not in VCaP (AR wild type)   patients had AR amplification at the time of baseline sample collection  patients who had baseline samples collected at disease progression, but in only 1 of the 19 (5.3%) patients whose samples were collected during the response to treatment. AR target sequencing in all cfDNA samples was successfully performed, and median coverage of each amplicon was >10000×. AR LBD mutations were identified in 25 of 102 (24.5%) patients  was not significantly different from that sampled during response to treatment . Most of the mutations were located at known hot spots. The F877L mutation is reportedly associated with resistance to enzalutamide15 and was detected in one sample collected upon development of resistance to enzalutamide. Four additional mutations  were identified. R630Q and V716M were identified at a relatively high VAF . Importantly, we determined that V716M was located near the known LBD hot spots in the 3D structure of the AR protein, suggesting that this amino acid change might alter ligand binding of AR and associates with treatment resistance  patients. All 5 patients with discordance showed AR amplification in gDNA from the prostate, but not in cfDNA. In AR mutation analysis, 10 of 12 (83.3%) patients showed completely concordant results. Patient KU-112 had W742L in cfDNA, but not in gDNA from the prostate, possibly reflecting tumor heterogeneity.AR status in cfDNA for each patient is presented in Fig. AR aberrations (amplification and/or mutations) in cfDNA. Eleven of these 61 patients had both AR amplification and mutations, which was inconsistent with other reports that indicated a tendency toward mutual exclusivity11. Clinicopathological factors associated with AR aberrations in cfDNA were analyzed in 83 patients who underwent blood collection at disease progression. Fifty-six of the 83 (67.5%) patients had AR aberrations, and we showed that Hb ≤ LLN, cfDNA concentration, increasing number of prior systemic treatments and resistance to estramustine phosphate, abiraterone, enzalutamide or docetaxel were significantly associated with the presence of AR aberrations in cfDNA by univariate analysis 9 or those with AR amplifications had poorer PSA response compared to patients without these aberrations , PSA was reduced by more than 80.0%. Median PSA-PFS in the patients with these aberrations also tended to be shorter than those without the aberrations    from baseline at 12 weeks (or earlier for those who discontinued treatment) and PSA progression-free survival (PSA-PFS) according to Prostate Cancer Working Group 2 Criteriaons Fig. . In the st) Fig. . On multst) Fig. . In the AR status , abiraterone (n = 1), docetaxel (n = 1) and cabazitaxel (n = 1). In one patient, AR amplification was newly detected at the time of response to treatment with paclitaxel and carboplatin. AR mutations were detected in 5 patients at baseline, all of which became undetectable during the patients’ responses to abiraterone (n = 2), enzalutamide (n = 1), docetaxel (n = 1) and paclitaxel and carboplatin (n = 1).In 41 patients, blood samples were collected multiple times during treatment to track tus Fig. . cfDNA sAR amplification at baseline, amplification remained detectable in 19 patients at the time of resistance, whereas AR amplification became undetectable in 3 patients . Among them, KU-053 and KU-055 were treated with enzalutamide after blood collection at baseline, and AR amplification became undetectable during their response, which remained undetectable at the time of progression. In patient KU-055, the AR mutation (H875Y), whose VAF at baseline was 21.3%, also became undetectable during treatment with enzalutamide and remained undetectable at progression. Intriguingly, this patient subsequently responded to abiraterone  was subsequently enrolled in a clinical trial for a vaccine therapy after exhibiting resistance to enzalutamide, and the mutation became undetectable upon progression to this therapy. T878A, L702H, H875Y, Q641R and D733N were newly detected in 3 patients  during treatment with abiratetone, docetaxel and estramustine phosphate, respectively.Of the 41 patients, we collected blood samples from 32 patients at baseline and at the time of resistance to treatment, including 4 patients who also had cfDNA analyzed while they were responding to therapy. Of the 22 patients with AR amplification can drive CRPC progression and is present in up to 50% of CRPC tissues. CN analysis in cfDNA has been performed by several methods; array comparative genomic hybridization, next generation sequencing (NGS), real time PCR and dPCR17. In the present study, we showed that AR amplification could be detected by dPCR even when VCaP gDNA was diluted to 1.0%. The rate of AR amplification in cfDNA from patients with CRPC (46.1%) was comparable to that in other reports, suggesting that our method that used dPCR was a simple and robust approach to analyze CN in cfDNA. However, the concordance rate of AR amplification status between cfDNA and matched tumor tissue gDNA was only 58.3%. All 5 patients with discordant statuses had AR amplification detected in gDNA from prostate tissues, but not from matched cfDNA. Notably, all the patients with discordance only had low volume bone metastases, raising the possibility that low yield ctDNA was undetectable in these cases. In addition, AR CN in 2 patients (KU-112 and KU-116) was near the cut-off line for determining amplification and might have been falsely considered no amplification by our criteria. Increasing the sensitivity of dPCR technology may aid in detecting AR amplification in future cases.AR mutations can also drive CRPC progression. Gain-of-function mutations such as L702H, W742C, W742L, H875Y and T878A can change ligand binding affinity, which results in increased sensitivity to steroid ligands or the conversion of anti-androgens to agonists2. AR mutations in cfDNA have been detected by NGS or dPCR13. NGS can evaluate targeted genes comprehensively; however, its sensitivity is restricted by the inherent detection limits of NGS technology which are around 1.0%. Digital PCR can detect rare mutations with a prevalence as low as 0.1%, however, it can evaluate only one mutation in one PCR reaction. In the present study, we combined 2 methods: initial screening of LBD mutations by multiplex PCR based target sequencing, followed by dPCR confirmation of hotspot mutations whose VAFs were between 0.5%-1.0%. By this approach, many hotspot mutations in the range of 0.5%-1.0% could be validated by dPCR. We did not validate other candidates outside of known hotspots; however, it is possible that these candidates also contain important biological information19. Therefore, efforts are underway to increase the sensitivity and specificity of NGS-based analysis using molecular barcodes whose utility have not yet been shown in cases of prostate cancer.AR amplification and mutations in this study was higher than previously reported. In contrast to many studies in which analysis was performed after 1st or 2nd line treatments for CRPC, most of the patients in the present study had been exposed to multiple lines of AR axis-targeted treatments which contained anti-androgen therapies hardly used in Western countries (i.e. estramustine phosphate). Exposure to multiple agents targeting the AR pathway might have produced a generation of diverse clones with different mechanisms of resistance. In the present study, the number of prior systemic treatments and resistance to estramustine phosphate abiraterone, enzalutamide, or docetaxel were significantly associated with the presence of AR aberrations in cfDNA; however, resistance to bicalutamide and flutamide were not. This indicates that AR aberrations tend to accumulate more after resistance to second line hormonal therapy, compared to first line complete androgen blockade.Interestingly, the number of patients with concomitant AR aberrations in cfDNA as predictors of responses to abiraterone and enzalutamide is still controversial. Initial studies have shown that AR aberrations in cfDNA are linked to poor outcomes with abiraterone and enzalutamide12, whereas a recent report showed that only strong AR gain (CN ≥ 8 copies) was associated with poor responses in patients unexposed to prior treatments for CRPC13. Despite our small sample size, we showed that AR amplification, L702H, T878A, or H875Y tended to be associated with poor response to abiraterone, and that AR aberrations were not associated with response to enzalutamide. The difference in outcomes between the 2 treatments is attributable to the different pharmacological mechanisms of each treatment; abiraterone mostly indirectly blocks the AR pathway via androgen biosynthesis, whereas enzalutamide blocks AR directly as an antagonist, and was developed to potently block the AR pathway within the context of AR overexpression21.The utility of AR status during treatment. Almost all amplifications and mutations at baseline became undetectable during the response to treatment, possibly reflecting decreased tumor burden11. In some patients, AR amplification and mutations at baseline disappeared by the time of progression, possibly indicating that the clones that have gained resistance to previous treatments by acquiring AR aberrations were mostly eliminated by the subsequent treatments and became non-dominant clones at later progression. Quite intriguingly, in one patient (KU-055) with an AR amplification and H875Y at baseline, these aberrations became undetectable by the time of response to enzalutamide and did not re-emerge upon acquiring resistance. It is possible that tumor clones with the AR aberrations regressed dramatically by exposure to enzalutamide, and the tumor may have eventually acquired resistance to enzalutamide by other mechanisms such as increased de novo steroidogenesis through overexpression or mutation of HSD3B122. The patient responded to subsequent treatment with abiraterone. Several studies have shown that response to abiraterone following enzalutamide is limited24; however, the present case raises the possibility that, on rare occasions, enzalutamide may eradicate the clones with AR aberrations associated with abiraterone resistance and re-sensitize the tumor to abiraterone. These observations, if further replicated in future studies, may lead to cfDNA based precision medicine in which optimal treatment sequence of CRPC could be guided by liquid biopsy.We serially analyzed cfDNA in 41 patients to evaluate dynamic changes in AR status in cfDNA from Japanese patients with CRPC, but we stress that this study has several notable limitations. The sample size was small and the heterogeneous patient cohort exhibited a variety of baseline characteristics. Larger prospective studies are required. Since the current study focused on cfDNA, we did not analyze AR splice variants. In particular, AR-V7 in CTC has been reported to be a prognostic biomarker associated with resistance to abiraterone and enzalutamide25. AR overexpression including AR amplification or AR genomic structural rearrangements are known to be involved in the expression of AR splice variants26. In the future, it will be important to analyze CTC and cfDNA concurrently. Additionaly, a recent study performed targeted 72-gene sequencing of cfDNA in patients with CRPC and identified BRCA2 and TP53 mutations in cfDNA as predictive biomarkers associated with poor outcomes of abiraterone and enzalutamide13. Genomic aberrations other than AR also need to be evaluated within the context of a large cohort of patients with CRPC.This is the first report to evaluate AR status in cfDNA, obtained from a minimally invasive blood sample, is feasible and informative for patients with CRPC. AR aberrations in cfDNA have the potential to become useful biomarkers in patients with CRPC.In conclusion, we have demonstrated that the analysis of One hundred two patients with CRPC and 15 healthy males were recruited at Kyoto university hospital between September 2015 and November 2017. Of the 15 healthy males, 6 underwent surgery for benign prostate hyperplasia before blood collection and were pathologically confirmed to be negative for PCa. The remaining 9 were healthy volunteers under the age of 40. All human experiments were approved by the ethical committees at Kyoto University Hospital (G1083). Written informed consent was obtained from all patients. All human experiments were performed in accordance with Japanese ethical guidelines for human genome/gene analysis research and ethical guidelines for medical and health research involving human subject.A blood volume of 8.5–10 ml was collected in an EDTA-containing tube, Cell-Free DNA BCT (Streck) or Cell-Free DNA Collection Tube (Roche). Plasma was isolated by 2 step centrifugation (1600 g × 15 min and 4100 g × 10 min) within 2 h of blood collection for EDTA-containing tubes, or within 7 days for the other tubes. Plasma was stored at −80 °C until cfDNA extraction.cfDNA was extracted from 4–6 ml plasma using the QIAamp Circulating Nuclear Acid Kit (Qiagen) according to the manufacturer’s protocol. cfDNA concentration was measured using the Qubit 3.0 Fluorometer (ThermoFisher Scientific). Extracted DNA in the first several samples were run on the Bioanalyzer 2100 (Agilent Techonologies) to evaluate successful extraction of cfDNA. The DNA exhibited a peak at 168 bp which was consistent with that of cfDNA  and the QIAamp DNA Mini Kit (Qiagen), respectively. gDNA concentration was measured using the Nanodrop 2000 spectrophotometer (ThermoFisher Scientific). cfDNA and gDNA were stored at −30 °C.AR CN was analyzed using the QuantStudio 3D Digital PCR system (ThermoFisher Scientific). PCR reaction was prepared with 7.5 μl of QuantStudio3D Digital PCR master mix (ThermoFisher Scientific), 0.75 μl of Taqman Copy Number Assay for AR (Assay ID: Hs04107225), 0.75 μl of Taqman Copy Number Reference assay for RNaseP (Assay ID: 4403326) and cfDNA or gDNA (about 5 ng) in a total volume of 15 μl. PCR reaction was loaded onto the QuantStudio 3D Digital PCR Chip (ThermoFisher Scientific) and amplified on ProFlex 2x Flat PCR System (ThermoFisher Scientific). The annealing and extension temperatures were set at 60 °C, and PCR was run for 39 cycles. After PCR amplification, chips were read on the QuantStudio 3D Digital PCR Instrument (ThermoFisher Scientific), and a secondary analysis was performed with QuanStudio 3D Analysis Suit Cloud software (ThermoFisher Scientific). AR CN was calculated using RNaseP as an internal control. The cut-off for indicating a positive AR amplification in cfDNA was AR CN > 1.54 copies/μl, which was the average plus 2 standard deviations of AR CN in cfDNA obtained from healthy males.AR mutations were evaluated by 2 approaches: dPCR and target sequencing. To detect AR L702H, W742C, W742L, H875Y and T878A mutations by dPCR, validated Taqman SNP genotyping assays  and custom-made Taqman SNP genotyping assays were used. The thermal cycling protocol was similar to that used in AR CN analysis; however, the annealing and extension temperatures and cycle number were changed to 56 °C and 39 cycles for L702H, W742C and W742L, 54 °C and 55 cycles for H875Y, and 62 °C and 39 cycles for T878A.AR mutations analysis by target sequencing was based on multiplex PCR based deep sequencing11. In brief, a total of 11 primer sets spanning the AR LBD were designed and amplified by 3 sets of multiplex PCR which was designed to contain non-overlapping target regions (first PCR). KAPA HiFi HotStart ReadyMix (KAPA BIOSYSTEMS) and 2 ng of cfDNA were used per reaction. Successful amplification was confirmed by agarose gel electrophoresis, and the PCR products were purified by AMPure XP beads (Beckman coulter). Next, overhang adapters specifically designed for Illumina sequencing were attached to purified first PCR amplicons using primers that were used for first PCR with an overhang adapter sequence (second PCR). After purification, the concentration of each set of second PCR amplicons was measured using the Qubit 3.0 Fluorometer, and the amplicons were pooled by sample. Finally, a limited cycle amplification was performed to attach sample-specific barcode to second PCR amplicons using KAPA HiFi HotStart ReadyMix and the Nextera XT Index Kit (Illumina) (third PCR). Agarose gel electrophoresis, purification, and concentration measurement were performed, and the PCR products from 96 samples were pooled. Library quality and quantity were evaluated using the Bioanalyzer 2100 and qPCR according to the Illumina qPCR Quantification Protocol Guide, and the libraries were normalized. Sequencing was performed on the Illumina Miseq according to the manufacturer’s instructions. Sequencing run included serially diluted LNCaP gDNA as a positive control, and WBC gDNA from 13 patients with CRPC and cfDNA from healthy males as negative controls.The Illumina Miseq generated raw images utilizing MiSeq Control Software v2.2 and base calling through an integrated primary analysis software called Real Time Analysis v1.18. The base calls binary was converted into FASTQ utilizing illumina package bcl2fastq v1.8.4. Adapters were trimmed away from the reads. Scythe v0.991 BETA and Sickle programs were used to remove adapter sequences. If the reads were shorter than 36 bp, those reads were dropped to produce cleaned data. FASTQ files were aligned against hg19 using Burrows-Wheeler Aligner, and the result files were converted to pileup format by samtools. Mutation candidates were determined according to the following criteria: (1) VAF of ≥0.5%; (2) minimum supporting reads of 5 at a variant position; (3) base quality and mapping quality of ≥20; (4) a minimum read depth of 1000; (5) if a variant was not consistent between paired-end reads, both reads were discarded. After mutation candidates calling, candidates were filtered based on the sequence data of WBC gDNA samples. Mean frequency of candidate single nucleotide variant (SNV) in WBC samples was considered as error rate. Each SNV allele frequency in cfDNA was compared with the error rate by one-sided binomial test, and if the p-value was higher than 0.05, we discarded the SNV. Additionally, we examined whether there was a strand bias at candidate SNV positions. We compared the genotypes inferred from the positive strand with those from negative strand using the chi-square test. If the p-value was lower than 0.05, the SNV was discarded. We further filtered mutation candidates by setting the cut-off VAF of 1.0%. However, for known hot spots , variants with VAF of 0.5–1.0% were regarded as ‘true mutations’ if the SNVs were subsequently validated by dPCR.AR, we estimated the location of V716M in an AR 3D protein structure  with a 3D permutation method28. We calculated the average distance between hot spots and V716M. We then randomly selected amino acid residues from the AR 3D protein structure and calculated the average distance. This procedure was repeated 10,000 times, and a null distribution was generated. The p-value of the average distance of the mutations was evaluated using the null distribution.To interpret a functional importance of the rare mutation V716M, which is not considered a hot spot in AR amplification, T878A, H875Y and L702H, respectively) were purchased from American Type Culture Collection . DNA from each cell line was used as a positive control for the development of AR CN and mutations analysis. All experiment using cell lines were performed in triplicate.VCaP, LNCaP, 22rv1 and MDA PCa 2b  mice  under anesthetization. 1.0 ml blood was collected in an EDTA-containing tube, and 2 step centrifugation was performed within 2 h. cfDNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen). All experiments involving laboratory animals were performed in accordance with the Kyoto University Guidelines for Animal Experiments and approved by the Animal Research Committee at Kyoto University Graduate School of Medicine.To confirm successful detection of AR CN between diluted VCaP gDNA and LNCaP gDNA, and the AR T878A mutation rate between diluted LNCaP gDNA and gDNA from healthy males were compared using the Student’s t-test. Frequency of AR amplification or mutations in cfDNA between samples collected at disease progression and while under response to treatment were compared using the chi-square test. Clinicopathologic factors associated with AR aberrations in cfDNA at disease progression were analyzed by univariate analysis . PSA-PFS rates after starting abiratetone and enzalutamide were estimated using the Kaplan-Meier method and the differences between groups were compared using the log-rank test and the Wilcoxon test. Univariate and multivariable Cox proportional hazard tests of PSA-PFS after starting abiraterone and enzalutamide were also performed. Differences were considered significant when a p-value < 0.05 was obtained. All statistical analyses were performed using JMP software version 13.0.0 for Windows .cfDNA concentration between patients with CRPC and healthy males was compared using the Mann-Whitney U test. To evaluate the sensitivity of dPCR, supplementary information


Background: The process of testicular descent requires androgen and insulin-like 3, hormones secreted by fetal Leydig cells. Knowledge concerning distinct and common functions of these hormones in regulating development of the fetal gubernaculum remains limited and/or conflicting. The current studies were designed to better define characteristics of androgen receptor (AR) expression, function and regulation, as well as the biomechanical properties of normal and cryptorchid gubernaculum during fetal development.Methods: We studied fetal gubernacula from Long Evans outbred (LE/wt) rats and an inbred (LE/orl) strain with an inherited form of cryptorchidism associated with an AR signaling defect. Gubernacular cells or whole organs obtained from LE/wt and LE/orl fetal gubernacula underwent AR immunostaining and quantitative image analysis. The effects of dihydrotestosterone (DHT) on AR expression, muscle fiber morphology, hyaluronan (HA) levels and glycosaminoglycan (GAG) content were measured in LE/wt gubernacula. Finally, the spatial mechanics of freshly harvested LE/wt and LE/orl fetal gubernacula were compared using micropipette aspiration.Results: AR is expressed in the nucleus of mesenchymal core, tip and cord cells of the embryonic (E) day 17 and 21 fetal gubernaculum, and is enhanced by DHT in primary cultures of gubernacular mesenchymal cells. Enhanced AR expression at the tip was observed in LE/wt but not LE/orl gubernacula. In in vitro studies of whole mount fetal gubernaculum, DHT did not alter muscle fiber morphology, HA content or GAG production. Progressive swelling with reduced cellular density of the LE/wt gubernaculum at E19–21 was associated with increased central stiffness in LE/wt but not in LE/orl fetuses.Conclusions: These data confirm nuclear AR expression in gubernacular mesenchyme with distal enhancement at the tip/cord region in LE/wt but not LE/orl rat fetuses. DHT enhanced cellular AR expression but had no major effects on muscle morphology or matrix composition in the rat fetal gubernaculum in vitro. Regional increased stiffness and decreased cell density between E19 and E21 were observed in LE/wt but not LE/orl fetal gubernacula. Developmental differences in cell-specific AR expression in LE/orl fetal gubernacula may contribute to the dysmorphism and aberrant function that underlies cryptorchidism susceptibility in this strain. Cryptorchidism or undescended testis, is the most common reproductive congenital anomaly , 2. TestInsl3, Rxfp2 or Ar (the GU-ARKO mouse) in the gubernaculum  of androgen action in testicular descent remain controversial. Hutson et al. reported absence of AR expression within the fetal gubernaculum, and hypothesized that prenatal androgens act indirectly via masculinization of the genitofemoral nerve (GFN) . Most reTfm mouse gubernacula suggest a role for neuro-hormonal interaction between androgens and CGRP in the process of testicular descent  of AR in testicular descent, in addition to cellular proliferation , 18, hav descent , 20.To more clearly define the role of AR in gubernacular development, we studied the effects of dihydrotestosterone (DHT) stimulation on AR expression, muscle fiber morphology, and ECM composition, and we compared the pattern of AR expression and the biomechanical properties of the gubernaculum in outbred and LE/orl fetal rats with evidence for impaired AR signaling , 22. PriWe obtained fetal gubernacula from timed-pregnant Long-Evans wild-type (LE/wt) and LE/orl (an LE substrain with inherited cryptorchidism) males by microdissection during Phase 1 (E17 and E19) and Phase 2 (E21) of testicular descent, at the onset of gubernacular inversion. Samples obtained from ≥3 fetuses and ≥2 separate litters were immediately processed for each experiment. All work was conducted under a protocol approved by the Nemours Institutional Animal Care and Use Committee (#NBR-2016-002).For tissue clearing we used the benzyl alcohol:benzyl benzoate (BABB) protocol  prior ton = 3–9 per group) using a custom analysis algorithm formulated in MATLAB . Each confocal volume was analyzed in full. Segmentation of the gubernacula was accomplished through manual elimination of any remaining the abdominal wall tissue and enhancement of the DRAQ5 channel. Briefly, DRAQ5 signal was median filtered with a 3.3 × 3.3 × 4.3 μm kernel, plane by plane rolling ball filtering (with a 66.4 μm radius) and morphological closing (with a 20 μm radius) to fill in gaps between the cells. The gubernaculum was finally segmented using hysteresis thresholding. Identification of the “tip” region was performed manually by a masked observer, and was defined as the region between the cord .E19 gubernaculum pairs were dissociated overnight at 4°C in 5 mg/ml collagenase with 2.5 mM CaCl. Samples were triturated and then plated on collagen or poly-L lysine (PLL)/laminin-coated 6-well tissue culture plates. Cells were then incubated for a total of 6 days in DME/F12 containing 18% fetal calf serum (FCS), rat basic fibroblast growth factor , rat epidermal growth factor , dexamethasone (0.4 ug/ml), and penicillin-streptomycin supplemented with 0, 0.1, 1.0, or 10.0 nM DHT. Media was changed on each of the 6 days of culture. Immunofluorescent staining of AR in gub cells was carried out using 1:200 dilution of the rabbit anti-AR antibody (Abcam cat# ab133273) in PBS at 4°C overnight. Cells were washed 3X with PBS and then incubated with donkey anti-rabbit Alexaflour 555 and Dapi (Fisher cat#EN62248) to visualize nuclei. On day 6, 5 random areas were captured from each treatment protocol using an Olympus BX-60 florescence microscope with an Evolution QEi 12-bit digital camera (Media Cybernectics) and a 20X objective. AR+ cells were counted manually using Image J software  in accordance with the manufacturer's instructions.Rat gubernacula were digested with 0.3 ml of 4 mg/ml Pronase  in PBS for 1.5 h at 37°C. Collagenase  0.1 ml, 2 mg/ml in PBS, was added to digestion samples, and samples were incubated at 37°C overnight. After centrifugation at 14,000 rpm for 10 min, the enzyme solution was removed. 150 μl of 50 mM Tris-HCl (pH = 7.0) was added to the pellets, and the sample solution was sonicated on ice for 15 s, 2 times. The sample solutions were stored at −20°C before pretreatment for GAG analysis. Further, preparation of gubernaculum and conditioned media samples for GAG analysis and quantification of GAG levels by liquid chromatography tandem mass spectrometry (LC-MS/MS) was previously described . In briebase , midportion (between 40 and 60% of the height), and tip (top 25% of the height) of the organ. Each aspiration was allowed to stabilize for 3 min to allow the tissue deformation to reach steady state and imaged with a camera mounted on the stereoscope . Apparent elastic modulus (stiffness) is defined by Equation 1  for no more than 6 h before measurement. Mechanical characterization of each intact gubernaculum was performed using micropipette aspiration on a stereoscope stage , similar to previous descriptions –33. Two uation 1 :(1)Eappa is the micropipette radius, Δp is the aspiration pressure, L is the length of aspirated tissue, and Φ is the wall function, a parameter to account for differences in pipette geometry, given by Equation 2  in SPSS® v25 (IBM Corporation) to test for differences between groups. Parametric or non-parametric p values < 0.05 were considered significant.We used ANOVA, UNIANOVA or independent sample Using a specific anti-AR antibody that recognizes the full-length form of the receptor and confocal imaging, we found that nuclear AR is expressed widely in mesenchymal cells of the fetal rat gubernaculum Figure , we obsein vivo distribution as shown in images of the intact mesenchymal core  for cells grown on either collagen type I or PLL/laminin substrata, but the response was more robust for PLL/laminin.In the process of developing fetal gubernaculum cell lines , we obsee Figure  and AR ee Figure . This efWe previously reported a fetal gubernaculum muscle development organ culture model, which utilizes intact E17 gubernacula embedded in Matrigel on PLL-coated plates . In thisUsing a sensitive rat-specific ELISA assay, we found that rat HA is easily measurable in the fetal gubernaculum, but levels are not influenced by exposure to DHT Figure . SimilarOur sensitive tandem mass spectrometry (LC-MS/MS) assay results Figure  suggest Eapp, in kilopasquales, kPa) of the fetal gubernaculum at E19 and E21. To account for potential spatial variation, we measured 3 locations along the outer curvature: base (near the pelvic floor), midportion, and tip (near the gubernacular cord); additionally we completed measurements at E19 and E21 to capture changes in mechanics as the gubernaculum undergoes developmental remodeling. In LE/wt rat fetuses, we observed increased stiffness in the midportion at both E19 and E21 compared to the other regions measured (p < 0.01), and increasing stiffness between E19 and E21  in vitro  and transinguinal (Phase 2) descent by INSL3 and androgens, respectively, is distinct . The majmulation . A “delamulation , 48 to Amulation , 50.Tfm) or transgenic (ARKO) Ar inactivation, or in the LE/orl rat, which shows reduced fetal androgen synthesis and action (The current experiments suggest that androgens do not have a major role in stimulating production of CM or defining muscle fiber size in the rat gubernaculum. These results are consistent with observations that swelling of the gubernaculum still occurs to some extent in mice with spontaneous (d action , 21, 37.d action . In largd action –54. The d action .in vitro model employed in the current study (A known role for androgen and INSL3 is stimulation of cellular proliferation via additive effects that were demonstrated using the nt study , 18. We nt study , a cell nt study . If the Syne2 and Ncoa4, two genes encoding AR-interacting proteins that are also implicated in myogenesis (Syne2 and Ncoa4, and by selective breeding to enrich for LE/orl Syne2 and Ncoa4 alleles in outbred strains, we were able to reproduce the cryptorchid phenotype (Our genetic and genomic studies of the LE/orl rat, a model of inherited cryptorchidism, suggest that dysfunctional AR signaling contributes to failure of testicular descent in this strain. In both LE/orl and flutamide-exposed rats, undescended testes become misdirected and reside in the superficial inguinal pouch , 36, 41.ogenesis –60. Our henotype . The mushenotype  are consOur data suggest regional differences in stiffness of the gubernaculum characterized by peak stiffness of the midportion, and increasing stiffness at the tip between E19 and E21. The reduced swelling that we observed in the LE/orl gubernaculum herein and in prior studies  may accoIn summary, current and prior data suggest that androgens enhance AR expression and proliferation of mesenchymal cells of the fetal gubernaculum, and that AR expression is lost with myogenic differentiation. Androgens may also have indirect effects on ECM production by stimulating the growth of AR-expressing myofibroblasts. Susceptibility to cryptorchidism in the LE/orl rat is associated with altered stiffness, but not with changes in ECM composition or muscle fiber morphology of the fetal gubernaculum. Regional differences in stiffness and localized ECM remodeling may be required for successful transinguinal passage of the gubernaculum and testis.This work was prepared while Dr. Julia Barthold was employed at Nemours. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government.This study was carried out in accordance in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, based on the Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Nemours Animal Care and Use Committee.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. JM and JG performed mechanical testing and quantitative image analysis. AR, AM, and DG performed dissection, cell and organ culture, and staining experiments. KS and ST performed matrix analysis. JSB, AR and JM were responsible for drafting the manuscript. All authors contributed to the development and interpretation of the experimental data as well as manuscript editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth  Prostate cancer (PCa) remains the most common cancer and the second leading cause of cancer related deaths in men in the United States. The American cancer society estimated that 161,360 new cases of PCa will be diagnosed and 26,730 patients with PCa will die in 2017 . One of In vitro, treatment of prostate cancer cells with selinexor resulted in XPO1 inhibition, which led to the nuclear retention of p53 and Foxo proteins and the degradation of cyclin D1, survivin and XPO1, consequently triggering apoptosis . The spectrophotometric absorbance of the samples was determined by using a plate reader SynergyHT  at 570 nm. The combination index (CI) value and isobologram were calculated and created by using CalcuSyn software .Cell apoptosis was detected using Annexin V FITC (Biovision Danvers MA) according to the manufacturer's protocol as we reported previously . PCa cel3, whichever comes first. Tumor were collected and fixed in 10% formalin for histopathology analysis.The study was designed to evaluate the efficacy of selinexor  and KPT-8602  in a 22Rv1 prostate cancer xenograft model in CB.17 SCID mice. Mouse weights and tumor size were monitored three times a week. Each mouse was euthanized at the end of study (Day 35) or when tumor volume reached 1000 mmn = 5), abiraterone acetate treated (n = 5), KPT-8602 treated (n = 5) and combination treatment (n = 6). Abiraterone (Selleckchem) was administered orally each day at the dose of 100 mg/kg for 3 weeks. KPT-8602 was administered orally at 20 mg/kg twice a week for a total of 7 doses. All mice were followed for measurement of S.C. tumors and observed for changes in body weight and any side effects. All tumors were collected at 24 hours after last dose of combination treatment and tumor picture was taken.The effects of combination treatment with KPT-8602 and abiraterone was also evaluated in the 22Rv1 prostate cancer xenograft model in ICR-SCID mice. Six days after 22Rv1 transplantation, mice were randomized into 4 groups: Untreated (A tissue microarray (TMA) was constructed with tumor samples collected from 22Rv1 mouse prostate cancer model. Paraffin sections of the TMA were processed and stained with antibodies using a Biogenex I6000 automated stainer. Digital images of the slides were obtained through an Aperio AT Turbo scanner at 20×. The following antibodies were used for immunohistochemistry staining: Cleaved Caspase 3 , p21 , APC , Ki67 , Rb , p53 , SMAD4 , AR .t-test using GraphPad Prism software . p < 0.05 was considered statistically significant.Wherever appropriate, the data were subjected to a Student's


Associations of AR with clinical and tumor features were determined using the Wilcoxon rank sum test (two-level factors) or the Kruskal–Wallis test (multi-level factors). We identified an optimal AR cut-point to maximize recurrence-free survival (RFS) differences between AR biomarker stratified groups, and assessed the association between the AR stratified groups and RFS using the Cox proportional hazard model. Pearson correlations between AR and selected genes were determined in I-SPY 1, METABRIC (n = 1992), and TCGA (n = 817). AR was lower in triple negative BC vs. hormone receptor positive (HR+)/HER2− and HER2+ disease (p < 0.00001), and lower in basal-like BC (p < 0.00001). AR was higher in grade I/II vs. III tumors (p < 0.00001), in patients >age 50 (p = 0.05), and in node negative disease (p = 0.006). Higher AR was associated with better RFS (p = 0.0007), which remained significant after receptor subtype adjustment (p = 0.01). AR correlated with expression of luminal, HER2, and steroid hormone genes. AR expression was related to clinicopathologic features, intrinsic subtype, and correlated with improved outcome.We studied androgen receptor (AR) gene expression in primary breast cancer (BC) to determine associations with clinical characteristics and outcomes in the I-SPY 1 study. AR was evaluated in I-SPY 1 ( Lehmann and colleagues identified a subtype of triple-negative breast cancer (TNBC) characterized by the presence of the androgen receptor (AR) and expression of other luminal genes termed the luminal-androgen receptor (LAR) subtype. This expression-based subtype was subsequently confirmed by other authors.5 It is likely that varying subtypes within each of the conventional types of breast cancer (hormone receptor (HR) positive, HER2+, TNBC) may have different clinical and prognostic features, and AR expression may factor into this heterogeneity, across breast cancer subtypes. In this study, we evaluated associations between AR gene expression in primary breast cancer, clinical characteristics, and patient outcomes in a well characterized subset of patients with early stage breast cancer in the I-SPY 1 cohort. We also explored gene correlations with AR and selected other genes involved in the development of breast cancer, using three publically available databases including I-SPY 1 ,7 METABRIC,4 and The Cancer Genome Atlas (TCGA).8A better understanding of the relationship of AR, clinical characteristics, and patient outcomes is needed to further individualize patient care based on tumor biology. A recent study demonstrated that varying subtypes of TNBC may respond differently to neoadjuvant therapy.Of the 149 patients with microarray data available, 25% (37) were TNBC, 30% (45) were HER2+, and 58% (86) were hormone receptor (HR)+. Of the patients with TNBC 86% were found to be basal by expression profiling.p < 0.00001), as expected and concordant with previous literature.9 When evaluated by intrinsic subtype, AR expression was the lowest in basal breast cancer . Significantly higher AR expression was also observed in grade I/II versus grade III tumors , and was associated with older age >50 . These findings were also observed in METABRIC. Additionally, in I-SPY 1 higher AR expression was found in node negative versus node positive disease ; no association was seen with menopausal status (p = 0.70), clinical stage (p = 0.31), or lymphovascular invasion (p = 0.22).In the I-SPY 1 dataset, AR expression was found to be significantly lower in TNBC (i.e. HR−/HER2−) than HR+/HER2− and HER2+ disease . At an optimal cut off point that maximizes survival differences between AR-stratified groups (−0.89), higher AR expression (>−0.89) was associated with better recurrence-free survival (RFS) . In a multivariable Cox model adjusting for HR/HER2 subtypes, high AR expression remained significantly associated with better RFS , suggesting that AR expression has prognostic value independent of receptor subtypes. The Kaplan–Meier survival plots of the AR-High vs. Low groups within each HR/HER2 subtypes are shown in Fig. No significant association was observed between AR expression and pathologic complete response (pCR) following neoadjuvant chemotherapy  cohorts of METABRIC and TCGA. Table AR gene expression positively correlated with the expression of multiple luminal genes including FOXA1, GATA3, AGR2, PIP, CA12, ESR1, PGR, TOX3, MUC1, DHRS2, ITGB5, CCND1, and RET as well as with the expression of steroid hormone genes such as ADRA2A, PTGER3, GHR, and ADRB2. In addition, AR gene expression positively correlated with the expression of HER2 pathway genes including ERBB2, ERBB3, and ERBB4, and the expression of TNFSF10, KRAS, and PTEN. Most these gene correlations were also apparent in the TN cohorts.Significant inverse gene correlations with AR were noted with the basal genes FOXC1, MYC, HORMAD1, SERPINB5, SOX10, ELF5, VTCN1, SOX6, SOX8, TUBB2B, KIT, KRT5, and KRT14; in most cases, these were observed in the TN cohorts as well. AR gene expression also inversely correlated with the expression of DNA damage repair genes, TOP2A and the PARPi-7 gene signature in the population as a whole, and in the TN cohorts alone. Finally, a significant inverse correlation between AR gene expression and the expression of CRYAB was observed, both in the population as a whole, and in the TN cohorts alone.In addition, significant inverse correlations of AR expression with mesenchymal genes, CDK6, EGFR, and PTGFR, and immune genes FOXP3, RARRES1, CXCL9, CXCL10, STAT5A, STAT1, CTLA4, PSMB9, LCK, CD2, and PDCD1 were noted; however, these correlations were generally not seen in the TN cohorts, suggesting that this may be related to HR/HER2 status rather than AR expression.13 and a lower expression of AR in TNBC.9The androgen receptor may characterize a discrete subtype of breast cancer. In this study, we analyzed gene expression in the AR pathway in patients enrolled in I-SPY 1, and utilized the METABRIC and TCGA datasets for validation. In both the I-SPY 1 and METABRIC datasets, we noted a lower expression of AR in TNBC than in HR+/HER2− and HER2+ disease, and the lowest expression in basal type breast cancer. Similarly, other authors have noted increased expression of AR in HR+ than ER- disease,14 and node negative disease.15 Additionally, higher AR expression correlated with older age (>50) in both I-SPY 1 and METABRIC, concordant with other datasets.16 We did not find a correlation of increased AR expression with lower disease stage in I-SPY 1, although others have observed this finding.15We also observed that AR expression correlated with features of less aggressive disease when evaluated across subtypes, with higher expression in grade I/II versus III tumors in both I-SPY 1 and METABRIC, and higher expression in node negative versus node positive tumors in I-SPY 1. Our findings are concordant with those of other authors who have demonstrated increased AR expression in well differentiated tumors6 Nevertheless, AR remained an independent prognostic factor in a Cox model adjusting for receptor subtypes.In concordance with the association of AR expression with lower risk disease, we demonstrated that higher AR expression correlated with better RFS in the I-SPY 1 dataset. The prognostic significance is particularly interesting as the patients in the I-SPY 1 cohort were treated with neoadjuvant chemotherapy, and AR expression was not associated with higher rates of pathologic complete response, suggesting that the prognostic impact may not be as tightly related to chemotherapy response. The association between AR and receptor subtypes may be a potential confounding factor as the subtypes have distinct pathologic complete response rates and prognosis. Indeed, AR expression was high in HR+/HER2− patients who had the lowest pathologic complete response rates but the best prognosis.n = 149), the number of patients with various subtypes is small, limiting our ability to draw firm conclusions about the impact of AR gene expression on outcomes. Additionally, the cut point used for AR gene expression in this analysis was data-derived and therefore the outcome data are hypothesis generating and require validation in a different cohort of patients. We recognize that I-SPY1 has a higher proportion of high risk patients , grade, and age were observed in both the I-SPY 1 and METABRIC, which has a much higher proportion (71%) of HR+/HER2− cases. Based on this assessment, it is unlikely these findings are attributable to the skewed subtype distribution of the I-SPY 1 cases.However, a caveat to these findings is that given the sample size of the I-SPY1 dataset , and worse DFS in AR + TNBC in 2 studies  and Jiang et al.23 (137 patient study)), with all of these studies utilizing immunohistochemical (IHC) testing for AR assessment. In addition to population differences and variations in treatment patterns, a possible explanation for these discordant findings is variability in the IHC methodology used to assess AR expression. The majority of the studies in the literature have relied upon IHC to assess tumor AR status. Kim’s meta-analysis17 exemplifies the variability that may occur in assessing AR positivity using IHC expression, as the 11 studies utilizing IHC that were included defined expression in several different ways. Clearly there is not a consensus definition for AR positivity by IHC, and in prospective clinical trials, the optimal approach to defining AR positivity is also still being debated, with recent clinical trials defining AR+ disease as ≥0–10% by IHC or by utilizing a genomic profiling assay.26 An advantage of the I-SPY 1 database is that AR positivity was defined using expression microarrays, which have less variability than non-standardized IHC analyses.Nevertheless, the majority of literature also suggests that AR positivity may be associated with better outcomes. Several recent studies have demonstrated an improvement in both disease free survival (DFS) and/or overall survival (OS) in AR positive tumors.10Consistent with our findings that AR expression is lower in triple negative and basal subtype breast cancer, using the I-SPY 1, METABRIC, and TCGA datasets (defining a positive gene correlation as one seen in ≥2 datasets), we were able to demonstrate that the gene expression of AR positively correlates with the expression of a multitude of luminal genes. As expected, AR expression also correlated with the gene expression of other steroid hormones. AR also anti-correlated with basal, mesenchymal, and immune genes. Many of these observations were also noted in the TNBC cohorts of METABRIC and TCGA, suggesting that AR receptor positivity may define a subset of TNBC with luminal features consistent with the previously described LAR subtype of TNBC.24 Altogether 424 patients with ER-/PR- breast cancer were screened of which 12% were AR positive. A clinical benefit rate (CBR) of 19% (95% CI: 7–39%) was noted in the 26 evaluable patients, and the median PFS was 12 weeks (95% CI: 11–22 weeks). A second phase II study evaluated enzalutamide, an androgen receptor inhibitor, in advanced AR + TNBC.26 In the 118 intent-to-treat population (AR >0% by IHC), the CBR at 16 weeks was 25% (95% CI: 17–33%) and median PFS was 2.9 months (95% CI: 1.9–3.7 months), and these endpoints were slightly improved in the evaluable population of 78 patients (AR ≥10% by IHC) where the CBR at 16 weeks was 33% (95% CI: 23–45%) and the median PFS was 3.3 months (95% CI: 1.9–4.1 months). Using a genomic assay for AR expression (PREDICT AR+), 56 patients with a positive PREDICT AR assay had a clinical benefit rate of 39% at 16 weeks, and a median PFS of 16.1 weeks.25 A third study evaluated seviteronel, a selective CYP17 lyase and AR inhibitor in advanced breast cancer, including TNBC, demonstrating the initial safety and efficacy in TNBC .27 These findings suggest potential value for therapies targeted to AR. While response rates may be low, some of them are durable which is encouraging for TNBC. Additional studies are exploring orteronel in TNBC disease,28 as well as combination therapy in AR+ TNBC, including a study of enzalutamide and paclitaxel in the pre-operative setting29 and one study evaluating enobosarm, a selective androgen receptor modulator, with pembrolizumab, an immunotherapy agent.30Understanding AR+ disease is particularly important in TNBC where the mainstay of treatment is chemotherapy, and few targeted therapies are available. A recent phase II study evaluated the use of bicalutamide, an androgen antagonist, in patients with AR+/ER- metastatic breast cancer, defining AR positivity as IHC >10%.However, better definitions of AR positivity are clearly required to identify patients likely to benefit from AR targeted therapies. Our work establishes the feasibility in utilizing gene expression microarrays for evaluating AR expression in tumor tissue, and this is one potential platform that could be considered in future trials, as it may circumvent the issue of non-standardization of immunohistochemical assays to identify patients who may benefit from AR blockade. However, further research is needed to compare AR IHC and genomic assays. Unfortunately, this could not be done using the I-SPY1 dataset as AR IHC results are not available for this cohort. However, we noted a 0.73 correlation coefficient between AR protein level as assessed using reverse phase protein array (RPPA) and AR gene expression levels in TCGA cohort. Additional research is needed to help AR gene expression levels translate into routine clinical practice, similar to other expression-based multi-gene signatures such as the Oncotype and MammaPrint which are used in clinical decision making. The development of a validated AR gene expression signature or validated AR IHC threshold may enable clinicians to identify those patients with metastatic breast cancer who may benefit from AR targeted therapies, and for earlier stage disease, to aid in risk prognostication.Due to the retrospective nature of this study using only publicly available data, ethics approval for the study was not required.7 In summary, I-SPY 1 enrolled patients with early stage breast cancer and ≥3.0 cm of disease in breast, without evidence of distant metastases. Patients underwent a core biopsy of their breast tumor at the time of enrollment, then during, and after completing neoadjuvant chemotherapy. Patients received 4 cycles of neoadjuvant anthracycline plus cyclophosphamide chemotherapy with 95% receiving a taxane prior to undergoing surgery. Starting in 2005, patients with HER2+ disease also received trastuzumab. Following surgery, patients received adjuvant chemotherapy, trastuzumab, hormonal therapy , and/or radiation per physician discretion.The I-SPY 1  study was a multicenter initiative performed by the American College of Radiology Imaging Network (ACRIN), Specialized Programs of Research Excellence (SPORE), and the Cancer and Leukemia Group B (CALGB). The design of this trial is described elsewhere.6 Specifically, the expression of AR was taken as the median-centered log2-scaled loess-normalized expression value of the A_23_P113111 probe.For 149 patients enrolled in I-SPY 1 (GSE22226), high quality gene expression data, assayed with the Agilent 44K arrays, was generated from the pre-neoadjuvant treatment biopsies; the clinical features of these 149 patients did not vary significantly from the total sample size of 221 patients evaluable. Previous studies have described the methodology by which the microarray data was generated and processed, and the manner in which molecular profiling was performed.http://www.ebi.ac.uk/ega/ (accession number EGAS00000000083).4 ComBat was used for batch adjustment when combining the discovery and validation cohorts to yield a dataset containing 1992 samples. Data was annotated using the corresponding array annotation file from GEO, and genes represented by multiple probes are collapsed by averaging. The normalized, batch-adjusted, gene-level expression data was median-centered prior to analysis.Normalized expression matrices of the METABRIC discovery and validation cohorts, generated on the HT-12 v3 platform were obtained from http://tcga—data.nci.nih.gov/tcga). Details of the data processing can be found in Ciriello et al.8Normalized gene-level expression data, assayed by RNA-sequencing, for 817 primary breast cancers analyzed as part of the TCGA program was obtained from the TCGA data portal website  or the Kruskal-Wallis test (for multi-level factors). The clinical characteristics assessed in both I-SPY and METABRIC datasets included: patient age (≤50 or >50), menopausal status, clinical stage (stage I/I vs. III or inflammatory), tumor HR and HER2 status, intrinsic subtype , nodal status (node positive vs. node negative), histologic grade (grade I/II vs. grade III).In addition, in the I-SPY 1 dataset, the associations between AR and tumor lymphovascular invasion (presence vs. absence), as well as chemotherapy response (pathologic complete response vs. not) were assessed. We also evaluated the association of AR expression and recurrence-free survival (RFS). First, we identified an optimal AR cut-point which maximizes the survival difference between the AR-stratified groups. Kaplan Meier survival curves were constructed to visualize the survival differences between AR-stratified groups in the overall I-SPY cohort and within HR/HER2 subtypes. Significance in curve separation was assessed using a log-rank test. A multivariate Cox analysis was used to evaluate the association between AR and RFS, adjusting for HR/HER2 subtype.2 as well as DNA damage repair deficiency genes including TDG and a seven gene signature shown to be associated with response to the poly ADP ribose polymerase (PARP) inhibitor olaparib, the PARPi-7.31 This signature was calculated on median-centered expression data in simplified form as PARPi-7 = −0.5320*BRCA1 + 0.5806*CHEK2 + 0.0713*MAPKAPK2 − 0.1396*MRE11A − 0.1976*NBN − 0.3937*TDG −0.2335*XPA. Gene correlations were determined in all 3 datasets, I-SPY 1, METABRIC, and TCGA, as well as in the triple negative (TN) subsets of the 2 larger datasets, METABRIC and TCGA. Gene associations were determined using Pearson correlations with the Benjamini Hochberg correction for multiple testing (p < 0.05).We determined correlations between the gene expression of AR and a selected set of genes that may be involved in the development of breast cancer, including genes shown to be associated with the luminal, mesenchymal, basal, and immune subtypes of TNBC,Further information on research design is available in the Reporting Summary


Background: The appropriate detection and therapy of concussion symptoms are of great importance to avoid long-term impairment and absence from pre-concussive activities, such as sport, school or work. Post-traumatic headache and dizziness are known as risk factors of persistent symptoms after a concussion. Dizziness has even been classified as a predictor for symptom persistence. One type of dizziness, which has never been considered is visually induced dizziness (VID) often develops as a consequence of vestibular impairment. This manuscript presents the clinical case of a 25-year-old male, professional ice hockey player, whereby a therapeutic approach to VID after concussion is demonstrated.Case: A detailed interdisciplinary clinical and laboratory-assisted neurological, neurovestibular and ocular-motor examination was performed 20 days post-concussion, which indicated VID symptoms. Thus, the player qualified for a 5-day combined vestibular, balance and optokinetic therapy, which aimed to reduce the player's increased sensitivity to visual information. Each treatment day consisted of two sessions: vestibular/ocular-motor training and exposure to optokinetic stimuli combined with postural control exercises. The optokinetic stimulus was delivered in the form of a rotating disk. VID symptoms were recorded daily via posturography and a visual analog scale prior to the optokinetic sessions. The player improved over the course of each treatment day and was able to return to ice hockey 15 days after the final treatment session. Three months later the player reported no symptoms in the follow up questionnaire.Conclusion: The combination of vestibular, balance and optokinetic therapy led to remission of VID symptoms in a professional ice hockey player after multiple concussions, within a short time frame after his last concussion. Thus, this case study highlights the significant benefit of treating post-concussive VID symptoms utilizing a multi-modal approach. Sport-related concussion (SRC) is defined as a traumatic brain injury induced by biomechanical forces , 2 and iIn the last few years the approach for the return to sport protocols has changed , 12, 15.Visually Induced Dizziness (VID) in adult athletes, have hardly been described in the literature. Given that approximately half of the brain circuits are involved in vision and eye movement control  , 32. WitA 25 years old, male, professional ice hockey player with a history of 3 concussions  was assessed on December 3rd, 2018 (20 days after the last head trauma). A written information & consent form was provided to the player upon his arrival. He was explained in details about the diagnostic procedure of evaluation for concussion as well as the therapeutic approaches. He was informed that he could stop the intervention at any time without any particular reasons or without damaging his relationship with his doctor and therapists. Risk, benefits as well as the use of his data in an anonymous form were described and explained to the player. The player provided the permission to use his data for the purpose of this clinical case.Initial symptoms immediately after the last hit were blurred vision fogginess, fatigue, nausea and sensitivity to light. After an initial period of 7 days of rest he started the RTS protocol but had to stop on the level of non-contact training on the ice [between level 3 and 4—RTS of McCrory et al. ].On the first day of assessment the predominant problems were attributed to visual and vestibular domains . With the information provided by the patient, it was not possible to exclude that the reported symptoms were pre-existing, residual symptoms from the previous concussions. Current complains resembled his symptoms during the previous concussions. Retrospectively the player admitted, that he never had full symptom relieve  and tried to avoid certain situations , since the previous trauma occurred (2nd concussion).During the clinical neurological examination impairments were identified in postural stability in the following conditions: with eyes closed, during ocular-motor tasks (convergence) and when exposed to OKN stimulation. Positioning maneuvers were performed for all six semi-circular canals without nystagmus/hints of Benign Paroxysmal Positional Vertigo (BPPV). However, the player was successfully treated for BPPV after his previous concussion. After the first concussion, the patient performed a structural magnetic resonance imaging (MRI), which was recommended by his team physician and resulted in no abnormalities. No specific imaging examinations  were performed. On the basis of the clinical neurological examination VID symptoms were identified by means of questionnaires: Situational Characteristic Questionnaire (SCQ) and specific Visual Vertigo Questionnaire using an Analog Scale (VVAS) .Laboratory assisted vestibular and ocular-motor tests were normal . For the oVEMP the bone-conducted burst type vibration of 2 ms was used (500 Hz sinus wave). For the cVEMP both air- and bone-conducted stimulations were used. Air-conducted stimulation tone burst was performed with a type of stimulation of 6 ms, 500 Hz sinus wave. As per routine the test always initiates with the air conducted stimulation at 95 dB when reflexes reported then the stimulation decreased at 90 dB and then 70 dB. If no reflexes observed, the stimuli increased to 100 dB. Although in this case, the air-conducted cVEMPs resulted normal, the bone-conducted was also performed (burst type 6 ms- 500 Hz sinus wave), since it was evaluated after the previous concussion. All results were normal.Vestibulo-oculography including: vestibular caloric irrigation test, and subjective visual vertical assessment. A pathological binocular cyclorotation to the left (fundus photography) was objectified. The result of the Sensory Organization Test (SOT) and the Head-Shake SOT objectified a marginal vestibular deficiency . The gaze stability test (GST) and active dynamic visual acuity (DVA) tests were normal.During the vestibular-ocular-motor screening (VOMS)  the patiCombining the results from the different examinations, the symptoms were classified as a visual-vestibular dysfunction with reduced gaze stabilization and VID.The therapeutic concept was based on the combination of vestibular, ocular-motor and OKN treatment. The player performed 45 min session of OKN exposure and 1 h of vestibular-ocular-motor exercises for 5 days over 2 calendar weeks. For the “resting” days, the player was instructed to perform exercises at home.The OKN treatment was performed according to a modified version of the treatment of VID patients performed in a previous study by Pavlou et al. . The treVerbal feedbacks on symptoms and VVAS questionnaires were recorded every day prior to the OKN treatment. To assess relationship between the VVAS scores and day of treatment, the spearman's rank correlation coefficient was also used.Postural stability was measured before every OKN session. The player was asked to stand upright for 1 min, with feet apart (hip width) on a Wii Balance board  , with eyThe symptoms reported by the player on the first treatment day  improved throughout the 5 days of treatment. The player mostly mentioned to have temporal headache, which usually vanished after a few hours after the sessions, and fatigue. This aspect could be a consequence of the OKN stimulation, however it remains unclear . Overallp < 0.001). More specifically, when examining each specific trigger, a statistically significant correlation was reported for four triggers .The Confidence Ellipse Area (CEA) extracted from the posturography data identified abnormal results (mean CEA + two standard deviation) on the first OKN treatment day   and completed successfully the RTS protocol according to the RTS protocol of McCrory et al. , 12, 36.During questionnaire based follow up examination, which was performed 3 months after completion of the treatment the player reported no symptoms during all previously symptomatic conditions .The presented case report describes the presence of VID symptoms following a concussion in a professional ice hockey player with a history of multiple SRCs and of treated post-traumatic BPPV . It has been recently discovered that SRC are capable to lead to a disruption of central and peripheral vestibular pathways. The interaction of vestibular symptoms in concussed patients was here highlighted . During The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.VM is the primary author and was involved in the creation of the protocol used to rehabilitate the player, she was involved in the data recording, analysis, and writing the manuscript. CM was involved in rehabilitating the patient. MB was involved in the rehabilitation program and follow up of the player. FR was involved in the data analysis and reviewing the manuscript. DA was involved in performing the patient vestibular test examination. AV reviewed the manuscript. GB was involved in reviewing the manuscript. NF-D diagnosed the patient, supervised the rehabilitation program, and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hence, a more accurate and reliable reconstruction of the AR is necessary for understanding the neurobiological substrates supporting audition and language processing mechanisms in both health and disease. This review aims to unite available information on the macroscopic anatomy and topography of the AR in humans and non-human primates. Particular attention is brought to the anatomical characteristics that make this bundle difficult to reconstruct using non-invasive techniques, such as diffusion-based tractography. Open questions in the field and possible future research directions are discussed.The auditory system of mammals is dedicated to encoding, elaborating and transporting acoustic information from the auditory nerve to the auditory cortex. The acoustic radiation (AR) constitutes the thalamo-cortical projection of this system, conveying the auditory signals from the medial geniculate nucleus (MGN) of the thalamus to the transverse temporal gyrus on the superior temporal lobe. While representing one of the major sensory pathways of the primate brain, the currently available anatomical information of this white matter bundle is quite limited in humans, thus constituting a notable omission in clinical and general studies on auditory processing and language perception. Tracing procedures in humans have restricted applications, and the  The connectivity pattern of these fibers has been described in some detail in cytoarchitectonic and myeloarchitectonic studies of non-human primates  and, at m brains , while lin vivo and non-invasively   and tracvasively . Howevervasively . This isvasively . Therefoin vivo reconstruction of the human auditory tract is of great importance for both clinical applications  and basic neurobiological research. Reliably revealing the 3D characteristics of this tract would help in correlating the anatomical and functional aspects of audition and in the study of human-only cognitive functions, such as language, both in healthy and pathological conditions.However, overcoming or circumventing these methodological considerations is essential, as successful The main aim of this review is to emphasize the paramount need for characterizing the human AR in both clinical and scientific contexts, which has yet to be done for a number of reasons that we discuss. This review collates the available information from primate studies on the anatomy, topography, and course of the AR, with particular emphasis on the anatomical features that make this tract extremely challenging to study, even for state-of-the-art dMRI tractography techniques. Additionally, recent attempts to reconstruct the AR using diffusion-based tractography methods will be discussed. Finally, open questions in the field will be presented and possible future research directions considered.The auditory system of mammals is a complex network of parallel and overlapping axonal projections that connect subcortical nuclei and cortical regions. It encodes and transmits stimuli coming from the acoustic environment, enabling an organism to detect a sound in its environment, determine the direction from which it originated, discriminate among potential sources, and thereby, react or communicate with conspecifics. In vivo diffusion imaging techniques constitute a powerful tool in investigating these topics.Anatomical knowledge of the human AR mainly comes from pioneering investigations at the beginning of the 20th century . Since tin vivo imaging literature, reviewing recent attempts to visualize this tract using tractography techniques.In the next section, we briefly review the AR microstructural topography, as described in animal studies, and its macrostructural anatomy, which has been gleaned from human research. Our goal is to provide a more complete anatomical profile of this bundle across species and to highlight the existing gap of topographical information between invasive and non-invasive studies. In particular, we focus on the AR The axonal connections between the thalamus and the auditory cortex have been investigated in animals using different invasive techniques. These fibers stem from the medial geniculate nucleus of the thalamus (MGN), as first described by Projections from the ventral MGN to the core region correspond to the most direct classic auditory pathway, also called the lemniscal pathway. These parallel connections are tonotopically organized and their neurons show sharp responses to tones . Direct Classical studies in non-human primates focus on the topography of the connectivity between the MGN and the auditory projection cortical territory but provide no information on either the course and extension of the AR tract itself or its relationship with the other WM pathways of the brain. Using Marchi axonal degeneration, carrefour sensitive” (sensory intersection). Classical topographical descriptions in humans  provide in vivo imaging studies of this brain region and provide the opportunity to investigate inter-subject variability and hemispheric asymmetry. Previous studies have found that the AR does not enter the lenticular nucleus, but rather runs dorsally to the OR, crossing the temporal isthmus as it ascends to the auditory cortex  tractography allows for the investigation of WM architecture in the human brain non-invasively ications , most ofications . Despiteications . In prinAs shown in the previous section see , 3, in iin vivo using multi-fiber-based models  diffusion protocols which makes it suitable for clinical investigations of the AR. b-value diffusion data (b ≥ 3000 s/mm2). In these models, the angular resolution of the reconstructed diffusion profiles is increased and the crossing fiber configurations are correctly represented  are very rarely achievable, even in research settings. More recently, b ≤ 3000 s/mm2) and the large sample (842 subjects) allowed them to reduce the rate of false positive artifacts. However, the AR profile shown in this study, while correctly originating at the posterior thalamus, does not reach the expected auditory cortex on the superior side of the temporal lobe.tinnitus . In thesresented , althougresented  used ultata-sets  and spheata-sets  to reconata-sets , a methoata-sets . This apata-sets . The obtOverall, the reconstructed 3D profiles shown in these studies are in accordance with the macrostructural landmarks defined by classic anatomical studies: streamlines originate in the posterior thalamus and course in an antero-lateral direction to terminate in the temporal lobe . HoweverWe suggest that the present variability and low reproducibility in the reconstructed AR profile are related to a combination of some of the specific characteristics of the AR that make its tractographic reconstruction quite challenging, even for state of the art tractography techniques: its anatomical location, small size, and inter-individual anatomical variability.b-value , it is far more challenging to automatically segment smaller structures, such as the MGN, due to both their size and their lower MRI contrast with neighboring WM. Alternative solutions exist but are also challenging. For example, subject-specific manual segmentations of MGN can lead to high anatomical accuracy, but they are very time intensive and, therefore, costly to do in large subject cohorts. Brain atlases also can be used to define the MGN, however given the small size and inter-subject variability, atlas-driven segmentations are unlikely to provide a good anatomical match for all subjects. The development of more accurate automatic parcellation techniques for the thalamic nuclei is expected to improve the accuracy of seed-to-target definition and, thus, of the resulting tractography reconstructions. Recently, a new and promising probabilistic atlas of the thalamic nuclei has been proposed based on a combination of ex vivo MRI and histology  is the standard of care for most brain tumors. However, XRT can damage normal brain tissue, causing neurocognitive deficits in different cognitive domains . The conAdditionally, studies have shown XRT side effects in both gray  and whitin vivo tractographic reconstruction of the human auditory tracts is of great importance for clinical applications , as well as for basic research . This review outlines how the characterization of the AR has been limited by the methods used in the past and how advances in MRI acquisition and diffusion tractography methods offer the possibility to improve the characterization of this important WM tract. A few exciting potential research areas are suggested that would investigate anatomy and function concurrently in the same individual, both in health  and in disease . However, in order to obtain reliable reconstructions of the AR across subjects and protocols, additional work is needed to better understand how diffusion MRI acquisition and tractography reconstruction strategies affect the AR 3D characterization and to validate tractography reconstructions at a micro-anatomical scale. Furthermore, as diffusion tractography is blind to the directionality of reconstructed fibers, the AR bundle could include both thalamo-cortical and cortico-thalamic projections, and therefore more studies are needed to differentiate between the afferent or efferent nature of these connections. Although these methodological challenges apply to diffusion MRI tractography in general, here we have focused on their relevance to the AR, a tract that has proven to be rather elusive for the reasons herein reviewed.The anatomical and functional organization of the auditory system is still not well understood, particularly in humans. Successful JJ conceived the review. SS revised the manuscript for neuro-anatomical content. CM drafted the manuscript and designed the figures. All authors contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.