Androgen Receptor Exon Research Articles

Genetic Approaches for Sex Determination of Chinese Female with Male Pseudohermaphroditism

Abstract Male pseudohermaphroditism is a rare disorders of sex development (DSD) that is manifested by a female-like appearance or incompletely differentiated external genitalia in an individual with a Y chromosome. In this paper, we report our investigation of the case of a 33-year-old Chinese female who was diagnosed with a malignant mixed germ-cell tumor of the ovary. To confirm the sex of the female, we utilized genetic approaches to detect amelogenin and Y-STR loci. Y chromosome microdeletion was performed to identify existing deletions in the AZF regions and SRY. Chromosome karyotyping and whole-exome sequencing (WES) were then applied to reveal the deletion of sex chromosome segments and pathogenic variations in DNA sequences. Using DNA-STR genotyping, we detected both AMEL-X and AMEL-Y fragments. We also found haplotype Y-STR loci and detected all alleles. Furthermore, no microdeletion was detected in the AZF regions and SRY. The chromosome karyotyping was 46, XY. WES revealed a transversion mutation of 58T → C in the androgen receptor exon 1, which could be the pathogenic variant in this case of abnormal sexual development. Sex determination in forensic DNA typing is based on the amelogenin system. It is important that forensic biologists should master various genetic approaches to overcome the issue of gender ambiguity caused by DSDs.

Impact of AR-V7 and other androgen receptor splice variant expression on outcomes of post-prostatectomy salvage therapy.

274 Background: Radiotherapy (RT) with or without androgen deprivation therapy (ADT) plays a key role in salvage therapy of prostate cancer recurrent after prostatectomy. However, not all patients benefit from salvage therapy, and there is an unmet need for biomarkers to distinguish responders from non-responders. Prostate cancer depends on androgen receptor (AR) signaling, and expression of AR splice variants (ARVs) that enable androgen-independent AR signaling is associated with resistance to ADT in the metastatic setting. Recent in vitro data suggest that ARVs also mediate DNA repair after irradiation, suggesting that ARV expression may also be a biomarker of RT resistance. However, the landscape of ARVs in primary prostate cancer and its effect on treatment outcomes remain unexplored. Here we hypothesize that ARVs are detectable in primary prostate cancer and may modulate response to salvage RT + ADT. Methods: We retrospectively identified 46 prostate cancer patients treated with prostatectomy followed by salvage RT+ ADT at a single institution from 1995 to 2012. The indication for salvage therapy was biochemical failure after undetectable post-operative PSA in 72%, gross local recurrence in 17%, and persistently elevated PSA after surgery in 11%. Median RT dose was 64.8 Gy, and all patients received concurrent ADT. We comprehensively interrogated the landscape of ARVs by performing ultra-deep targeted RNA-seq of archival formalin-fixed paraffin-embedded prostatectomy samples. Using a custom library of > 3000 primers spanning all AR exons and introns, we evaluated 21 native splice junction sites and 20 splice variants with a mean depth of coverage of > 5000x. Decipher score was also evaluated. We tested for association between splice variant expression and clinical outcomes using the log-rank test and Cox proportional hazards model. Results: In total, 76% of patients had one or more detectable AR splice variants. The most commonly detected variants were AR-45 in 41%, AR-V9 in 20%, and AR-V7 in 13%. At a median follow-up of 33.8 months, biochemical progression-free survival (BPFS) at 3 years was 60%, distant metastasis-free survival was 90%, and overall survival was 100%. Among detected splice variants, only AR-V7 was associated with differential clinical outcomes, with a median BPFS of 10.9 months in AR-V7 positive vs 73.4 months in AR-V7 negative patients ( p = 0.0020, HR 5.23, 95% CI 1.62-16.87). Conclusions: Using ultra-deep targeted RNA-Seq, we provide among the first comprehensive descriptions of the ARV landscape in primary prostate cancer. Moreover, we show that detectable AR-V7 in prostatectomy specimens was associated with inferior outcomes following salvage RT+ADT, suggesting for the first time that AR-V7 may modulate outcomes for localized as well as metastatic disease. Ongoing work includes comparison with Decipher score and validation in independent cohorts.

Impact of AR-V7 and Other Androgen Receptor Splice Variant Expression on Outcomes of Post-Prostatectomy Salvage Therapy

Radiotherapy (RT) with or without androgen deprivation therapy (ADT) plays a key role in the salvage treatment of prostate cancer recurrent after prostatectomy. However, not all patients benefit from salvage therapy, and there is an unmet need for biomarkers to distinguish responders from non-responders. Prostate cancer depends on androgen receptor (AR) signaling, and expression of AR splice variants (ARVs) that enable androgen-independent AR signaling is associated with resistance to ADT in the metastatic setting. Recent in vitro data suggest that ARVs also mediate DNA repair after irradiation, suggesting that ARV expression may also be a biomarker of resistance to RT. However, the landscape of ARV expression in primary prostate cancer and its effect on treatment outcomes remain unexplored. Here, we hypothesized that ARVs may be detectable in primary prostate cancer and furthermore may modulate response to salvage RT+ADT.We retrospectively identified 46 prostate cancer patients treated with prostatectomy followed by salvage RT+ ADT at a single institution from 1995 to 2012. Median age at salvage was 64.5 years. The indication for salvage therapy was biochemical failure after an undetectable post-operative PSA in 72%, gross local recurrence in 17%, and persistently elevated PSA after surgery in 11%. Median RT dose was 64.8 Gy, and all patients received concurrent ADT. We comprehensively interrogated the landscape of ARVs by performing ultra-deep targeted RNA-seq of archival formalin-fixed paraffin-embedded prostatectomy samples. Using a custom library of > 3000 primers spanning all AR exons and introns, we evaluated 21 native splice junction sites and 20 splice variants with a mean depth of coverage of > 5000x. We tested for association between splice variant expression and clinical outcomes using the log-rank test and Cox proportional hazards model.In total, 76% of patients had one or more detectable AR splice variants. The most commonly detected variants were AR-45 (exon 1b-2) in 41%, AR-V9 (exon 3-cryptic exon (CE) 5) in 20%, and AR-V7 (exon 3-CE3) in 13%. At a median follow-up of 33.8 months, biochemical progression-free survival (BPFS) at 3 years was 60%, distant metastasis-free survival was 90%, and overall survival was 100%. Among detected splice variants, only AR-V7 was associated with differential clinical outcomes, with a median BPFS of 10.9 months in AR-V7 positive vs 73.4 months in AR-V7 negative patients (P = 0.0020, HR 5.23, 95% CI 1.62-16.87).Using a custom ultra-deep targeted RNA-Seq approach, we provide among the first comprehensive descriptions of the AR splice variant landscape in primary prostate cancer. Moreover, we show that detectable AR-V7 expression in prostatectomy specimens was associated with inferior outcomes following subsequent salvage RT+ADT, suggesting for the first time that AR-V7 may modulate outcomes for localized as well as metastatic disease.

Clinical characteristics and molecular genetics of complete androgen insensitivity syndrome patients: a series study of 30 cases from a Chinese tertiary medical center

To study the clinical characteristics and genetic basis of complete androgen insensitivity syndrome (CAIS) in patients from the People's Republic of China. CAIS patients with 46 XY karyotype produce male levels of androgens but present with female external genitalia and secondary sex characteristics. The majority of affected individuals have androgen receptor (AR) gene mutations. This case series explored clinical and molecular characteristics of CAIS patients from the People's Republic of China. Genomic DNA from peripheral blood of clinically diagnosed CAIS patients was sequenced for mutation in the androgen receptor (AR) gene and steroid 5α-reductase type 2 gene (SRD5A2). Participants were recruited from Peking Union Medical College Hospital when they came in for consultation. Thirty patients from unrelated families were recruited. Data from medical documents recording diagnosis and treatment of these patients were retrospectively collected. Patient genotypes were determined by sequencing the AR and SRD5A2 genes. Their clinical characteristics were summarized based on symptoms, hormone profiles, operative findings, and pathological results. Twenty-one patients diagnosed with CAIS had mutations in AR exons. Analysis of AR exons revealed the presence of seven novel mutations (c.58C>T, c.645_652delGGGGGCTC, c.910G>T, c.1078C>T, c.1786T>A, c.2230G>T, and c.2522G>C); of these mutations, 47.6% (10/21) were located in the ligand-binding domain. Gonadal insufficiency was found in one case of CAIS. Among the remaining nine patients, three had SRD5A2 mutations and therefore a steroid 5α-reductase deficiency. No AR or SRD5A2 mutations were detected in the other six patients. This study broadens the spectrum of known AR gene mutations responsible for CAIS, and implies that there can be more complex underlying causes of CAIS.

Abstract 4319: Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer

Abstract Background: Androgen receptor (AR) amplification is a common genomic event in advanced prostate cancer, in particular, the metastatic castration-resistant prostate cancer (mCRPC) and is associated with poor treatment response and short overall survival (OS). Recent studies also show more frequent amplification at AR enhancer region in the mCRPC patients. The aim of this study is to evaluate clinical utility of multiplex digital PCR assay to detect amplification at AR locus in cell-free DNA (cfDNA) for prognostication of mCRPC. Methods: Plasma samples were collected from 108 patients with mCRPC before stage-specific treatment. The target regions were selected based on our previous sequencing data showing frequent amplification in mCRPC patients at AR loci, including AR enhancer, AR (AR exon 1 and exon 8) and OPHN1 (downstream of AR). Multiplex digital PCR assays to detect four targets were developed by adjusting the concentrations of primers and probes. The Kaplan-Meier (KM) estimate was used for OS analysis. Results: We first examined existing databases to determine relative stable genomic regions in prostate cancer. This analysis showed most stable regions in three gene loci including C2orf16 at 2p23.2, FAM111B at 11q12.1, and GRIA3 at Xq25, which were then used as reference controls for copy number normalization. We then designed two multiplex digital PCR assays to quantify copy number of selected targets. To separate two different targets with same fluorescence, we adjusted ratio of FAM-labeled probes with 1: 0.4 and HEX-labeled probes with 1:0.4. The ratio of FAM to HEX probes was 1:1.5 in final assays. The combination of four different targets in one reaction gave 9 possible clusters in the 2D amplitude space. We defined the amplification as the normalized ratio ≥ 1.8 between target and reference loci. By applying this cutoff in 108 mCRPC samples, we detected the amplification of AR-enhancer, AR and OPHN1 in 38, 34, and 25 samples, respectively. KM analysis showed the significant association of amplification in AR-enhancer (p=0.0053, HR=2.16[1.41-6.53]), AR (p=0.04, HR=1.74[1.04-4.03]) and OPHN1 (p=0.031, HR=1.91[1.07-5.53]) with OS. This result was consistent with whole genome sequencing data in the same group of patients. Conclusions: We established a highly sensitive multiplex digital PCR assay to detect copy number changes of AR, AR enhancer and OPHN1 in plasma cfDNA and demonstrated its potential clinical use as non-invasive testing tool for mCRPC prognosis. The easy-to-use and low-cost multiplex digital PCR assay covers multiple gene loci, which can aid in improving the sensitivity of detecting amplification at AR locus in the highly heterogeneous mCRPC patients. Citation Format: Meijun Du, Chiang-Ching Huang, Winston Tan, Manish Kohli, Liang Wang. Clinical utility of multiplex digital PCR to detect androgen receptor amplification in cell-free DNA for prognosis of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4319.

Multiplex Digital PCR to Detect Amplifications of Specific Androgen Receptor Loci in Cell-Free DNA for Prognosis of Metastatic Castration-Resistant Prostate Cancer.

Amplification of androgen receptor (AR) is a common genomic event in metastatic castration-resistant prostate cancer (mCRPC). To evaluate the prognostic value of the amplifications of specific loci in the AR gene in cell-free DNA, we developed a multiplex digital PCR (dPCR) assay that targeted AR enhancer (AR-En), AR exon 1 (AR-E1), AR exon 8 (AR-E8) and OPHN1 (downstream of AR). We selected three relatively stable genes, C2orf16, FAM111B, and GRIA3, as reference controls for copy number normalization. One hundred and eight mCRPC patients were recruited to test the association of specific AR loci amplification with clinical outcome. Using a normalized ratio ≥ 1.92 as cutoff, amplification of AR-En, AR-E1, AR-E8 and OPHN1 was observed in 28, 25, 24 and 19 of 108 mCRPC patients, respectively. Among the 41 patients with AR region amplification, 9 (21.9%) showed amplification at all four selected regions and 15 (36.6%) showed amplification at AR-En, AR-E1, and AR-E8. Six (14.6%) patients showed independent AR-En amplification, while the remaining 3 (7.3%) demonstrated AR-E8 amplification only. Kaplan–Meier analysis showed overall survival’s association with the amplification of AR-En (p = 0.02, HR = 1.68 (1.07–2.65)), AR-E8 (p = 0.02, HR = 1.78 (1.08–2.92)) and AR-En-E8 (the combination of AR-En and AR-E8 (p = 0.009, HR = 1.77 (1.15–2.73)). Multivariate models that included AR-En-E8 amplification and clinical factors significantly improved prognostic performance (p = 0.0001). With further validation, the multiplex dPCR assay may assist in prognostication of mCRPC patients.

BDNF as a novel therapeutic candidate for Kennedy's disease.

BDNF as a novel therapeutic candidate for Kennedy's disease.

The association between glutamine repeats in the androgen receptor gene and personality traits in dromedary camel (Camelus dromedarius).

Temperament traits such as fearfulness are important as they define an animal’s responses to its environment and handling. The increasing automation of daily tasks and growing population limits contact between camels and humans. Such limitations contribute to fear of humans and changes in physical environment. Monoamine oxidase A (MAOA) and androgen receptor (AR) genes are important candidates associated with mammal personality. In our analysis, MAOA exon 15 showed no polymorphism but a novel polymorphism was seen in the camel AR exon 1; 16, 17, 18, and 19 glutamine repeats were detected. We genotyped 138 camels belonging to four Egyptian breeds: Maghrabi (n = 90), Sudani (n = 15), Somali (n = 23), and Baladi (n = 10) for AR. Out of the 90 genotyped Maghrabi camels, we evaluated responses of 33 and 32 mature females to a novel object and exposure to an unfamiliar person, respectively. AR gene showed a significant association based on the principal component (PC) score, which indicated the fear of human touch, and the PC score indicates fear during interaction with novel objects. Individuals carrying a shorter genotype in homozygote (S/S) were found to be more fearful. Furthermore, we found that Sudani and Somali breeds had a higher frequency of shorter genotype (S/S), which was associated with increased fearfulness. These findings reflect the behavioral tendency and consequently, affect the use of this breed. This is the first report showing the role of AR glutamine repeats influencing a behavioral trait in dromedary camels and leading to inter-breed differences. Fear-related traits reported here are important because camels cope with various types of stresses and fear, resulting from the demands of intensive production systems and racing events. However, further studies, employing functional genomics and linkage analysis are necessary for confirming the relationship between fearfulness and genetic variation.

DNA methylation inhibitor mitigates spinal and bulbar muscular atrophy model mouse

Background: Spinal and bulbar muscular atrophy (SBMA) is a late-onset neuromuscular disease caused by the expansion of a CAG repeat within the first exon of androgen receptor (AR) gene. Transcriptional dysfunction plays a critical role in the pathogenesis of this disease.

A recurrent synonymous mutation in the human androgen receptor gene causing complete androgen insensitivity syndrome

Androgen insensitivity syndrome (AIS) is the most common cause of 46,XY disorders of sex development (46,XY DSD). This syndrome is an X-linked inheritance disease and it is caused by mutations in the human androgen receptor (AR) gene. Non-synonymous point AR mutations are frequently described in this disease, including in the complete phenotype. We present a novel synonymous mutation in the human AR gene (c.1530C > T) in four 46,XY patients from two unrelated families associated with complete androgen insensitivity syndrome (CAIS). The analysis of mRNA from testis showed that synonymous AR mutation changed the natural exon 1 donor splice site, with deletion of the last 92 nucleotides of the AR exon 1 leading to a premature stop codon 12 positions ahead resulting in a truncate AR protein. Linkage analyses suggested a probable founder effect for this mutation. In conclusion, we described the first synonymous AR mutation associated with CAIS phenotype, reinforcing the disease-causing role of synonymous mutations

Abstract 425: SMRT® Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants

Abstract Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known. Here we utilized long read sequencing to identify and quantify AR isoforms expressed in CRPC. To unambiguously characterize all AR isoforms, we prepared Iso-Seq™ libraries via 3’ rapid amplification of cDNA ends (RACE) with RNA isolated from prostate cancer cell lines and xenograft tissues using a forward primer anchored in AR exon 1. 3’RACE reactions were subjected to single molecule, real-time (SMRT®) long-read sequencing with a Pacific Biosciences RSII System. Our work identified AR-V9 as a truncated isoform that is frequently co-expressed with AR-V7 in CRPC. Mechanistically, our work re-annotated AR-V7 and AR-V9 mRNAs, showing these two species shared a common 3’ terminal exon containing separate splice acceptor sites. Taking into account this new information, novel siRNAs and antibodies which could distinguish between AR-V7 and AR-V9 were designed, validated and used to measure the relative expression of these two AR isoforms in CRPC cells with a view to determining the potential of AR-V9 as a predictive biomarker of primary resistance to AR-targeted therapies. Citation Format: Manish Kohli, Yeung Ho, David W. Hillman, Jamie L. Van Etten, Christine Henzler, Rendong Yang, Yingming Li, Elizabeth Tseng, Ting Hon, Tyson A. Clark, Liguo Wang, Kevin Silverstein, Liewei Wang, Scott M. Dehm. SMRT® Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2017-425

Abstract 3731: A novel multidimensional cfDNA assay for real time analysis of chemotherapy response

Abstract Due to its non-invasive nature and transient lifespan (~30 mins), circulating cell free DNA (cfDNA) may provide novel insights into cancer evolution and response to therapy. Here, we have developed a multidimensional cfDNA assay to measure patients’ responses to chemotherapy on a time scale that would be impossible using traditional biopsy methodologies. We extracted cfDNA from plasma collected immediately before and after infusion of cytotoxic chemotherapy, and 24 hours after treatment. We developed assays to (1) quantify cfDNA via qPCR of a 288 base pair amplicon of Long Interspersed Nuclear Elements (LINE1), (2) determine the DNA Integrity Index based on additional analysis of 115bp and 516bp LINE1 amplicons, and (3) analyze androgen receptor exon 8 via targeted deep sequencing. We analyzed cfDNA extracted from 17 patients with metastatic castration-resistant prostate cancer through sequential application of these 3 assays. Median PSA levels were 69 (0-2223) ng/dl, Hgb 12 (8.2-15) g/L, Alk Phos 117 (35-879) IU/mL. Deep sequencing of AR exon 8 revealed 6 of the 17 patients presented with 1-2 SNVs (H874Y, T877A, D890H or D879Y) at ≥1% frequency. Considering LINE1 quantification alone, the average change pre/post chemotherapy was 38.78 pg/mL (0.5-292.0 pg/mL). A variety of responses was noted including: 8 of the patients expressed elevated LINE1 levels post-treatment when compared to baseline, and 5 expressed depressed levels. Importantly, therapy did not affect tumor subclones equally, as demonstrated by sequencing and quantitation of mutant AR exon 8 species. Additional data on changes in DNA Integrity Index will be presented. We conclude that cytotoxic chemotherapy produces immediately observable effects on cfDNA. The effect of docetaxel chemotherapy on distinct clones within the tumor can be measured within hours. Now that a short-time dynamics response has been demonstrated, follow up studies will be performed involving more patients to determine the prognostic value. Citation Format: Pavan P. Shah, Katherin Patsch, Naim Matasci, Anjana Soudararajan, Patricia Diaz, David B. Agus, Daniel Ruderman, Mitchell E. Gross. A novel multidimensional cfDNA assay for real time analysis of chemotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3731. doi:10.1158/1538-7445.AM2017-3731

AR mutations in 28 patients with androgen insensitivity syndrome (Prader grade 0-3).

We investigated the androgen receptor (AR) gene mutation profiles of Chinese patients exhibiting severe androgen insensitivity syndrome (AIS) phenotypes. The present study enrolled 28 patients with genetically diagnosed AIS, who presented with severe phenotypes (Prader grade 0-3). Patients and some family members were screened via amplification and sequencing of their AR exons 1-8, including the corresponding intronic flanking regions. Luteinizing (LH), follicle-stimulating (FSH), and testosterone (T) hormone levels were found to be slightly, but not significantly, higher in patients with complete androgen insensitivity syndrome (CAIS) than in patients with partial androgen insensitivity syndrome (PAIS) (P>0.05). We identified 24 different AR mutations, including 12 that were novel. Ten patients (cases 2, 3, 10, 28, 11, 12, 19, 20, 24, and 25) were found to carry five recurrent mutations (p.Y572S, p.P914S, p.S176R, p.Y782N, and p.R841H); of these, p.Y572S, p.S176R, and p.Y782N were novel. Among the mutations identified in patients with CAIS, six (66.7%) were characterized as single-nucleotide missense mutations, and six (66.7%) were found to be located in the AR ligand-binding domain (LBD). Among the mutations identified in patients with PAIS, 15 (93.8%) were found to be missense, and 11 (68.8%) were found to be located in the LBD. Patients 10 and 28 were determined to harbor the same missense mutation (p.P914S), but were diagnosed with CAIS and PAIS, respectively. Sex hormone levels were slightly, but not significantly, elevated in patients with CAIS compared to those with PAIS. Missense mutations spanning AR exons 1-8 were the predominant form of identified mutations, and these were mostly located in the AR LBD. Approximately 50% of the identified mutations were novel, and have enriched the AR gene-mutation database. Patients harboring identical mutations were in some instances found to exhibit divergent phenotypes.

Abstract A20: Use of cell-free plasma DNA for dynamic monitoring of response to cytotoxic chemotherapy

Abstract Background: There is great interest in utilizing blood-based circulating biomarkers to gain molecular insight into cancer. Most of the published data examines circulating free DNA (cfDNA) obtained at a single time-point. Here, we developed and utilized an methodology for monitoring cfDNA at multiple time-points within 24 hours of cytotoxic chemotherapy infusions over sequential cycles. Methods: To monitor the short- and long-term responses to treatment, we analyzed cfDNA from plasma drawn immediately before (baseline), 5 hours and 24 hours after administration of docetaxel chemotherapy. We quantified the extracted cfDNA via qPCR of Long interspersed nuclear elements (LINE1). We then amplified and performed targeted deep sequencing (Illumina Miseq) of androgen receptor (AR) exon 8. To reduce PCR variability, we pooled 10 technical replicate amplifications. Results: Detailed data and time courses are presented for a patient with castrate resistant prostate cancer metastatic to bone treated with docetaxel/everolimus/bevacizumab according to a recently published trial (J Clin Oncol 33, 2015 suppl; abstr 5066). LINE1 was measured at 34.4 pg/mcl at baseline prior to treatment. Targeted deep sequencing of AR exon 8 revealed two independent mutations at 11% (H874A) and 13% (T877A), respectively. 5 hours after start of docetaxel infusion, total DNA content measured by LINE1 decreased to 0.4 pg/mcl. However, H874A and T877A mutations were observed at frequencies similar to the baseline values. A similar pattern was observed for samples obtained at Cycle 2 (21 days later). Specifically, baseline values for LINE1 (67.4 pg/mcl), H874A (3%), and T877A (7%) were noted to change to LINE1 (8.5 pg/mcl), H874A (6%), and T877A (17%) 5 hours later. Conclusion: We conclude that cytotoxic chemotherapy can produce effects immediately observable on cfDNA markers. The generalizability and clinical relevance of these findings will require follow up studies involving more patients. Citation Format: Daniel Ruderman, Katherin Patsch, Naim Matasci, Anjana Soundararajan, Patricia Diaz, Mitchell E. Gross. Use of cell-free plasma DNA for dynamic monitoring of response to cytotoxic chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A20.

A novel variant of androgen receptor is associated with idiopathic azoospermia.

A variety of genetic variants can lead to abnormal human spermatogenesis. The androgen receptor (AR) is an important steroid hormone receptor that is critical for male sexual differentiation and the maintenance of normal spermatogenesis. In the present study, each exon of AR in 776 patients diagnosed with idiopathic azoospermia (IA) and 709 proven fertile men were sequenced using use panel re-sequencing methods to examine whether AR is involved in the pathogenesis of IA. Two synonymous variants and seven missense variants were detected. Of the missense variants, a luciferase assay demonstrated that the R630W variant reduced the transcriptional regulatory function of AR. This novel variant (p. R630W) of AR is the first to be identified in association with IA, thereby highlighting the importance of AR during spermatogenesis.

Abstract 238: Androgen insensitivity syndrome germline loss-of-function mutations in the androgen receptor that acquire somatic gain-of-function in prostate cancer

Abstract Genomic alterations in the androgen receptor (AR) commonly occur in patients with advanced prostate cancer resistant to androgen deprivation therapies. While still retaining androgen sensitivity, recurring AR mutations become drivers of antiandrogen resistance by causing alterations in the ligand binding pocket that enables one specific antiandrogen to bind that particular mutant receptor in an agonist conformation, thereby aberrantly activating AR transcriptional activity. We chronically treated antiandrogen-sensitive preclinical prostate cancer models (LNCaP-AR and CWR22Pc) with antiandrogens followed by high-throughput sequencing of the AR exons 2-8 to search for novel mutations that may be associated with antiandrogen resistance. The mutation A597T in the DNA binding domain was found in four LNCaP-AR xenograft tumors from mice receiving enzalutamide or ARN-509, while the ligand binding domain mutation P893S was uncovered in CWR22Pc cells treated in vitro with combined androgen depletion and bicalutamide. Recent whole exome sequencing studies of clinical prostate cancer identified single patients with each of these mutations at high allele frequencies and without concurrent AR amplification (www.cBioPortal.org). A597T occurred in a Gleason 6 primary tumor, whereas P893S was found in a patient with metastatic castration resistant prostate cancer who had received bicalutamide during the course of treatment. Remarkably, both A597T and P893S were earlier reported as germline loss-of-function mutations in patients with androgen insensitivity syndrome. Using an AR reporter assay in AR-null cells coupled with overexpression of these AR mutants, we confirmed that P893S is not activated by the androgen dihydrotestosterone (DHT), but nevertheless is potently stimulated by bicalutamide. In silico modeling showed that in contrast to the wild-type AR, cofactor-recruiting helix 12 of the ligand binding domain of P893S substantially drifted away from an agonist conformation when bound to DHT but was able to adopt the active conformation when simulated with bicalutamide. Importantly, overexpression of AR-P893S in CWR22Pc cells conferred bicalutamide resistance in vivo. These findings highlight an unexpected contextual element to the function of AR mutants, and suggest that rare or private AR mutations may nonetheless act as drivers of clinical progression. Citation Format: Philip A. Watson, Minna D. Balbas, Zeda Zhang, Taslima F. Ishmael, Kayla E. Lawrence, John Wongvipat, Sophie D. Sawyers, Yi-Mi Wu, Dan Robinson, Yang Shen, Arul M. Chinnaiyan, Charles L. Sawyers. Androgen insensitivity syndrome germline loss-of-function mutations in the androgen receptor that acquire somatic gain-of-function in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 238.

Abstract B18: Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent

Abstract African American (AA) men exhibit a nearly 2-fold higher incidence and 3-fold higher mortality rate from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. Previous work from our laboratory and others has revealed differences in gene expression that contribute to prostate cancer health disparities among AAs. Oncogenic signaling pathways exhibiting up-regulation more often in AA prostate cancer include androgen receptor (AR) and epidermal growth factor receptor (EGFR). Thus, novel therapeutic strategies capable of driving production of inhibitory AR and EGFR isoforms and capable of limiting aberrant constitutively active AR isoforms are urgently needed. Such strategies will increase our understanding of these molecular mechanisms underlying prostate cancer in AA men. In addition, such strategies have the potential to ultimately result in novel specific approaches for treatment that will help reduce prostate cancer disparities for AAs and will improve treatment of advanced stage disease in all men with aggressive disease driven by these mechanisms. Splice-switching oligonucleotides (SSOs) represent a novel therapeutic strategy to combat prostate cancer. Unlike the strategy of using RNA interference to inhibit the expression of a gene, SSOs simultaneously limit the production of pathogenic proteins and induce the expression of protein variants with therapeutic value. SSOs modulate pre-mRNA splicing by binding to target pre-mRNAs and blocking access of the splicing machinery to a particular splice site, and can be used to produce novel splice variants or correct aberrant splicing. Herein we have designed and synthesized novel SSOs targeting AR and EGFR pre-mRNAs to produce inhibitory splice variants and correct aberrant splicing as follows. We have synthesized a SSO to correct the aberrant splicing that leads to production of the constitutively active AR-V7 variant leading to restoration of ligand dependency. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the cryptic exon included in AR-V7, decreases AR-V7 RNA. In addition, we have synthesized a SSO to drive production of a naturally occurring dominant-negative AR variant (AR45) leading to interference with wild type AR transactivation activity. Transfection of prostate cancer cells with this chemically modified SSO, which targets the splice site of the first exon of wild type AR and leads to inclusion of an alternative first exon encoding the unique N-terminal extension characterizing AR45, increases AR45 RNA. Furthermore, we have synthesized SSOs targeting exons in the transmembrane domain and the tyrosine kinase domain of EGFR to drive production of a soluble, naturally expressed inhibitory EGFR isoform and a dominant-negative EGFR isoform, respectively. Transfection of prostate cancer cells with these chemically modified SSOs increase RNA corresponding to these isoforms. Studies are underway to examine the effects of these SSOs on AR and EGFR signaling, respectively, and the effects of these SSOs on prostate tumor cell biology. These studies suggest that SSOs can be developed to modulate AR and EGFR signaling. Such SSOs have the potential to further our understanding of the contribution of the targeted molecular mechanisms to AA prostate cancer and have the potential to yield novel therapeutic modalities to combat prostate cancer in AA men as well as men of all races with aggressive disease driven by these mechanisms. Citation Format: Jennifer A. Freedman, Timothy J. Robinson, Bonnie LaCroix, Brendon M. Patierno, Daniel J. George, Bruce A. Sullenger, Steven R. Patierno. Development of novel therapeutic splice-switching oligonucleotides against aggressive prostate cancer in men of African descent. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B18.

A Protective Role for Androgen Receptor in Clear Cell Renal Cell Carcinoma Based on Mining TCGA Data.

Androgen receptor (AR) is expressed in normal murine and human kidneys of both genders, but its physiologic role is uncertain. Several studies showed loss of AR in renal cell carcinoma (RCC) in conjunction with increasing clinical stage and pathological grade, but others found that higher AR expression correlated with worse outcomes. Limited functional studies with renal cell lines suggested tumor-promoting activity of AR. In this study, we queried transcriptomic, proteomic, epigenetic and survival data from The Cancer Genome Atlas (TCGA) to evaluate AR expression and its association with overall survival in three subtypes of RCC (clear cell [ccRCC], papillary [pRCC], and chromophobe [chRCC]). We found that although there was no significant difference in AR mRNA expression in ccRCC of males vs. females, AR protein expression in ccRCC was significantly higher in male compared to female patients. More importantly, higher expression of AR at both transcript and protein levels was associated with improved overall survival in both genders with ccRCC, but did not predict survival of either gender with pRCC or chRCC. Genes whose transcript levels were associated with AR mRNA levels significantly overlapped between ccRCC and pRCC, but not with chRCC, suggesting a similar transcriptional program mediated by AR in ccRCC and pRCC. Ingenuity pathway analysis also identified overlapping pathways and upstream regulators enriched in AR-associated genes in ccRCC and pRCC. Hypermethylation of CpG sites located in the promoter and first exon of AR was associated with loss of AR expression and poor overall survival. Our findings support a tumor suppressor role for AR in both genders that might be exploited to decrease the incidence or progression of ccRCC.

Androgen Receptor Gene Aberrations in Circulating Cell-Free DNA: Biomarkers of Therapeutic Resistance in Castration-Resistant Prostate Cancer.

Although novel agents targeting the androgen-androgen receptor (AR) axis have altered the treatment paradigm of metastatic castration-resistant prostate cancer (mCRPC), development of therapeutic resistance is inevitable. In this study, we examined whether AR gene aberrations detectable in circulating cell-free DNA (cfDNA) are associated with resistance to abiraterone acetate and enzalutamide in mCRPC patients. Plasma was collected from 62 mCRPC patients ceasing abiraterone acetate (n = 29), enzalutamide (n = 19), or other agents (n = 14) due to disease progression. DNA was extracted and subjected to array comparative genomic hybridization (aCGH) for chromosome copy number analysis, and Roche 454 targeted next-generation sequencing of exon 8 in the AR. On aCGH, AR amplification was significantly more common in patients progressing on enzalutamide than on abiraterone or other agents (53% vs. 17% vs. 21%, P = 0.02, χ(2)). Missense AR exon 8 mutations were detected in 11 of 62 patients (18%), including the first reported case of an F876L mutation in an enzalutamide-resistant patient and H874Y and T877A mutations in 7 abiraterone-resistant patients. In patients switched onto enzalutamide after cfDNA collection (n = 39), an AR gene aberration (copy number increase and/or an exon 8 mutation) in pretreatment cfDNA was associated with adverse outcomes, including lower rates of PSA decline ≥ 30% (P = 0.013, χ(2)) and shorter time to radiographic/clinical progression (P = 0.010, Cox proportional hazards regression). AR gene aberrations in cfDNA are associated with resistance to enzalutamide and abiraterone in mCRPC. Our data illustrate that genomic analysis of cfDNA is a minimally invasive method for interrogating mechanisms of therapeutic resistance in mCRPC.

Mitochondrial dysfunction in Kennedy's disease: a new pharmacological target?

Mitochondrial impairment and elevated oxidative stress have been implicated in the pathogenesis of Kennedy's disease. However, there is still no study describing the mitochondrial nutrient management in patients with Kennedy's disease. We assessed the clinical and electrophysiological features in a patient with Kennedy's disease. This patient was diagnosed by genetic analysis. We also measured the plasma 8-hydroxydeoxyguanosine (8-OHdG) levels of the patient and his family members using commercial enzyme-linked immunosorbent assay (ELISA). Treatment with intravenous L-carnitine (2 g/day) for the patient was started on admission, followed by two weeks. Routine laboratory tests revealed a severe elevation of creatine kinase (CK) (606.5 U/L; normal value: 15-170 U/L). Sequencing of the first exon of androgen receptor (AR) revealed an increased number of CAG repeat (50; the normal range from 10-36) in the patient. Plasma 8-OHdG level in the patient was relatively elevated (34.68±1.01 ng/mL) compared with the female carriers and non-carriers. Two weeks after L-carnitine treatment, we observed a reduction of approximately 40% in CK level (391 U/L) in the patient. Oxidative stress resulting from mitochondrial dysfunction could be involved in Kennedy's disease. Targeting the mitochondrial dysfunction in Kennedy's disease may have significant therapeutic potential.