Androgen Receptor Staining Research Articles

Abstract 5080: Decursinol intercepts LNCaP human prostate cancer xenograft growth bypassing androgen receptor-prostate specific antigen axis

Abstract The slow-growing nature of most prostate cancers (PCa) provides multiple windows of opportunity to block or delay disease progression to reduce patient suffering and mortality. This is especially true following the failure of radical prostatectomy (RP) and radiation therapy (RT) of localized PCa with biochemical recurrence detectable by a rise in plasma/serum prostate specific antigen (PSA) and prior to androgen deprivation therapy (ADT). Costly aside, ADT is not curative and causes many adverse effects which negatively affect the quality of life of the patients. Currently there is no standard of care regimen for post-RP/RT patients with rising PSA to stump the trajectory. Many patients seek food or supplement-based interventions with a hope to delay ADT. The root of Korean Angelica gigas Nakai (AGN) contains decursin (D) and its isomer decursinol angelate (DA) as the major pyranocoumarins. We have previously shown that daily gavage of AGN and D/DA suppressed the growth of androgen receptor (AR)+ and AR- xenograft tumors and TRAMP neuroendocrine carcinomas. We also showed that D/DA rapidly converted to decursinol (DOH) in rodents and humans, and DOH inhibited LNCaP-AR xenograft growth, therefore the likely in vivo active compound. To assess merit of DOH to intercept recurrent PCa, we tested in NSG SCID mice bearing s.c. inoculated human LNCaP PCa xenograft (a) its efficacy to inhibit tumor growth and b) serum PSA as an efficacy metric. A day prior to start of daily DOH gavage (120 mg/kg body weight) for the tumor bearing mice, blood was drawn through tail vein for baseline PSA. After 4 weeks, the mice were bled for post-PSA and their tumors/prostate were dissected and weighed. Gavage delivered DOH did not affect body weight, but significantly reduced the tumor volume and weight without decreasing the weight of the prostate, distinct from ADT drugs. The post-PSA showed a significant and tight linear correlation with the tumor weight for mice in the vehicle group (r2=0.94, n=6). Five of 7 DOH-treated mice showed post-PSA/tumor weight plot on the same regression line but two non-responders displayed exaggerated post-PSA/weight ratio. The much slower PSA rise in the DOH-responders (2.9 fold, n=5) than vehicle group (7.8 fold, n=6, p=0.031) directly reflected a reduction of tumor burden by DOH treatment. Histological (H&E) analysis showed that DOH responder tumors had greater necrosis and less hemorrhage than non-responders and control tumors. Immunohistochemistry staining for AR and PSA did not detect any reduction of either protein in the responder tumors which contained reduced mitotic marker phospho-histone 3. Our preclinical modeling therefore suggests serum PSA as a reliable efficacy metric for DOH-mediated PCa interception in post-PR/RT patients, involving mitosis inhibition and necrosis without affecting the cancer AR/PSA axis. Citation Format: Sangyub Kim, Chongtao Qin, Deepkamal N. Karelia, Arati Sharma, Cheng Jiang, Junxuan Lu. Decursinol intercepts LNCaP human prostate cancer xenograft growth bypassing androgen receptor-prostate specific antigen axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5080.

The impact of androgen receptor (AR) expression on the prognosis of ductal carcinoma in situ (DCIS).

e14731 Background: Ductal carcinoma in situ (DCIS) display favorable outcome while little is known about the factors associated with invasive recurrence. To identify better prognostic biomarkers we performed gene expression analysis followed by immunohistochemistry (IHC) staining validation. Methods: Differential gene expression analysis of 29 pure DCIS patients was performed using nanostring platform. RNA was extracted from paraffin blocks from age/size matched 11 recurrence-free and 18 invasive-recurrence cases (disease free interval > 5 years). Gene annotation enrichment analysis was done for differentially expressed genes (DEG) using DAVID. Eighty-two pure DCIS cases were selected for external validation by IHC staining. Allred score cutoff 1 was used for survival analysis. Results: Ninety-nine differentially expressed genes were found statistically significant (p-value < 0.05). Androgen receptor (AR) gene, which encodes a transcription factor AR, has recently been highlighted as a favorable prognostic marker and a therapeutic target in invasive tumor (fold change = - 1.35, p < 0.001). AR protein expression was externally validated by IHC staining of 82 pure DCIS cases (24 invasive-recurrence versus 58 recurrence-free). Similar to gene expression analysis result, patients with invasive recurrence showed lower AR staining score than recurrence-free patients (p = 0.007). Cox regression analysis showed lower AR level as an independent risk factor of long-term invasive recurrence (HR 7.43, 95%CI 1.50 – 36.62). Gene enrichment analysis revealed enrichment of kinase pathway and cell cycle pathway in recurred cases (Enrichment Score = 2.43, 2.41 respectively). Conclusions: DEG pattern was observed among pure DCIS cases. AR may serve as a prognostic biomarker and targeting kinase, cell proliferation may be effective for higher risk DCIS patients.

SAT-328 Androgens Modulate the Expression of Prostatic Acid Phosphatase

INTRODUCTION: Prostatic acid phosphatase (PAP) is an acid phosphatase synthesized in prostate epithelial cells that served as the biochemical diagnostic mainstay for prostate cancer for several decades. There are two forms of PAP, secretory(sPAP) and transmembrane (TM-PAP), that are derived from alternative transcripts of the gene. PAP is highly expressed by epithelial cells of the adult prostate, however, the androgenic regulation of prostatic acid phosphatase is not well understood. HYPOTHESIS AND OBJECTIVES: We hypothesized that androgens do not increase PAP expression. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human bone metastases, and intact and castrated xenograft tumors of human VCaP prostate cancer cells. In addition, the effects of dihydrotestosterone (DHT) onTM- and sPAP expression in vitro were quantified. METHODS: Immunohistochemical staining for PAP, androgen receptor (AR) and Prostate Specific Antigen (PSA) protein expression was carried out on human fetal prostate (9.5, 11.5, 13, 16.5 and 18 weeks of gestational age) and human castration-resistant prostate cancer bone metastases archival specimens obtained with IRB approval. Tissue from human benign prostate and breast cancer metastases were used as controls. For xenograft studies, VCaP cells were inoculated subcutaneously into SCID mice and surgical or sham castration was performed after tumor development. Mouse tumors and weights were measured biweekly and tumor tissue isolated after mouse sacrifice. Isolated tumors were stained for PAP, AR and PSA. RESULTS: PAP expression was an early marker of prostate development and observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was observed in all human castration-resistant prostate cancer bone metastases examined (4/4 specimens). The AR was absent in two of four specimens. PSA was expressed in human castration-resistant bone metastatic prostate specimens; however, the expression was not as strong as PAP. In vivo, castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact animals but increased PAP expression was noted after castration. In contrast, PSA and AR expression was reduced in castrated VCaP tumors compared to tumors from intact mice. DHT treatment of VCaP cells increased AR expression while decreasing both sPAP and TM-PAP at doses ranging from 10-7-10-14M for 24 h. CONCLUSIONS: Our findings demonstrate that PAP is expressed early in normal human fetal prostate development and may be a marker of the early stages of differentiation. DHT decreased the expression of both isoforms of PAP in vitro and castration increased PAP expression in vivo. TM-PAP may serve as a potential target for castration-resistant prostate cancer as its expression increases with castration.

Correlation of Gleason Grading and Prognostic Immunohistochemistry Markers (Human Epidermal Growth Factor Receptor 2/neu and Androgen Receptor) in Prostatic Core Needle Biopsy: A Study in a Tertiary Care Center

Abstract Background: Worldwide prostate cancer is the most common cause of cancer and the second leading cause of cancer death among men. Transrectal ultrasound-guided core needle biopsies are the diagnostic modalities which help in proper categorization and grading of prostatic carcinoma, thus facilitating individualized treatment. These biopsies are the primary source for performance of additional diagnostic immunohistochemical testing for basal cell-associated markers to rule out the morphological mimicker and prognostic markers such as androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2)/neu. Materials and Methods: A prospective, observational study was conducted in the department of pathology in collaboration with department of urosurgery of a tertiary care hospital. One hundred and nineteen patients diagnosed with prostatic nodules were included in this study. Values of serum prostate-specific antigen were recorded. Tissue for histopathological study was obtained in the form of core needle biopsy, and Gleason grade was calculated in all malignant cases. Immunohistochemistry for p63 and alpha-methylacyl-CoA racemase was performed as an additional test in premalignant cases. Prognostication of the prostate cancer cases was done using AR and HER2/neu. Results: A total of 119 cases aged between 20 and 90 years were included in this study. Malignant lesions reveal an increase in the percentage of AR staining in comparison to the benign glandular structure. The Gleason score with higher value (8–9) showed increased expression of HER2/neu receptor. Conclusion: Critical histopathological analysis of core needle biopsy along with immunohistochemical evaluation maximizes the diagnostic accuracy in prostate cancer cases and also helps in prognosis assessment.

Imaging features of triple-negative breast cancers according to androgen receptor status

ObjectiveDifferent molecular subtypes of triple-negative breast cancer (TNBC) have previously been identified through analysis of gene expression profiles. The luminal androgen receptor (LAR) subtype has been shown to have a lower rate of pathologic complete response to neoadjuvant chemotherapy than other TNBC subtypes. The purpose of this study was to determine if the imaging features of TNBCs differ by AR (androgen receptor) status, which is a surrogate immunohistochemical (IHC) marker for the chemoresistant LAR subtype of TNBC. Materials and methodsThis sub-study was part of a clinical trial in patients with stage I-III TNBC who were prospectively monitored for response while receiving neoadjuvant systemic therapy (NAST) at a single comprehensive cancer center. This interim imaging analysis included 144 patients with known AR status measured by IHC. AR-positive (AR+) tumors were defined as those in which at least 10% of tumor cells had positive nuclear AR staining. Two experienced, fellowship-trained breast radiologists who were blinded to the IHC results retrospectively reviewed and reached consensus on all imaging studies for the index lesion (i.e., mammogram, ultrasound, and breast magnetic resonance imaging). The index lesion for each patient was reviewed and described according to the fifth edition of the Breast Imaging Reporting and Data System lexicon. Logistic regression modeling was used to identify imaging features predictive of AR status. p ≤ 0.05 was considered statistically significant. ResultsUnivariate logistic regression models for AR status showed that AR+ TNBC was significantly associated with heterogeneously dense breast composition on mammography (p = 0.02), mass with calcifications (p = 0.05), irregular mass shape on mammography (p = 0.03), and irregular mass shape on sonography (p = 0.003). Multivariate logistic regression models for AR status showed that AR+ TNBC was significantly associated with heterogeneously dense breast composition on mammography (p = 0.01), high mass density on mammography (p = 0.003), and irregular mass shape on sonography (p = 0.0004). ConclusionThe imaging features of TNBCs differ by AR status. Multimodality breast imaging may help identify the LAR subtype of TNBC, which has been shown to be a subtype that is relatively resistant to neoadjuvant chemotherapy.

Morphology-predicted, large-scale transition (LST) number in circulating tumor cells (CTC) as a biomarker of chromosomal instability (CIN) to assess early resistance to standard of care (SOC) drugs in mCRPC.

225 Background: CIN is a mechanism by which tumor cells evolve, adapt, and evade therapeutic insults. We explored the relationship between the presence of LST as a biomarker of CIN through distinct CTC phenotypes, and sensitivity of tumors with the biomarker to standard of care (SOC) drugs. Methods: CTCs from 634 mCRPC blood samples were morphologically characterized by the Epic Sciences platform [Cytokeratin, CD45, DAPI, Androgen Receptor (AR) staining]. Training: 20 digital single cell image features were extracted from 608 CTC images from 26 patients (Pts) and single cell sequenced to determine the number of genomic LSTs. An image feature based algorithm to predict whether the number of LSTs was above the normal range defined by analysis of leukocytes was trained using this cohort (pLST) and parameters were locked. Analytical Validation (AV): the pLST algorithm was applied to a separate cohort of 570 CTCs from 54 Pts that were also sequenced to test single cell analytical performance. Clinical Validation: The pLST algorithm was then applied to CTC detected in 554 Pt samples obtained at prior to (BL, n= 324) and matched on-therapy (OnTx, n= 230) ~1 mo after starting SOC [including AR directed Tx and taxane]. A pre-defined patient scoring of > 3 pLST+ CTC/mL was related to PSA kinetics, time on drug, and overall survival (OS). Results: pLST identifies CTCs with a high number of genomic LSTs. Pts with pLST+ at BL or OnTx had poor survival (see table): 12/13 pts receiving ARSi’s and 17/18 pts receiving taxanes who were pLST+ On-Tx required a change of Tx in a median of 1.3 mo (0.9 to 2.1) or died within 6 mo. Conclusions: CTC morphology can be used to predict CIN as defined by high LSTs. CTC pLST is a strong marker of resistance to SOC Tx in mCRPC pts and treatment failure requiring a change in Tx earlier than standard criteria.[Table: see text]

Abstract P3-08-01: Characteristics, outcomes and prognostic factors of luminal androgen receptor (LAR) triple-negative breast cancer (TNBC)

Abstract Background: The LAR subtype is a genomically distinct subset of TNBC. Using a large cohort of non-metastatic TNBC patients (pts) with long term follow-up, we sought to further characterize the clinicopathologic features and outcomes of LAR vs non-LAR TNBC. Methods: From a cohort of 9982 women with surgically-treated non-metastatic breast cancer, 605 met criteria for TNBC (ER/PR<1% and HER2-negative) by central pathology. RNA extracted from 304 FFPE tumor specimens using the HighPure RNA extraction kit was subjected to TruSeq RNA Access library preparation and sequencing on a HiSeq2500. Adequate RNA was available for 283 pts. Tumors were classified as LAR or non-LAR using a shrunken centroid model, CABAL (Clustering Among BAsal and Luminal androgen receptor). In addition to previously described analyses [Leon-Ferre et al, Breast Cancer Res Treat 2017], immunohistochemical (IHC) androgen receptor (AR) staining was performed and the impact of various parameters on invasive disease-free survival (IDFS) and overall survival (OS) was assessed using Cox proportional hazards models. Results: 58 (20%) tumors were classified as LAR and 225 (80%) as non-LAR. Compared to non-LAR, LAR pts were older (mean age 65 vs 54) and more often postmenopausal (79%vs53%), both p=0.01. Apocrine histology was more common among LAR tumors (21%vs0%), which were also lower grade (grade3: 69%vs95%) and had lower Ki-67 (Ki-67>15%: 64%vs82%), all p<0.01. Additionally, LAR tumors had lower median stromal tumor infiltrating lymphocytes (TILs, 20%vs25%) and were less frequently lymphocyte-predominant [≥50% stromal or intratumoral TILs (19%vs32%)], although neither reached statistical significance. AR IHC was available for 223 of 283 tumors. Median AR IHC score in LAR was 65% (range 0-100%) vs 0% (range 0-90%) in non-LAR. T/N stage, surgery type, and receipt of adjuvant chemotherapy (AdjCT) or radiotherapy were similar between LAR and non-LAR. LAR pts had shorter IDFS and OS compared to non-LAR (5.6 vs 11.8 yrs and 10.8 vs 20.8 yrs, respectively), although this did not reach statistical significance. Test of proportional hazard assumption was not significant for IDFS or OS (p = 0.30 and 0.09). IDFS estimates were numerically higher in LAR vs non-LAR (80.2%vs70.5%,p = 0.92) at 3yrs post-diagnosis; whereas the opposite was true (40.9%vs55.6%,p = 0.07) after 10yrs. OS estimates at 3 and 5yrs were similar between LAR and non-LAR, but at 10yrs OS was inferior in LAR (40.9%vs66.4%,p = 0.24). In a univariate analysis including both LAR and non-LAR, older age, higher N stage, lower TILs and absence of AdjCT were associated with poorer IDFS and OS. In a multivariate analysis, higher N stage and absence of AdjCT remained associated with both poorer IDFS and OS; while lower stromal TILs were associated with poorer IDFS (p=0.01), and with a trend towards poorer OS (p=0.07). Conclusions: LAR TNBCs occurred in older women, were lower grade, and had lower TIL density than nonLAR tumors. While significant differences in IDFS or OS were not demonstrated, LAR pts exhibited a numerically lower risk of a disease event at 3yrs, but higher risk by 10yrs compared to nonLAR pts. In the entire cohort, higher N stage, absence of AdjCT and lower TILs were independently associated with poorer outcomes. Citation Format: Leon-Ferre RA, Polley M-Y, Liu H, Kalari KR, Boughey JC, Liu MC, Cafourek V, Negron V, Ingle JN, Thompson KJ, Tang X, Barman P, Carlson E, Visscher DW, Carter JC, Couch FJ, Goetz MP. Characteristics, outcomes and prognostic factors of luminal androgen receptor (LAR) triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-08-01.

Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics.

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent.Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-β-gal activity (senescence) and analyzed by digital autoradiography (DAR).Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- β-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017).Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint.

Evaluation estrogen, progesteron and androgen receptor expressions in corneal epithelium in keratoconus

PurposeTo evaluate the role of sex steroid hormone receptors in corneal epithelium in etiopathogenesis of keratoconus (KC). MethodsThirty patients with KC who were planned for corneal collagen-crosslinking and 20 patients who were planned for excimer laser for refractive errors included in this study. Corneal epitheliums were curated mechanically during surgeries. Right eyes were evaluated immunohistochemically and left eyes were evaluated by quantitative polymerase chain reaction (qPCR) to investigate estrogenα, estrogenβ, progesterone and androgen receptors. ResultsImmunohistochemically, staining for progesterone and androgen receptors did not significantly differ between KC and control groups (p > 0.05). None of the cases had staining for estrogenα and estrogenβ receptors. qPCR showed that mRNA expressions of estrogenα and androgen receptors were significantly higher in the KC group (p < 0.001). ConclusionA significantly higher rate of estrogenα and androgen receptor expressions in corneal epithelium from patients with KC through qPCR supports a possible relation between KC and sex steroid hormones.

Molecular characterization of metaplastic breast carcinoma via next-generation sequencing

Metaplastic breast carcinoma (MBC) is a rare subtype of breast cancer with variable morphology. MBC is more often triple negative (ER-, PR-, HER2-) and is associated with poorer clinical outcome when compared with infiltrating ductal carcinoma. The purpose of our study is to identify molecular alterations in MBC using next-generation sequencing (NGS), which may aid chemotherapy selection and use of targeted therapy. A cohort of 18 patients with MBC yielded adequate DNA from microdissected formalin-fixed and paraffin-embedded tumor blocks. NGS was performed using the Ion AmpliSeq cancer hotspot mutation panel version 2 kit, which targets hotspot regions in 50 genes. Immunohistochemical stains for androgen receptor (AR), and programmed cell death ligand-1 were performed. A total of 23 genetic alterations were identified in 15 (83.3%) of 18 patients. Eleven genetic alterations in the PI3K signaling pathway were identified in 9 (50.0%) of 18 patients, including 7 PIK3CA mutations (38.9%), 3 PTEN genetic alterations (16.7%), and 1 AKT1 mutation (5.6%). Ten (55.6%) of 18 patients each harbored 1 TP53 genetic alteration. Additional genetic alterations identified were 1 HRAS mutation and 1 ATM mutation. AR immunoreactivity was identified in 2 (11.1%) of 18 patients. Programmed cell death ligand-1 was negative in all patients. NGS analysis demonstrated that PI3K pathway-related genetic alterations were detected in a high percentage of MBCs, suggesting that targeting the PI3K/mTOR pathway may be promising in patients with MBC. In addition, patients with AR expressing MBC may benefit from androgen antagonist treatment.

Ascorbic acid supplementation ameliorates testicular hormonal signaling, sperm production and oxidative stress in male rats exposed to rosuvastatin during pre-puberty.

Dyslipidemias are occurring earlier in the population due to the augmentation of obesity. Rosuvastatin reduces cholesterol and triglycerides; however, previous studies have shown that it may affect male reproduction. Ascorbic acid (AA), an antioxidant compound, plays a protective role in the male reproductive system. This study aimed to evaluate whether pre-pubertal exposure to rosuvastatin may impair testicular structure and antioxidant status in male rats and if supplementation with AA may alleviate these damages. Male rats were randomly divided into six experimental groups (n=10) on postnatal day (PND) 23 and received the different treatments by gavage from PND 23 to 53. The experimental groups received vehicle (saline solution 0.9%), 3 or 10mg/kg/day of rosuvastatin diluted in saline solution 0.9%, supplementation with 150mg/day of AA, 3mg/kg/day of rosuvastatin in association with 150mg/day of AA or 10mg/kg/day of rosuvastatin associated with 150mg/day of AA. Testicular parameters were assessed on PND 53 and 110. There were diminished androgen receptors staining in the Sertoli cells and increased germ cell death in rosuvastatin-exposed groups, in both periods. Spermatids showed lower estrogen alpha-receptors staining in the group exposed to 10mg of statin at adulthood. There were androgen depletion and increased lipid peroxidation and catalase activity in statin-exposed groups. Rosuvastatin exposure during pre-puberty impaired testicular structure, steroid receptor distribution and increased oxidative stress; however, AA was able to ameliorate the impairment provoked by statin exposure.

Expression of androgen receptor in invasive ductal breast carcinomas: a clinicopathological study from Jordan.

BACKGROUNDThe clinical relevance of androgen receptors (ARs) expressed in breast cancer cells and the suggested prognostic impact has been an area of active research. The prevalence rate of AR expression in breast cancer has never been reported among Jordanian patients.OBJECTIVEDetermine the expression rate of ARs among invasive ductal breast cancer cases of different stages and molecular subtypes. Also, analyze the relationship between AR expression and clinicopathologic and immunohistochemical criteria, and assess the impact of AR expression on survival.DESIGNRetrospective medical record review.SETTINGTertiary care hospital in Amman, Jordan.PATIENTS AND METHODSOur study comprised only of cases of invasive ductal breast carcinoma of no special type among females from records during a 10-year period between 2006 and 2015. Immunohistochemical staining was considered positive if more than 10% of tumor nuclei showed positive staining.MAIN OUTCOME MEASURESThe expression rate of ARs and the association of the expression rate with the clinicopathologic features of invasive breast cancer.SAMPLE SIZE293.RESULTSImmunohistochemical staining for AR revealed positive staining in 180 (61.4%) cases, including approximately 50% of triple-negative breast cancer cases. AR positivity correlated with estrogen receptor (ER) status (P=.007) and smaller T size (P=.014). However, no significant association was found with any of the other variables. AR expression was positively associated with overall survival (P=.022) in general and in ER-positive cases (P=.012). However, in the multivariate Cox regression model, AR was not independently associated with survival.CONCLUSIONSThese results were consistent with international reports showing a significant relationship of AR expression with ER status. In addition, AR expression was significantly associated with smaller tumor size. Although AR status was not independently associated with survival, our data suggest AR is a good prognostic factor.LIMITATIONSSome clinical data were missing.

Nuclear loss and cytoplasmic expression of androgen receptor in penile carcinomas: role as a driver event and as a prognosis factor.

Androgen receptor (AR) is a member of the steroid and nuclear family receptor that acts as transcription factor. AR signaling plays pivotal role in the development and progression of prostate cancer. However, the role of AR in penile cancer (PeCa) is poorly explored. Our previous molecular studies unveiled frequent AR mRNA loss in PeCa, which was further predicted as a major driver alteration in this neoplasm. Herein, we assessed the AR protein expression in 59 usual PeCa tissues and 42 surrounding normal tissues (SNT) by immunohistochemistry using a tissue microarray. In a paired analysis, we found a total absence of nuclear AR expression in PeCa while 95.2% of SNT samples presented strong nuclear AR expression (P < 0.001). Interestingly, 17 of 42 PeCa presented weak or moderate cytoplasmic AR staining, contrasting with 5 of 42 SNT (P = 0.008). Increased levels of AR cytoplasmic expression were related with poor prognosis features including advanced clinical staging (P = 0.044), compromised surgical margins (P = 0.005), and pathological inguinal node status (P = 0.047). Furthermore, AR cytoplasmic expression was also related with shorter overall survival (P = 0.032). In conclusion, the frequent loss of nuclear AR protein levels suggests a potential function in PeCa development. Based on this result, the androgen deprivation therapy is not indicated for PeCa patients. In addition, the AR cytoplasmic expression found in a significant number of cases (40.5%) showed prognostic value and pathways activated by the non-genomic AR signaling may represent a promising therapeutic strategy.

Intrafollicular level of steroid hormones and the expression of androgen receptor in the equine ovary at puberty

Steroidogenic activity in the equine ovary from birth to puberty has been poorly investigated. This study aimed to examine the capability of the ovarian follicles of prepubertal and pubertal fillies to produce steroid hormones and to evaluate the expression and cellular localization of androgen receptor (AR) in their ovaries. The ovaries of 6–18 month-old fillies were divided into two groups: prepubertal (PrP) – without preovulatory follicle (pF) and corpus luteum (CL), and ovulating/postpubertal (Ov/pB) – with pF and/or CL in at least one of the gonads. Adult mares (Me) were used as a control. The concentration of progesterone (P4), testosterone (T) and estradiol (E2) in follicular fluid (FF) was measured by radioimmunoassay. AR distribution was assessed by immunohistochemistry, while AR protein expression was examined by Western blot analysis. In the female groups, E2 concentration in FF of small follicles (<10 mm) was low and increased with the diameter of the follicle reaching the greatest value in pF (Ov/pB and Me group). In follicles (11–30 mm) of PrP fillies, the concentration of E2 was similar to that from Ov/pB fillies, but less than half (P < 0.05) than in Me follicles. In FF from all classes of follicles of Ov/pB fillies, the concentration of all steroids was similar to that in Me. AR immunolocalization, predominantly nuclear, was observed in all types of follicular cells (granulosa and theca cells) as well as in stroma and luteal cells. The pattern of staining was dependent on the follicle size and the group of females. In smaller antral follicles and in pF, the nuclear AR staining in granulosa cells was stronger than that found in follicles of 21–25 mm. In theca interna cells of pF, both nuclear and faint cytoplasmic reactions were seen. In luteal cells, AR labeling was noted in the nuclei and the cytoplasm: the strongest one in the early CL and almost negative in the late CL. AR protein expression in filly and mare ovarian tissues was confirmed by Western blot analysis and detected as a single band at approximately 110 kDa. In summary, the ovaries of fillies aged at least 6 months are capable of active steroidogenesis. ARs are present either in the cell nuclei or cytoplasm of all compartments of the equine ovary. AR expression in follicular and stroma cells may indicate the sensitivity of the filly ovarian tissue to androgens, the impact of androgens on folliculogenesis and the development of the equine ovary via a receptor-mediated pathway.

Abstract 2640: Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma by immunohistochemistry

Abstract Background: Small cell prostate carcinoma (SCPC) is a rare and highly malignant subtype of prostate cancer. SCPC only accounts for 0.5%-2% of untreated primary prostate cancer, but can present in up to 25% lethal metastatic prostate cancer autopsies. Small cell prostate carcinoma frequently lacks androgen receptor (AR) and prostate specific antigen (PSA) expression, and often responds poorly to androgen deprivation therapy (ADT). However, the expression pattern of different AR isoforms in SCPC after ADT remains poorly characterized. In this study, we have validated and developed a new immunohistochemical assay for full length AR (AR-FL) and AR splice variant-7 (AR-V7) protein expression in prostate cancer specimens. The expression of AR-FL and AR-V7 protein were determined in post-ADT radical prostatectomy samples from twelve SCPC patients. In this study, we developed and validated a new immunohistochemical assay for the full-length AR (AR-FL) and AR splice variant-7 (AR-V7) proteins. We conducted an IHC study in post-ADT surgical specimens from twelve SCPC patients treated with neoadjuvant ADT. Methods: AR-FL, AR-V7, and total AR proteins were specifically detected using AR C-terminal domain antibody (Sigma-Aldrich), AR-V7 antibody (RevMab), and AR N-terminal domain antibody (AR441, Santa Cruz), respectively. These antibodies were validated using prostate cancer cell lines with known AR profiles, including LNCaP95 cells which express both AR-FL and AR-V7 and the AR-negative PC3 cells. The IHC protocols were optimized for each individual antibody, and applied to the 12 SC/NE tissue specimens. Results: Twelve surgical (RP or TURP) prostate small cell carcinoma specimens were collected from patients after neoadjuvant hormonal therapy at Indiana University School of Medicine. Seventy five percent (9/12) of the specimens showed positive staining for the full-length AR with various intensities. Thirty three percent (4/12) of the specimens showed positive staining for AR-V7. Among the specimens with positive AR-V7 staining, two samples displayed very weak staining, one sample showed weak to moderate staining, and one sample showed strong staining. All positive specimens displayed a heterogeneous pattern of AR/AR-V7 staining. All specimens positive for AR-V7 were also positive for the full length AR. Conclusion and future direction: Our preliminary results indicate that small cell prostate carcinoma may continue to express AR and AR-V7. The functional and clinical relevance of AR/AR-V7 detection in this setting remains unknown. Future investigation will focus on clinical correlation in an expanded patient cohort. Citation Format: Pei Zhao, Yezi Zhu, Liang Cheng, Jun Luo. Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma by immunohistochemistry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2640.

Abstract PR02: β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor

Abstract Background: Androgen receptor (AR) has emerged as a new target for treating TNBC. AR is expressed in 10-43% of TNBCs. Although there are conflicting reports in the literature about the effect of AR status on TNBC prognosis, agents targeting AR signaling (enzalutamide) are already being evaluated in AR-positive TNBCs in early-stage clinical trials. However, no study so far has evaluated the association/correlation of AR status with ethnicity in TNBCs and downstream effects of AR loss in TNBCs. Given the association of AR loss with poor prognosis in breast cancer and that the African American (AA) with TNBC suffers aggressive disease course when compared to European American (EA) TNBCs, we hypothesized that AR loss might be an underlying cause of aggressive disease course in AR-negative TNBCs. Thus, in this project we aimed to study if loss or gain of AR in AA and EA TNBCs regulates the expression of β-catenin and leads to more aggressive disease course by activating downstream canonical Wnt-beta catenin signaling. Methods: We evaluated AR expression immunohistochemically in 424 formalin-fixed, paraffin-embedded samples from TNBC patients for whom complete clinicopathologic and overall survival (OS) data were available. Samples with &amp;lt;1% nuclear-stained cells were considered quadruple-negative. We also performed gene set enrichment analysis for the AR low and high group in AA and EA TNBCs using the publicly available TCGA gene dataset. Finally, we compared levels of 187 proteins with suspected involvement in breast tumorigenesis in AR-high vs AR-low TNBCs (using median AR expression as a cutpoint, as the IHC-derived AR status of these samples was unknown). Results: IHC staining of AR indicated that 79.5% of AA TNBCs (n=214) and 70% of EA TNBCs (n=210) were AR negative. Loss of AR was associated with poor overall survival in adjuvant-treated high Ki67 (&amp;gt;14%) (HR=1.72; p=0.095) AA TNBC (n=98) when compared to EA TNBCs (n=80). These data were validated by our in silico findings, which suggested that EA TNBCs (n=81) exhibited higher levels of AR mRNA compared to AA TNBCs (n=41) (p&amp;lt;0.05). Similarly, AR protein expression was higher in EA TNBCs (n=75) than AA TNBCs (n=32) (p&amp;lt;0.05). We further observed that β-catenin protein levels are higher in AA AR-low TNBCs compared with AA AR-high TNBCs (median AR expression used as cut point; p&amp;lt;0.05). This was strengthened by our observation in a cohort of 142 TNBCs wherein AAs with AR-negative TNBC showed a preponderance of cells with nuclear β-catenin staining via IHC compared with EA TNBCs that lack AR. Our GSEA analysis results indicated that Wnt/β-catenin signaling was the top-enriched gene ontology in the AR-low subgroup compared to AR-high subgroup of TNBCs. Furthermore, proteomic data revealed that β-catenin and Dvl3 expression was significantly upregulated in the AR-low subgroup when compared with AR-high group, suggesting that Wnt signaling is in an overdrive in the AR-low subgroup, especially in AA TNBCs. Conclusion: This study suggests that increased expression of β-catenin coupled with AR loss in AAs may underlie the ethnic disparity in outcomes among TNBC patients and strongly supports the prognostic role of AR and β-catenin in this breast cancer subtype. Citation Format: Karuna Mittal, Shristi Bhattarai, Sergey Klimov, Uma Krishnamurthi, Xiaoxian Li, Ceyda Sonmez Wetherilt, Mohammad A. Aleskandaran, Andrew A. Green, Emad A. Rakha, Ian O. Ellis, Guilherme Cantuaria, Guanhao Wei, Remus Mihai Osan, Meenakshi V. Gupta, Upender Manne, Padmashree C.G Rida, Ritu Aneja. β-Catenin overexpression underlies the aggressive disease course in African American triple-negative breast cancer patients who lack androgen receptor [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr PR02.

Abstract 1561: Increased phenotypic depth for automated visual identification of biomarkers on circulating tumor cells by cocktailing epithelial markers EpCAM and cytokeratin

Abstract Background: Canonical methods for CTC identification (such as CellSearch®) have typically relied on co-expression of surface (EpCAM) and cytoplasmic (cytokeratin, CK) epithelial markers to separate them from white blood cells. RareCyte has developed AccuCyte®-CyteFinder®, a platform for automated visual identification and retrieval of rare cells in blood by immunofluorescence (IF). For CTC detection, EpCAM and CK are placed in individual fluorescence channels, allowing for identification of CTCs that may be EpCAM(-) /CK(+) as well as EpCAM(+)/CK(-). Recently we have developed technology that increases the number of markers from 4 to 6 to allow for biomarker phenotypic analysis in the additional two channels. Given that our platform does not require co-expression of EpCAM and CK, we hypothesized that we could gain a third biomarker channel by combining detection reagents for EpCAM and CK into a cocktail within a single channel. We tested feasibility of epithelial marker cocktailing with an assay to detect androgen receptor (AR) in prostate CTCs. Methods: Normal human whole blood samples were spiked with prostate cancer lines PC3, LNCaP, and 22RV1 as model CTCs (mCTCs). Blood samples from University of Washington patients with advanced cancer were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer using a CTC assay for detection of AR in which anti-cytokeratin and anti-EpCAM antibodies were combined in a reagent cocktail into a single channel; the CD45 WBC marker and a nuclear dye were included in the assay. After development using spike-in models, the assay was applied to a patient sample. Results: No CTCs were identified in non-spiked normal donor blood samples. In spike-in models, the epithelial cocktail assay performed as well as the non-cocktail assay in identifying CTCs. AR fluorescence signal was present in LNCaP and 22RV1 mCTCs (which express AR) and absent in PC3 mCTCs (which do not express AR), confirming specificity of AR staining. When the assay was applied to a blood sample from a prostate cancer patient with known high CTC count, 160 CTCs per mL were identified; of these, 114 were observed to be AR-positive (71%). Conclusions: Combining the epithelial markers cytokeratin and EpCAM into a single-wavelength cocktail is feasible for IF identification of CTCs, allowing an additional channel for phenotypic investigation. In a patient with advanced prostate cancer with high CTC count, there was high incidence (71%) of AR expression in identified CTCs. Citation Format: Yao Sun, Arturo Ramirez, Daniel Campton, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Eric Kaldjian. Increased phenotypic depth for automated visual identification of biomarkers on circulating tumor cells by cocktailing epithelial markers EpCAM and cytokeratin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1561.

CD10, TDAG51, CK20, AR, INSM1, and Nestin Expression in the Differential Diagnosis of Trichoblastoma and Basal Cell Carcinoma.

Trichoblastoma (TB) and basal cell carcinoma (BCC) are 2 different neoplasms composed of basaloid cells and have overlapping histopathological features. We compared the immunoexpression of CD10, T-cell death-associated gene 51 (TDAG51), cytokeratin 20 (CK20), androgen receptor (AR), insulinoma-associated protein 1 (INSM1), and nestin for the differential diagnosis of these tumors. We assessed a total of 27 BCC and 27 TB cases, including 4 TB lesions in nevus sebaceous and 3 malignant TB lesions for CD10, TDAG51, CK20, AR, INSM1, and nestin expression. Staining for CK20, TDAG51, INSM1, and stromal CD10 was significantly more common in TB cases than in BCC cases ( P < .001). Epithelial CD10 and AR staining was significantly more common in BCC cases than in TB cases ( P < .001). The difference between the groups for nestin staining was not significant ( P > .05). Stromal CD10 staining was the most sensitive marker (96.3%) and INSM1 the least sensitive (55.6%) marker for TB. TDAG51 showed 100% specificity for TB. A larger number of CK20 positive cells was found in the cases associated with nevus sebaceous than in the other TBs. All the selected markers except nestin were useful for the differential diagnosis between TB and BCC. CD10 and TDAG51 were more useful than the other markers. The use of CK20 could be preferred in nevus sebaceous lesions. INSM1 was less effective in highlighting Merkel cells within the lesion than CK20.

PO-178 Androstenedione (4AD) activates an androgen receptor (AR) mediated transcriptome and AR interactions associated with cell-cell adhesion in aromatase inhibitor (AI) resistant breast cancer cells

Introduction AI’s are the front line treatment for endocrine postmenopausal breast cancer. However, response rates vary from 35%–70% and recurrence occurs in almost all advanced disease. In order to identify a target for overcoming AI treatment resistance, research has turned to the most widely expressed sex steroid receptor in breast cancer the AR. AI therapies, by their mechanism of action, create an unopposed androgenic steroid environment. 4AD is the major circulating steroid in postmenopausal women and AI therapy prevents it’s conversion to estrogens. We hypothesise that cancer cells adapt to utilise 4AD to promote resistance and survival in AI resistance. Material and methods Molecular changes in a breast cancer cell model which have transitioned from AI sensitive (MCF7 cells) to AI resistant (LetR cells) were assessed using RNA sequencing. Alterations in the steroid microenvironment occur as a result of AI therapy and responses to these steroids was confirmed using MTS, mammosphere and invasion assays. Mass spectrometry (LC–MS/MS) analysis identified androgen-mediated, AR protein interactions.The clinical relevance of AR in AI resistance was also evaluated in a cohort of primary breast cancer patients (n=375). Results and discussions LetR cell growth is AR dependent as the anti-AR drug Enzalutamide inhibits cell growth. Furthermore, Enzalutamide abrogates mammosphere formation and self-renewal of LetR cells. We have found LetR cells to be responsive to 4AD, and treatment with this steroid drives a more aggressive phenotype in vitro. RNA sequencing data highlighted a non-canonical cytoplasmic AR signalling mechanism. Our research suggests that 4AD initiates rapid second messenger signalling in the AI resistant setting. LC–MS/MS analysis identified androgen-mediated, AR protein interactors unique to our resistant model particularly associated with cell-cell adhesion and invasion. Evaluation of AR protein in a cohort of primary breast cancer patients found AR expression in 90% of patients, with 25% expressing high intensity AR staining. Analysis is ongoing into this subset and its association with clinical pathological parameters. Conclusion AR and its ligand 4AD may be a significant driving factor in AI resistance and may play a role in facilitating a more invasive phenotype. Further investigations into AR interactors identified will help elucidate mechanisms of resistance to AI therapy, and these novel AR protein partners may aid the identification of patients who would benefit from anti-AR therapy.

Androgen Receptor Expression in Endometrial Carcinoma.

Endometrial carcinoma (ECA) is frequently hormonally driven and can be treated with endocrine-based therapy, yet hormone receptor status is not routinely assessed. In particular, little is known about the significance of androgen receptor (AR) in ECA. Androgen has antiproliferative effects in the healthy endometrium and could serve a similar role to progesterone in curbing the progression of estrogen-dependent neoplasia. There may also be a subset of ECA that benefits from androgen antagonistic therapy. We herein investigate AR expression across ECA subtypes and compare its expression to estrogen receptor (ER) and progesterone receptor (PR). Immunohistochemical staining for AR, ER, and PR was performed on an endometrial tissue microarray containing 50 ECA with a variety of morphologic subtypes as well as 20 benign and 9 atypical hyperplastic endometria. AR was expressed by 54% (27/50) of ECA including 60% of low grade endometrioid carcinomas, 70% high grade endometrioid carcinomas, 70% serous carcinomas, 50% carcinosarcomas, and 20% clear cell carcinomas. High AR expression was chiefly restricted to a subset of serous carcinomas (50%). AR expression occurred most often in concert with ER staining, although 5 high grade cancers (1 serous carcinoma, 4 carcinosarcomas) showed AR expression in the absence of ER. In summary, AR positivity is seen in over half of ECA in our study, including the majority of low grade endometrioid carcinomas, high grade endometrioid carcinomas, and serous carcinomas. High level expression is seen in half of serous carcinomas and a subset of serous carcinomas and carcinosarcomas show some degree of AR staining in the absence of ER, suggesting a possible role for androgen inhibition in treatment of these cases.