High Androgen Receptor Expression Research Articles

Molecular and immunological characterization of androgen receptor expression in different breast cancer subtypes.

1114 Background: While estrogen receptor (ER) is well-studied in breast cancer (BC), the role of androgen receptor (AR) in prognosis and therapy response is less understood. Here, we aimed to characterize the clinicopathologic and molecular features of AR gene expression in BC subtypes. Methods: 10,728 BC samples were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). MSI was tested by IHC and NGS. TMB-high was designated as >10 somatic mutations/MB. Tumors with AR-high(H) and AR-low(L) RNA expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) and treatment-associated survival was obtained from insurance claims and calculated from treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined by chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons ( q<.05). Results: Lobular carcinoma had higher AR expression (2.37-fold) and AR positivity by IHC (93.6% vs 63.6%) compared to ductal carcinoma ( q<.05). Luminal A (LumA) had higher AR expression and IHC positivity compared to HER2-enriched, basal-like, and normal-like tumors ( q<.05). AR-H tumors were significantly ( p<.05) enriched for PIK3CA mutations in LumA (60.3% vs 29.8%), LumB (48.1% vs 23.2%), HER2-enriched (51.2% vs 39.3%), and basal-like (23.8% vs 8.3%); CDH1 mutations in LumA (26% vs 10.3%) and normal-like (55.5% vs 4.8%); MAP3K1 in LumA (17.1% vs 5.3%); ESR1 in LumB (17.9% vs 10.7%); and NF1 in HER2-enriched (19.3% vs 7.1%). AR-H had higher immune infiltration for LumA (B cells, M2 Mφ, neutrophils, NK cells, DC), LumB (B cells, M2 Mφ, neutrophils, NK cells, CD4+ T cells, DC), basal-like (NK cells, CD4+ T cells), and normal-like (NK cells, DC; q<.05) than AR-L. Conversely, AR-H HER2-enriched tumors had lower immune infiltration (B cells, M1 and M2 Mφ, NK cells, CD8+ T cells, DC) and fewer TMB-high (15.1% vs 23.2%), dMMR/MSI-high (0.3% vs 2%), and PD-L1+ tumors (15% vs 40.8%; p<.05) compared to AR-L (all q<.05). Post-doxorubicin survival was longer for patients with AR-H tumors; however, opposing trends were observed for patients with LumB (75.5 vs 56.3 m; p=.081) and HER2-enriched tumors (47.9 vs 65.2 m; p.071). AR-H was associated with longer post-dox survival in patients with LumB PIK3CA-mt tumors (81.7 vs 39.3 m; p=.036), but shorter post-dox survival in HER2-enriched PIK3CA-wt (50.4 vs 70.4 m) and PIK3CA-mt cohorts (40.8 vs 58.0 m; p=.18). Conclusions: Findings from our updated analysis based on PAM50 designation suggest a strong association between AR and PIK3CA mutations across subtypes and suggest that distinct AR-associated alterations in the immune microenvironment require further study. Exploration of specific mutations and immune-oncology markers associated with AR-H may aid in molecularly selected clinical trial design for advanced BC patients.

Abstract 5462: Single-cell spatial proteomic analysis to explore the spatial heterogeneity of desmoplastic small round cell tumors

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare and usually incurable aggressive sarcoma subtype. All malignant cells harbor a pathognomonic EWSR1::WT1 fusion protein (FP). However, FP-targeted agents are nonexistent; less than 20% of patients survive beyond five years. The spatial organization of DSRCT, consisting of tumor nests surrounded by desmoplastic stroma, is a hallmark of this disease. Given this context, we seek to understand the spatial heterogeneity within DSRCT and the tumor-stroma interactions to reveal potential therapeutic vulnerabilities. We generated a 20-panel marker developed to interrogate the cellular constituents of the tumor microenvironment (TME) and to characterize the multilineage expression DSRCT, including androgen receptor (AR) and neuroendocrine signatures (NE). The following markers were used: DSRCT (ST6GALNAC, pan-Cytokeratin), Fibroblasts (Collagen, α-smooth muscle actin), Endothelial Cells (CD31, α-smooth muscle actin), T-cells (CD45, CD4, CD8), Macrophages (CD45, CD68, CD163, CD11c), AR/NE markers (AR, neural-specific enolase, synaptophysin). We explored the expression of these markers in a nine-patient cohort from 9 x 9 mm2 tissue sections imaged by the Lunaphore COMET. We used a deep learning model in Visiopharm to extract high-quality areas and segment cells based on their nucleus. The spatial image data was converted into matrix data, which could be further analyzed in R. We confirmed that the tumor nests are surrounded by dense desmoplasia by collagen expression. We identified tumor cells, fibroblasts, immune cells, and endothelial cells by thresholding for classical cell markers. ST6GALNAC, a sialyltransferase that was found to be highly expressed in DSRCT based on RNA-seq data, marked the tumor nests. Previous data from single-nucleus RNA-sequencing (snRNA-seq) showed that DSRCT exhibited three possible subtypes: AR+/NE-, AR-/NE+, and Hybrid AR+/NE+. We found concordance between the prior transcriptomic signatures and the proteomic markers. In tumor nests that were positive for AR, we found AR localized to the nucleus, suggesting downstream activation of this pathway. High AR expression was also correlated with positive expression of pan-cytokeratin in the tumor nests. Future work will focus on quantifying spatial relationships and patterns. This includes characterizing the spatial distribution of all cell types found in DSRCT, including fibroblasts, endothelial cells, and immune cells. We will also address whether tumor nest characteristics are associated with cell phenotype or DSRCT subtype. Citation Format: Danh Truong, Sandhya Krishnan, Clement Agyemang, Davis Ingram, Rossana Lascano, Akshay Basi, Javier Gomez, Jared Burks, Alexander Lazar, Joseph Ludwig. Single-cell spatial proteomic analysis to explore the spatial heterogeneity of desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5462.

Diagnostic role and prognostic impact of PSAP immunohistochemistry: A tissue microarray study on 31,358 cancer tissues from 127 different tumor types

Abstract Introduction/Objective Prostatic specific acid phosphatase (PSAP) protein is produced in prostate epithelial cells and it is used as an immunohistochemical marker for prostate cancer. However, studies have reported PSAP expression to occur in various other tumor entities as well. Methods/Case Report Prostatic specific acid phosphatase (PSAP) protein is produced in prostate epithelial cells and it is used as an immunohistochemical marker for prostate cancer. However, studies have reported PSAP expression to occur in various other tumor entities as well. Results (if a Case Study enter NA) In prostate cancer, PSAP staining was seen in 100% of Gleason 3 + 3, 95.5% Gleason 4 + 4, 93.8% recurrent prostate cancer under androgen deprivation therapy, 91.0% Gleason 5 + 5 and 31.2% small cell neuroendocrine prostate cancer. Reduced PSAP staining was strongly linked to high pT stage, high Gleason grade, lymph node metastasis, early PSA-recurrence (p<0.0001 each),high androgen receptor expression and TMPRSS2:ERG fusions. In multivariate analyses, low PSAP expression was able to predict PSA recurrence independent of pre- and postoperative prognostic markers in ERG negative cancers. In extra-prostatic cancers, PSAP immunostaining was only seen in 3 of 94 (3.2%) neuroendocrine tumors of the pancreas and in 1 of 129 (0.8%) diffuse type gastric adenocarcinomas. Conclusion A positive PSAP immunostaining is highly specific for prostate cancer and reduced PSAP expression is associated with an aggressive prostate cancer phenotype. The independent association of reduced PSAP expression with poor prognosis in ERG negative prostate cancer makes PSAP measurement a candidate marker for prognostic multiparameter panels for prostate cancer.

Diagnostic Role and Prognostic Impact of PSAP Immunohistochemistry: A Tissue Microarray Study on 31,358 Cancer Tissues.

Prostate-specific acid phosphatase (PSAP) is a marker for prostate cancer. To assess the specificity and prognostic impact of PSAP, 14,137 samples from 127 different tumor (sub)types, 17,747 prostate cancers, and 76 different normal tissue types were analyzed via immunohistochemistry in a tissue microarray format. In normal tissues, PSAP staining was limited to the prostate epithelial cells. In prostate cancers, PSAP was seen in 100% of Gleason 3 + 3, 95.5% of Gleason 4 + 4, 93.8% of recurrent cancer under androgen deprivation therapy, 91.0% of Gleason 5 + 5, and 31.2% of small cell neuroendocrine cancer. In non-prostatic tumors, PSAP immunostaining was only found in 3.2% of pancreatic neuroendocrine tumors and in 0.8% of diffuse-type gastric adenocarcinomas. In prostate cancer, reduced PSAP staining was strongly linked to an advanced pT stage, a high classical and quantitative Gleason score, lymph node metastasis, high pre-operative PSA levels, early PSA recurrence (p < 0.0001 each), high androgen receptor expression, and TMPRSS2:ERG fusions. A low level of PSAP expression was linked to PSA recurrence independent of pre- and postoperative prognostic markers in ERG-negative cancers. Positive PSAP immunostaining is highly specific for prostate cancer. Reduced PSAP expression is associated with aggressive prostate cancers. These findings make PSAP a candidate marker for prognostic multiparameter panels in ERG-negative prostate cancers.

Cognitive Dysfunction in Patients Treated with Androgen Deprivation Therapy: A Multimodality Functional Imaging Study to Evaluate Neuroinflammation.

Androgen deprivation therapy (ADT) for prostate cancer is implicated as a possible cause of cognitive impairment (CI). CI in dementia and Alzheimer's disease is associated with neuroinflammation. In this study, we investigated a potential role of neuroinflammation in ADT-related CI. Patients with prostate cancer on ADT for ≥3 months were categorized as having ADT-emergent CI or normal cognition (NC) based on self-report at interview. Neuroinflammation was evaluated using positron emission tomography (PET) with the translocator protein (TSPO) radioligand [11C]-PBR28. [11C]-PBR28 uptake in various brain regions was quantified as standardized uptake value (SUVR, normalized to cerebellum) and related to blood oxygen level-dependent functional magnetic resonance imaging (BOLD-fMRI) choice-reaction time task (CRT) activation maps. Eleven patients underwent PET: four with reported CI (rCI), six with reported NC (rNC), and one status unrecorded. PET did not reveal any between-group differences in SUVR regionally or globally. There was no difference between groups on brain activation to the CRT. Regardless of the reported cognitive status, there was strong correlation between PET-TSPO signal and CRT activation in the hippocampus, amygdala, and medial cortex. We found no difference in neuroinflammation measured by PET-TSPO between patients with rCI and rNC. However, we speculate that the strong correlation between TSPO uptake and BOLD-fMRI activation in brain regions involved in memory and known to have high androgen-receptor expression mediating plasticity (hippocampus and amygdala) might reflect inflammatory effects of ADT with compensatory upregulated/increased synaptic functions. Further studies of this imaging readout are warranted to investigate ADT-related CI.

OR16-04 Androgen Deprivation Restricts Anti-tumor Immunity In Adrenocortical Carcinoma

Abstract Disclosure: K.M. Warde: None. L. Smith: None. C.J. Stubben: None. P.W. Willet: None. G. Castaneda-Hernandez: None. L. Liu: None. K. Basham: None. Adrenocortical carcinoma (ACC) is a rare cancer that is often diagnosed late and progresses rapidly, resulting in routinely poor outcomes. Through The Cancer Genome Atlas (TCGA) and other genomic studies, homozygous loss of ZNRF3 was newly identified as a frequent genetic alteration in human ACC tumors. ZNRF3 is an E3 ubiquitin ligase that negatively regulates Wnt signaling by degrading Wnt receptors. To examine the role of ZNRF3 in adrenal biology, we generated a Znrf3 conditional knockout (cKO) mouse model. Despite roust early hyperplasia, we found that Znrf3 cKO mice only develop adrenal tumors with advanced aging. Moreover, tumors emerge in a sex dimorphic manner whereby males, who exhibit a high myeloid immune response, predominately get benign tumors and females, who have a dampened myeloid response, develop metastatic ACC. This phenomenon is reflected clinically in patients whereby the incidence of ACC is up to 2.5x higher in women compare to men. In human ACC patients, females exhibit a lower myeloid signature compared to males. Moreover, a low myeloid response signature is associated with shorter progression-free and overall survival. These data suggest a potential relationship between anti-tumor immunity and sex in ACC, yet the underlying mechanisms remain unclear. Sex differences are common in cancer, and although there are many potential factors, gonadal hormones including androgens and estrogens, have been implicated as major biological drivers. Previous studies of the adrenal cortex highlight a specific role for androgens in repressing stem cell recruitment and proliferation. We hypothesized androgens promote anti-tumor immunity to help protect against ACC. To test this hypothesis, we castrated male Znrf3 cKO mice to lower androgen levels. We found that androgen deprivation significantly reduced myeloid cell infiltration, resulting in significantly larger adrenals in castrated as compared to sham controls. RNA-seq analysis revealed a decrease in immune activation pathways and monocyte-derived myeloid cell populations. Additionally, pro-inflammatory cytokines, including TNF, INFG, and IL-6, were significantly inhibited in castrated animals compared to controls. These data support a protective role for androgens in ACC through driving an intra-adrenal myeloid immune response. Consistent with these results, analysis of the TCGA-ACC patient cohort demonstrated that high androgen receptor expression is associated with better overall survival. Taken together, our results reveal an important role for androgens in promoting anti-tumor immunity in a mouse model of ACC, and we highlight the prognostic value of androgen receptor expression in patients. Further understanding of androgen signaling and downstream biological mechanisms that regulate the immune response may ultimately offer novel therapeutics and improve patient outcomes in ACC. Presentation: Friday, June 16, 2023

Overexpression of BQ323636.1 contributes to anastrozole resistance in AR+ve/ER+ve breast cancer.

Aromatase inhibitors (Ais) are used as adjuvant endocrine therapy for oestrogen receptor-positive (ER+ve) post-menopausal breast cancer patients. Ais, by inhibiting the enzyme aromatase, block the conversion of androgen to oestrogen, reducing oestrogen levels. Resistance to Ais limits their clinical utilisation. Here, we show that overexpression of BQ323636.1 (BQ), a novel splice variant of nuclear co-repressor NCOR2, is associated with resistance to the non-steroidal aromatase inhibitor anastrozole in ER+ve post-menopausal breast cancer. Mechanistic study indicates that BQ overexpression enhances androgen receptor (AR) activity and in the presence of anastrozole, causes hyper-activation of AR signalling, which unexpectedly enhanced cell proliferation, through increased expression of CDK2, CDK4, and CCNE1. BQ overexpression reverses the effect of anastrozole in ER+ve breast cancer in an AR-dependent manner, whilst co-treatment with the AR antagonist bicalutamide recovered its therapeutic effect both in vitro and in vivo. Thus, for BQ-overexpressing breast cancer, targeting AR can combat anastrozole resistance. Clinical study of 268 primary breast cancer samples of ER+ve patients who had been treated with non-steroidal Ais showed 32.5% (38/117) of cases with combined high nuclear expression of BQ and AR, which were found to be significantly associated with Ai resistance. Non-steroidal Ai-treated patients with high nuclear expression of both BQ and AR had poorer overall, disease-specific, and disease-free survival. These findings suggest the importance of assessing BQ and AR expression status in the primary ER+ve breast tumour prior to Ai treatment. This may save patients from inappropriate treatment and enable effective therapy to be given at an early stage. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Expression of androgen receptor in bladder cancer: A tertiary care center study

IntroductionThe significance of androgen receptor (AR) expression in bladder cancer is the focus of current research. This study aimed to assess the significance of AR expression in bladder urothelial carcinoma. MethodsA total of 206 cases of urothelial carcinoma of the urinary bladder were reviewed and immunohistochemical staining for AR was performed. The histological score was calculated as the product of the staining intensity and percentage of cells with positive nuclear staining. The staining results were correlated with demographic parameters, tumor grade, and detrusor muscle invasion status. ResultsSeventy-eight percent (n = 161) of cases tested positive for AR expression. AR was significantly overexpressed in non-muscle-invasive tumors (p = 0.042). Moreover, A higher AR expression was associated with increased disease-free survival (p = 0.019). However, AR expression was not significantly associated with cancer-specific survival, patient sex, or tumor grade (0.359, 0.747, and 0.867, respectively). AR expression is inversely related to muscle invasion and recurrence in bladder cancer. ConclusionThe results of this study support those of several reports worldwide. Further molecular studies are required to elucidate the role of androgens in bladder cancer pathogenesis and therapy.

Abstract A082: The epigenetic impact and therapeutic opportunity of AR-directed therapy for desmoplastic small round cell tumor

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare, usually incurable, aggressive sarcoma subtype that occurs predominantly in adolescent and young adult (AYA) males. At diagnosis, most present with hundreds of intraabdominal nodules composed of malignant cells that harbor a EWSR1-WT1 chromosomal translocation and resultant fusion protein (FP). It was shown that a subset of DSRCT cells (mostly of epithelial histotype) highly express the androgen receptor (AR), a key epigenetic driver of prostate cancer (PC). Given the male predilection and the high expression of AR, we sought to assess the role of AR more comprehensively in DSRCT and ask if this tumor shared AR resistance mechanism to PC. We tested if AR antagonists curb cell proliferation and tumor growth, where both enzalutamide and AR-antisense therapy blocked DHT-induced cell proliferation and reduced xenograft tumor burden. Next, to mechanistically interrogate AR’s oncogenic effects, we performed single-nuclei RNA-seq (snRNA-eq) and chromatin immunoprecipitation sequencing (ChIP-seq) on frozen patient specimens. Those studies revealed a surprising epigenetic similarity between DSRCT to PC. Though overlap exists in the DNA binding MOTIFs of AR in DSRCT and PC, our ChIP-seq resultsrevealed DSRCT-specific AR DNA binding sites adjacent to key oncogenic regulators, including FOXF1 and WT1 (the C-terminal partner of the pathognomonic FP). To our surprise, we identified a subset of DSRCT samples that underwent partial neuroendocrine (NE) epigenetic reprogramming, akin to what occurs in ~1/3 of castrate-resistant PC (CRPC). Since androgen deprivation therapy (ADT) isn’t yet a standard treatment for DSRCT, it remains an enigma why DSRCT would undergo NE reprogramming or how this may affect ADT sensitivity. To address this, nine DSRCT patient samples were profiled using snRNA-seq. Sub-clustering revealed epithelial, mesenchymal, or NE signatures and marked inter-patient and intra-tumoral heterogeneity. Notably, five of nine patients exhibited NE markers (SYP, ENO2, CHGA, FOXA2, ASCL1, and SOX2), while three expressed epithelial markers (MUC1, MUC6, KRT18, KRT23, CDH1). One patient exhibited a hybrid AR indifferent phenotype. While strikingly different from PC morphologically and phenotypically, our data suggest that DSRCT is a second androgen-stimulated malignancy (third if we consider breast cancers that rely on shared nuclear receptor family signaling). Shared dependence upon AR for tumor growth and survival provides an exciting opportunity to prospectively study AR signaling in a different cancer type and younger DSRCT-stricken patient population. Ongoing work will determine if DSRCT undergoes dedifferentiation towards a more stem-like cell as an intermediary step before undergoing neural lineage commitment or transdifferentiation directly from an epithelial cell type towards a neuroendocrine cell fate. We are also exploring if the FP acts as a Pioneer factor to direct AR towards DSRCT-specific androgen response elements that explain this sarcoma subtype’s unique clinical presentation. Citation Format: Danh D. Truong, Salah-Eddine Lamhamedi-Cherradi, Mayinuer Maitituoheti, Hannah C. Beird, Chia-Chin Wu, Sandhya Krishnan, Davis Ingram, P. Andrew Futreal, Mark Titus, Alexander Lazar, Kunal Rai, A. Robert MacLeod, Najat C. Daw, Andrea Hayes, Joseph Ludwig. The epigenetic impact and therapeutic opportunity of AR-directed therapy for desmoplastic small round cell tumor [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A082.

Abstract A012: Adaptive and non-adaptive gene expression responses in prostate cancer during androgen deprivation

Abstract Development of novel and efficient treatments for advanced prostate cancer (PCa) requires better understanding of the cellular mechanisms behind the transition to castration resistant prostate cancer (CRPC). We created long-term cell cultures to model the development of castration resistance. Using a low passage VCaP cells as an initial model, we first created a highly testosterone-dependent PCa cell line (VCaP-T) and then a subclone cell line developing the ability to grow at low testosterone concentration (VCaP-CT). Adaptation to low testosterone was solely due to long term culturing and experimental work confirmed for example higher AR expression and antiandrogen resistance in VCaP-CT compared to VCaP-T. With the developed models we aimed to uncover persistent and adaptive responses to testosterone level and to identify genes that potentially contribute to transition and proliferation of the castration resistant cells through androgen receptor (AR) regulation. Expression levels were measured using RNA-seq. We defined AR-associated genes as the ones with changes due to testosterone depletion in VCaP-T (418 genes) and further adaptive genes as the ones that restored expression level in VCaP-CT (134 genes). Interestingly, we identified 151 genes that were non-AR-associated in VCaP-T but gained the association in VCaP-CT cells. Genes gaining the AR-association were also hypothesized to be important for CRPC progression and castration resistant cells. We validated and assessed the clinical significance of the model and the findings using The Cancer Genome Atlas Prostate Adenocarcinoma data and multivariate Cox regression analysis. Expressions of 47 AR-associated or association gaining genes were statistically significant markers for progression-free survival. These included genes related to immune response, adhesion and transport. Genes ABCC5, SPRED3, TMPRSS11F, SPARC, SLC1A1, SATB1, PDGFRB and C1QTNF6 showed strong evidence being associated with tumor stage and aggressiveness in addition to progression-free survival. Taken together, we identified and validated multiple genes being linked with progression of prostate cancer and proposed several novel risk genes. These findings increase knowledge on mechanisms of castration resistance and may open new possibilities for treatments. Identified genes have potential to be used as biomarkers or therapeutic targets after further studies. Citation Format: Reetta Nätkin, Pasi Pennanen, Heimo Syvälä, Merja Bläuer, Juha Kesseli, Teuvo L.J. Tammela, Matti Nykter, Teemu J. Murtola. Adaptive and non-adaptive gene expression responses in prostate cancer during androgen deprivation [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A012.

Characterizing the role of PIM kinases in the prostate tumor immune microenvironment.

e17080 Background: The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, a family of pro-survival kinases ( PIM1, PIM2, and PIM3), are frequently overexpressed in prostate cancer (PC) and associated with PC aggressiveness and immune evasion. Preclinical data indicate that PIM1 overexpression increases macrophage infiltration and survival using in vivo models of PC. Here, we characterized the association of PIM1, PIM2, and PIM3 with PC immune signatures. Methods: 77 primary prostate and 11 metastatic lymph node samples from treatment-naive metastatic hormone-sensitive PC pts were analyzed by DNA next-generation sequencing (592-gene, NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC with PIM1/PIM2/PIM3-high(H) and -low(L) expression were classified by top and bottom quartile, respectively. Androgen receptor (AR) signature and Neuroendocrine Prostate Cancer (NEPC) score were calculated based on the expression level of previously defined gene signatures (Hieronymus et al. 2006, Beltran et al. 2016). Pathway enrichment was determined by GSEA (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS using Quantiseq. Statistical significance was determined by chi-square and Mann-Whitney U (p &lt; 0.05) and adjusted for multiple comparisons (q &lt; 0.05). Results: PIM1/PIM2/PIM3-H PC had a higher median MAPK activation score (MPAS) compared to PIM1/PIM2/PIM3-L PC (0.9, 1.3, 0 vs -1.6, -1.4, -1.1 respectively, all p &lt; 0.05). There was no difference in AR signature or NEPC score between PIM-H and PIM-L PC. PIM3-H PC exhibited higher PSA expression (3.15-fold, p &lt; 0.05). PIM1/PIM2-H PC had higher AR expression (1.9 and 3.5-fold, respectively, all p &lt; 0.05). PIM2/PIM3-H PC had enrichment of PI3K/AKT/mTOR, IL2/STAT5, IL6/JAK/STAT3, KRAS, TGFβ, NOTCH, hypoxia signaling, and ROS, P53, OXPHOS pathway and EMT (NES: 1.3-1.5, all FDR &lt; 0.25). PIM1/PIM2-H PC had higher expression of immunostimulatory genes ( IL1β, TNF, and TNFSF13, FC: 1.1-3.1, p &lt; 0.05). PIM1/PIM2/PIM3-H PC had higher expression of MHC class I ( HLA-A, HLA-B, HLA-C, B2M, FC: 1.3-2.5, all p &lt; 0.05) and MHC class II ( HLA-DPA1, HLA-DRB1, HLA-DPB2, TAP1, TAP2, FC: 1.1-3.2, all p &lt; 0.05) genes. PIM3-H PC had increased infiltration of M2 macrophages (8% vs 5.2%) and NK cells (5.2% vs 3.3%), while PIM2-H PC had increased Tregs (1.7% vs 0%) (all p &lt; 0.05). Moreover , PIM1/PIM2/PIM3-H PC had a high T cell inflamed score compared to PIM1/PIM2/PIM3-L PC (66, 80, 56 vs -143, -157, -136, respectively, all p &lt; 0.05). Conclusions: Our data suggest a strong association between PIM expression and increased MAPK activation score, T cell inflamed score, inflammatory, MHC class I and MHC class II gene expression, and differential immune cell infiltration. A better understanding of these differences will provide a rationale for tailored therapeutic approaches for PIM-expressing PC.

Abstract PD5-08: PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation

Abstract Title: Drug target activation mapping of matched triple negative primary and axillary LN metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation Background: Triple negative breast cancer (TNBC) is comprised of heterogeneous subtypes that arise from various molecular alterations that result in TNBC having the poorest overall prognosis. There is growing interest in more sensitive ways to measure estrogen receptor (ER) and HER2 levels in the ER “negative” and HER2 “low/negative” settings, as there are patients with TNBC whose tumors are actually ER and/or HER2 “expressing/active”, and who may benefit from ER- and/or HER2-targeted therapies. Given the limitations of IHC in reliably quantitating ER and HER2 in low (0-1+) expressing tumors, we utilized highly sensitive reverse phase protein array (RPPA) to quantitate expression of these therapeutic targets along with downstream signaling activation mapping in a unique pilot set of patient-matched primary (P) and axillary lymph node (LN) metastases obtained synchronously. The quantitative expression of steroid hormone receptor expression/activation (ER, glucocorticoid receptor (GR), androgen receptor (AR)) along with their signaling architecture and dynamics were analyzed. Methods: A 12 P-LN paired tumor set was chosen for analysis (N=24). Manual macrodissection was used to enrich for tumor epithelium prior to RPPA analysis that quantitatively measured 155 proteins/phosphoprotein drug targets in key signaling pathways known to be involved in steroid hormone receptor biology and tumorigenesis. Statistical analysis using paired sample t-testing or Wilcoxon rank testing was performed (p&amp;lt; 0.05 uncorrected). Results: Protein pathway activation mapping and unsupervised clustering analysis revealed a subset of TNBCs from 3 of the 12 (25%) patients (N=4: 1 LN and 1 P, and one patient-matched LN and P) that was characterized by high relative levels of total ER. Interestingly, these same tumors also had amongst the highest relative levels of both total AR and activated (phosphorylated) GR compared to the rest of the 20 tumor samples. Downstream signaling analysis found significant (p&amp;lt; 0.05) activation/phosphorylation of AKT (S473 and/or T308) associated with the ER-AR-GR/steroid hormone activated/”high” TNBC signature compared to the 20 tumors that were steroid hormone “low”. Conclusions: Functional protein pathway activation mapping of a unique study set of synchronously obtained TNBC primary and LN metastasis revealed a steroid hormone receptor expression/activation signature of co-incidentally activated/expressed ER, GR and AR, that was also characterized by increased AKT activation (p&amp;lt; 0.05). This steroid hormone receptor “activated” signature was not observed to be significantly differentially expressed in LN compared to P tissues in cohort or matched pair analysis, suggesting that this signature may be present as an intrinsic event in the P, and not acquired in LN metastases. Recent data from stage I-III TNBC patients treated in the neoadjuvant setting with an AKT inhibitor1 revealed that responding TNBC patients had pre-treatment tumor biopsies that were enriched for both high AR expression and high phosphoAKT (S473 and/or T308) levels. If these observations that a subset of TNBCs are steroid hormone receptor “activated” despite being ER “negative” are confirmed in larger study sets, we hypothesize that this subgroup, identified by RPPA analysis of both P and LN metastases, could be potentially sensitive to AKT inhibition in combination with agents that target steroid hormone receptors. 1Shi Z, et al. Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer. Clin Cancer Res. 2022 Mar 1;28(5):993-1003 Citation Format: Julia Wulfkuhle, Page Blas, Heather Williams, Claudius Mueller, Rosa I. Gallagher, Mariaelena Pierobon, Joyce O’Shaughnessy, Emanuel F. Petricoin. PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-08.

Abstract C036: Correlation of Kaiso and androgen receptor expression in women of African ancestry with triple-negative breast cancer

Abstract Breast cancer (BCa) is the most frequently diagnosed female cancer and a leading cause of female deaths worldwide, regardless of increased awareness and improved therapies. Triple-negative breast cancer (TNBC) is one of the most challenging BCa subtypes as it is highly heterogeneous, metastatic in nature and has limited targeted therapies. TNBC is most prevalent in young women of African ancestry (WAA), who despite having lower BCa rates, have a disproportionately higher mortality rate compared to women of European ancestry (WEA). However, the cause for this racial disparity is currently unknown, thus highlighting the importance of unraveling the genetic and molecular factors that contribute to TNBC in WAA. Our lab previously showed that the transcription factor Kaiso may be linked to this disparity as it contributes to increased metastasis and mortality in WAA TNBC patients. Interestingly, multiple studies have indicated that WAA TNBC tissues also express less Androgen Receptor (AR) than WEA TNBC tissues, supporting the existence of a novel BCa subtype – quadruple-negative breast cancer (QNBC). Notably, in silico analysis revealed several Kaiso binding sites in the AR promoter region, and that high Kaiso and low AR expression correlated with poorer overall survival in BCa patients. Thus, we hypothesized that high Kaiso and low AR expression could be contributing to the increased mortality in WAA with TNBC. This study seeks to examine the relationship between Kaiso and AR, the clinical significance of this relationship and its role, if any, in TNBC racial disparities. Using tissue microarrays (TMAs) and immunohistochemistry (IHC), we found reduced and cytoplasmic AR expression in WAA compared to WEA. Moreover, preliminary findings from western blot analyses showed an increase in AR expression in response to Kaiso depletion in TNBC cells. These findings suggest that AR could be a bona fide Kaiso target gene and that there may be clinical relevance of high Kaiso, and low AR expression in BCa survival, especially in patients with an African heritage. Ongoing experiments are focused on determining if Kaiso directly associates with the endogenous AR promoter region and discerning the link between high Kaiso and low AR expression in TNBC racial disparities. A significant correlation between Kaiso and AR expression will help to validate the existence of QNBC in WAA and support Kaiso and AR as clinically relevant prognostic markers for TNBC, especially in WAA. Citation Format: Stephanie Ali Fairbairn, Robert Cowan, Juliet M. Daniel, Shawn M. Hercules. Correlation of Kaiso and androgen receptor expression in women of African ancestry with triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C036.

AMACR Expression is a Potential Diagnostic Marker in Apocrine Lesions of Breast, and is Associated with High Histologic Grade and Lymph Node Metastases in Some Invasive Apocrine Breast Cancers

BackgroundCarcinoma with apocrine differentiation (AC) is a subtype of breast carcinoma with apocrine features in >90% of the tumor. Molecular studies demonstrate AC has high expression of androgen receptor (AR) mRNA. Pure AC lack estrogen receptor (ER), progesterone receptor (PR), and express AR, with variable human epidermal growth factor 2 (HER2) status. Currently, in triple negative AC, no targetable therapies or specific diagnostic markers exist. Materials and Methodsα-Methylacyl CoA racemase (AMACR) expression was investigated as a marker of apocrine differentiation using a single-plex immunoperoxidase stain, and a novel AMACR/p63 dual stain in a subset of cases, across 1) benign apocrine lesions (apocrine metaplasia, adenosis) 2) apocrine DCIS (ADCIS), 3) AC/ invasive ductal carcinoma (IDC) with apocrine features, 4) non-apocrine triple negative breast cancer (TNBC) and 5) IDC, no special type. A sub-set of cases were evaluated by tissue microarray. ResultsAMACR expression was increased in both AC and ADCIS, with minimal expression in benign breast tissue, TNBC and IDC, NST cases. In invasive cases, those with positive AMACR (>5% positivity) were significantly associated with higher histologic grade (P = .006), initial N stage (chi squared 0.044), and lack of ER or PR expression (both P < .001), with no correlation with overall survival. Analysis of TCGA breast cancer datasets revealed AMACR expression was significantly higher in molecularly defined apocrine carcinomas relative to basal and luminal subtypes. Moreover, high AMACR expression predicted worse relapse-free and distant-metastasis free survival, among both ER-/PR-/Her2- and ER-/PR-/Her2+ breast cancer cohorts (log-rank P = .081 and .00011, respectively). ConclusionAMACR represents a promising diagnostic and prognostic marker in apocrine breast lesions. Further study is needed to determine the biologic and clinical significance of this protein in AC.

Correlation between Androgen Receptor Expression in Luminal B (HER-2 Negative) Breast Cancer and Disease Outcomes.

(1) Background: Hormone receptor positive breast cancer is a subtype of breast cancer with relatively good prognosis, but luminal B (HER-2 negative) breast cancer has a higher risk of recurrence and metastasis. Patients with endocrine therapy resistance and chemotherapy insensitivity have poor prognosis. Androgen receptor (AR) is widely expressed in breast cancer, but there is no clear conclusion about its function and correlation with prognosis in luminal B breast cancer. Further research is needed to reveal the role of AR in luminal B (HER-2 negative) breast cancer. (2) Methods: Retrospectively analyzed patients with early-stage luminal B breast cancer. The correlation between AR and its associated indexes with long-term survival was determined. (3) Results: A total of 985 patients were included with 143 treated by neoadjuvant therapy. Of these, 83.5% of the patients had AR expression ≥65%. High AR expression was associated with good disease-free survival (DFS) and overall survival (OS). In the neoadjuvant population, AR/estrogen receptor (ER) &gt; 1.06 and residual tumor Ki67 &gt; 23% had significantly worse DFS. (4) Conclusion: Low AR (&lt;65%) expression is associated with poor prognosis in luminal B (HER-2 negative) breast cancer patients. High AR/ER and residual tumor Ki67 were associated with poor DFS in neoadjuvant group with a cutoff value of AR/ER &gt; 1.06 and residual tumor Ki67 &gt; 23%.

Androgen Receptor Expression in the Various Regions of Resected Glioblastoma Multiforme Tumors and in an In Vitro Model.

Glioblastoma multiforme (GBM) is a malignant glioma, difficult to detect and with the lowest survival rates among gliomas. Its greater incidence among men and its higher survival rate among premenopausal women suggest that it may be associated with the levels of androgens. As androgens stimulate the androgen receptor (AR), which acts as a transcription factor, the aim of this study was the investigate the role of AR in the progression of GBM. The study was conducted on tissues collected from three regions of GBM tumors (tumor core, enhancing tumor region, and peritumoral area). In addition, an in vitro experiment was conducted on U-87 cells under various culture conditions (necrotic, hypoxic, and nutrient-deficient), mimicking the conditions in a tumor. In both of the models, androgen receptor expression was determined at the gene and protein levels, and the results were confirmed by confocal microscopy and immunohistochemistry. AR mRNA expression was higher under nutrient-deficient conditions and lower under hypoxic conditions in vitro. However, there were no differences in AR protein expression. No differences in AR mRNA expression were observed between the tested tumor structures taken from patients. No differences in AR mRNA expression were observed between the men and women. However, AR protein expression in tumors resected from patients was higher in the enhancing tumor region and in the peritumoral area than in the tumor core. In women, higher AR expression was observed in the peritumoral area than in the tumor core. AR expression in GBM tumors did not differ significantly between men and women, which suggests that the higher incidence of GBM in men is not associated with AR expression. In the group consisting of men and women, AR expression varied between the regions of the tumor: AR expression was higher in the enhancing tumor region and in the peritumoral area than in the tumor core, showing a dependence on tumor conditions (hypoxia and insufficient nutrient supply).

Higher Expressions of SHH and AR Are Associated with a Positive Receptor Status and Have Impact on Survival in a Cohort of Croatian Breast Cancer Patients.

Breast cancers (BC) are usually classified into four molecular subtypes according to the expression of estrogen (ER), progesterone (PR), and human epidermal growth factor 2 (HER2) receptors and proliferation marker Ki-67. Despite available anti-hormonal therapies and due to the inherent propensity of some subtypes to develop metastasis, there is a permanent need to discover new prognostic and predictive biomarkers, as well as therapeutic targets for BC. In this study, we used immunohistochemical staining to determine the expression of androgen receptor (AR) and sonic hedgehog protein (SHH), the main ligand of the Hedgehog-GLI (HH-GLI) signaling pathway, in 185 archival primary BC tissue samples and correlated it with clinicopathological characteristics, molecular subtypes, receptors statuses, and survival in a cohort of Croatian BC patients. Results showed that higher SHH and AR expressions were associated with positive receptor status, but increased SHH expression had a negative impact on survival in receptor-negative BCs. On the contrary, higher AR expression was mostly protective. However, multivariate analysis showed that only higher AR expression could be considered as an independent prognostic biomarker for poorer overall survival in triple-negative breast cancer patients (TNBC) (HR 10.9, 95% CI 1.43–83.67; p = 0.021), what could be Croatian population-related. SHH could be a potential target for treating TNBCs and HER2-enriched BCs, in cases where HH-GLI signaling is canonical (SHH-dependent).

Transcriptional Profile Associated with Clinical Outcomes in Metastatic Hormone-Sensitive Prostate Cancer Treated with Androgen Deprivation and Docetaxel.

Simple SummaryThe combination of androgen deprivation therapy (ADT) with docetaxel (DX) or/and with novel anti-androgen receptor therapies have become standards for the treatment of patients with metastatic hormone-sensitive prostate cancer (mHSPC). However, metastatic PC remains incurable, and biomarkers for individual treatment selection are needed. We propose here that molecular alterations associated with castration resistance may predict the clinical evolution of mHSPC patients. To test this hypothesis, we designed a custom expression panel of 184 genes and tested it in tumor biopsies from patients with mHSPC treated with ADT+DX. We found that AR and ESR signatures and ESR2 gene expression correlate with a good prognosis. The lower expression of TSG (PTEN, TP53 and RB1) signature, as well as high ARV7 and low RB1 gene expression, were associated with adverse clinical outcomes. The usefulness of transcriptomic analysis of such signatures as a strategy for personalized treatment selection should be further explored.(1) Background: Androgen deprivation therapy (ADT) and docetaxel (DX) combination is a standard therapy for metastatic hormone-sensitive prostate cancer (mHSPC) patients. (2) Methods: We investigate if tumor transcriptomic analysis predicts mHSPC evolution in a multicenter retrospective biomarker study. A customized panel of 184 genes was tested in mRNA from tumor samples by the nCounter platform in 125 mHSPC patients treated with ADT+DX. Gene expression was correlated with castration-resistant prostate cancer-free survival (CRPC-FS) and overall survival (OS). (3) Results: High expression of androgen receptor (AR) signature was independently associated with longer CRPC-FS (hazard ratio (HR) 0.6, 95% confidence interval (CI) 0.3–0.9; p = 0.015), high expression of estrogen receptor (ESR) signature with longer CRPC-FS (HR 0.6, 95% CI 0.4–0.9; p = 0.019) and OS (HR 0.5, 95% CI 0.2–0.9, p = 0.024), and lower expression of tumor suppressor genes (TSG) (RB1, PTEN and TP53) with shorter OS (HR 2, 95% CI 1–3.8; p = 0.044). ARV7 expression was independently associated with shorter CRPC-FS (HR 1.5, 95% CI 1.1–2.1, p = 0.008) and OS (HR 1.8, 95% CI 1.2–2.6, p = 0.004), high ESR2 was associated with longer OS (HR 0.5, 95% CI 0.2–1, p = 0.048) and low expression of RB1 was independently associated with shorter OS (HR 1.9, 95% CI 1.1–3.2, p = 0.014). (4) Conclusions: AR, ESR, and TSG expression signatures, as well as ARV7, RB1, and ESR2 expression, have a prognostic value in mHSPC patients treated with ADT+DX.

The sex‐dependent role of the androgen receptor in glioblastoma: results of molecular analyses

We sought to analyse the androgen receptor (AR) in glioblastoma (GBM) due to the location of the AR gene on chromosome X, often reported with shorter survival and higher prevalence of GBM among males. Copy number (CN) and mRNA expression of AR were tested with droplet digital PCR in 91 fresh‐frozen GBM samples and 170 formalin‐fixed, paraffin‐embedded samples collected at Linköping University Hospital. The fresh‐frozen cohort was also subjected to pyrosequencing methylation analysis of 17 CpG sites in the AR promoter. Additionally, the gene expression of AR was analysed in the fresh‐frozen cohort and The Cancer Genome Atlas (TCGA) cohort of isocitrate dehydrogenase wild‐type primary GBM (135 females and 219 males). The association of AR expression and overall survival (OS) was tested with Kaplan–Meier log rank analysis after dichotomisation by maximally selected rank statistics. We found that AR CN alterations were more common in female GBM. AR gene expression correlated with methylation levels of different CpG sites in males and females but there was no difference in expression between sexes. Survival analysis of TCGA cohort revealed the opposite effect of AR overexpression on OS of males and females, with high AR expression correlating with shorter OS in females and longer OS in males. Additional gene set enrichment analysis showed that AR expression correlated with DNA repair response, especially in the male group. In summary, we found that high AR gene expression in GBM exhibits sex‐dependent effects on patient survival, which, for males, is linked to DNA repair response.

Abstract 507: Trop-2 expression in triple-negative breast cancer according to histological subtype: correlations with tumor infiltrating lymphocytes (TILs) and survival

Abstract Background: Trop-2 is a transmembrane calcium signal transducer highly expressed in multiple solid tumors including breast cancer. Limited data exist about associations between Trop-2 protein expression, clinicopathological characteristics and outcome in triple-negative breast cancers (TNBC). Given the approval of sacituzumab govitecan in unselected TNBC, its evaluation in other breast cancer settings and the emergence of other Trop-2 targeted antibody-drug conjugates, Trop-2 emerges as an important drug target in solid tumors. Methods: Trop-2-expression of patients with TNBC diagnosed between 2000-2017 at UZ Leuven was determined with IHC (ab227689, Abcam) (continuous and categorical variables, high 201-300, medium 100-200 and low &amp;lt;100 H-score). Associations of Trop-2-expression with age, BMI, germline BRCA status, tumor grade and size, histological subtype, lymphovascular invasion (LVI), presence of DCIS, nodal status, stromal TILs, androgen receptor (AR), standardized mitotic index, and outcome (invasive disease-free survival (iDFS), distant recurrence-free survival (D-RFi) and breast cancer-specific survival (BCSS)) were assessed. Associations with continuous variables were assessed by the Spearman correlation coefficient (ρ). Group differences were assessed by the Kruskal Wallis test for multiple groups or Mann-Whitney U test for two groups. Cox proportional hazards models were used to assess the association between Trop-2 expression and outcome. Results: Total of 658 patients with a median age at diagnosis of 53y (range 22-89y) were included, with median follow-up 9.6y. Low, medium and high Trop-2 expression was seen in 58.2%, 25.3% and 16.5% of cases. AR was positive in 25.5% of cases (10%-cutoff). Highest Trop-2 expression was observed in tumors with apocrine histology (median 180 score), and lowest expression in tumors with metaplastic histology (median 45 score) (p&amp;lt;.001). Higher Trop-2 expression was correlated with higher AR expression (10%-cutoff) (p=0.009), more presence of nodal involvement (p=0.016), associated DCIS (p=0.004) and LVI (p=0.006). Trop-2 expression was not associated with pathological complete response (pCR) observed in 22 of 64 patients treated with neoadjuvant chemotherapy (p=0.96). There was an iDFS events frequency of 199 out of 658 (30.2%). There was no correlation between Trop-2 expression and sTILs or outcome. Conclusion: In patients with TNBC in a large tertiary center, higher Trop-2 expression was correlated with apocrine histology, higher AR expression, more associated DCIS, more LVI and nodal involvement. There was no correlation between Trop-2 expression and sTILs or outcome. Citation Format: Hava Izci, Kevin Punie, Lise Waumans, Annouschka Laenen, Hans Wildiers, Freija Verdoodt, Christine Desmedt, Jan Ardui, Ann Smeets, Sileny Han, Ines Nevelsteen, Patrick Neven, Giuseppe Floris. Trop-2 expression in triple-negative breast cancer according to histological subtype: correlations with tumor infiltrating lymphocytes (TILs) and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 507.