Detection Of Androgen Receptor Splice Variant Research Articles

Simultaneous detection of tumor-derived mutations in DNA and RNA extracted from a single circulating tumor cell based on droplet digital polymerase chain reaction.

e15053 Background: Androgen receptor splice variant 7 (AR-V7) is a constitutively activated isoform of AR and has been associated with resistance to AR-targeted therapies and development of metastatic castration-resistant prostate cancer. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the appropriate drug, however, the detection of AR-V7 with tissues has limitations, such as the clinical challenge to poorly accessible tumor tissues before and after therapy. In addition, KRAS mutations serve as a prognostic biomarker for the administration of appropriate anti-cancer drugs in patients with various cancers, including non-small cell lung cancer (NSCLC). Here, we developed the process for the simultaneous detection of AR-V7 and KRAS mutation in CTCs using isolation with CytoGen’s Smart Biopsy™ System with droplet digital polymerase chain reaction (ddPCR) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Methods: ddPCR assays were performed to detect the KRAS G12C mutation using CTC gDNA and to evaluate the absolute levels of AR-V7 using CTC mRNA. Analytical validation was performed using NSCLC NCI-H358 and prostate cancer 22Rv1 cells (10 and 5 cells each), which harbor KRAS G12C and AR-V7, respectively, spiked into 5 mL of healthy blood to mimic CTC. CTCs were isolated using CytoGen's Smart Biopsy™ System. Oligo-dT magnetic beads were used to isolate mRNA from CTCs, and gDNA was isolated from the mRNA-depleted CTC lysates using the AllPrep DNA/mRNA Nano Kit. PCR and droplet reading for each assay were performed on the QX200 AutoDG Droplet Digital PCR System. Results: As a result of CTC KRAS G12C detection using the ddPCR™ KRAS G12/G13 Screening Kit, the mutated KRAS gene was successfully detected in as few as 5 NCI-H358 cells. In the results of CTC AR-V7 detection using the custom TaqMan ddPCR assay, transcripts (AR-V7 and HPRT1) were successfully detected even with only 5 22Rv1 cells. These results suggest that CytoGen's Smart Biopsy™ System could be applied to the simultaneous detection of DNA/RNA mutated genes in clinical NSCLC and prostate cancer patients with detection technology requiring high sensitivity and specificity. Conclusions: Accurately detecting multiple genetic mutations in CTCs is a challenging because CTCs are very rare in the blood. In this study, we developed the process to simultaneously extract and detect DNA mutations and RNA splicing variants in CTCs using CytoGen's Smart Biopsy™ System. This process, along with the results from our ongoing clinical trials, could provide information for the diagnosis and appropriate treatment of patients with various cancers, including NSCLC and prostate cancer.

Abstract 7505: Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays

Abstract Introduction: Androgen-receptor splice variant 7 (AR-V7) is constitutively activated isoform of AR and has been associated with resistance towards AR targeting therapies and development into mCRPC. Confirmation of AR-V7 expression is important step in prostate cancer diagnosis and determining the drug, but the detection of AR-V7 with tissues has limitations such as clinical challenge to poorly accessible tumor tissues before and after treatment. Here, we developed the method of AR-V7 detection in circulating tumor cells (CTC) after isolation with CytoGen’s Smart BiopsyTM CTC isolator by in vitro assays of qPCR and immunofluorescence (IF) with high sensitivity and specificity, overcoming the limitations of tissue biopsy. Method: qPCR and IF methods were developed for high sensitivity and specificity of AR-V7 detection. Sensitivity and specificity were tested using AR-V7 positive cells (VCaP and 22Rv1) and negative cells (PC-3 and Raji). Limit of detection (LOD) test of AR-V7 expression was performed with AR-V7 positive cells (200 cells to 50 cells, 3 points) mixed with Raji cells (1 × 10^5cells). Then, AR-V7 positive cells (200 cells to 50 cells, 3 points) were spiked into peripheral blood mononuclear cells (PBMC) of blood sample (5mL, healthy donor) to mimic CTC after isolation by CytoGen’s Smart BiopsyTM CTC isolator, subsequently AR-V7 expression was tested with qPCR and IF assay. Results: AR-V7 expression was clearly detected by qPCR and IF in the LOD test of AR-V7 positive cells mixed with Raji cells and was not detected at all in the negative controls. The similar clear results were also observed in the spiking test of AR-V7 positive cells into PBMC isolated by CytoGen’s Smart BiopsyTM CTC isolator, suggesting that clinical samples of prostate cancer could be applied with excellent sensitivity and specificity for AR-V7. Conclusions: AR-V7 expresses only a part of cancer tissue, so it is difficult to accurately diagnose with traditional histological assays. Detection of AR-V7 in intact live CTC isolated by CytoGen’s Smart BiopsyTM CTC isolator might overcome limitations of tissue biopsy. In this study, we developed the method to detect AR-V7 in CTC with high sensitivity and specificity with qPCR and IF assay, and presented the results of verifying AR-V7 expression by double check using two kinds of assays. We plan to conduct clinical validation of the developed method through additional study using patient samples. Citation Format: Hoin Kang, Jihyun Lee, Min Hyung Kim, Soee Kim, Hyoyong Kim, Jubi Lee, Hae Ung Lee, Hai-Chon Lee, Jung Won Kim. Detection validation of androgen-receptor splice variant 7 in circulating tumor cells of prostate cancer by double-check assays [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 7505.

1222P Detection of androgen-receptor splice variant 7 messenger RNA in circulating tumor cells of prostate cancer by in vitro assay

1222P Detection of androgen-receptor splice variant 7 messenger RNA in circulating tumor cells of prostate cancer by in vitro assay

Abstract 5139: Development of AR-V7 blood-based detection test by digital PCR in castration-resistant prostate cancer patients undergoing hormonal therapy

Abstract Introduction: Androgen receptor splice variant 7 (AR-V7) is found to be associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). This study aims to develop a minimally-invasive approach for the direct quantification of AR-V7 in blood and evaluates whether AR-V7 can act as a predictor of progression in CRPC patients undergoing hormonal therapy. Materials and Methods: A prostate cancer cell line (VCaP) was used to test the assay’s limit of detection. The detection of AR-V7 was then evaluated in 126 serial plasma samples and 77 serial platelet samples, prospectively collected from 30 patients with CRPC before and during hormonal therapy (abiraterone or enzalutamide) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital polymerase chain reaction (ddPCR). Results: RT-qPCR and ddPCR were first performed on VCaP to measure a range of 20 fg to 20 ng VCaP cDNA. ddPCR was able to detect down to 2 pg while RT-qPCR was only able to detect 200 pg for AR-V7 and total AR. RT-qPCR and ddPCR were then performed on the plasma samples of CRPC patients. Our results observed that RT-qPCR was unable to detect AR-V7 from these plasma samples, whereas ddPCR yielded a higher detection rate of 13%. Plasma with 22Rv1 and VCaP RNA spike-in revealed that RNase in plasma degraded spike-in RNA. We further detected AR-V7 in platelet samples, our results found that RT-qPCR was able to detect AR-V7 from 56% of the platelet samples and ddPCR yielded a much higher detection rate of 97%. Finally, we quantified the AR-V7 levels of 22 available platelet samples taken at the point closest to the patients’ progression time to evaluate the correlation of AR-V7 with the patient’s progression records. In receiver operating characteristic curve analysis, the area under the curve was 0.656. Conclusion: Our results showed that platelet samples had higher detection sensitivity than plasma samples, this might be due to the possibility of RNA degradation by RNase during processing in plasma samples. Performing RT-qPCR and ddPCR on platelet samples, a higher detection rate was observed in ddPCR when compared with RT-qPCR. We successfully develop a detection test to assess AR-V7 by highly sensitive ddPCR in the platelet of CRPC patients, with a high detection rate of 97%. We also reveal that there is a certain correlation between the ddPCR detection for AR-V7 in patients’ platelet samples with the patients’ progression status, indicating that AR-V7 may act as a potential predictor of progression in CRPC patients undergoing hormonal therapy. Citation Format: William C. Cho, King Y. Fung, Alvin H. Fong, Timothy T. Chan. Development of AR-V7 blood-based detection test by digital PCR in castration-resistant prostate cancer patients undergoing hormonal therapy [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 5139.

A phase III trial of docetaxel versus docetaxel and radium-223 (Ra-223) in patients with metastatic castration-resistant prostate cancer (mCRPC): DORA.

TPS5091 Background: Ra-223, a bone-targeted alpha therapy, prolongs survival in patients (pts) with symptomatic mCRPC to bone. Docetaxel targets microtubule trafficking improving survival in the mCRPC and metastatic hormone-sensitive settings. We hypothesized that simultaneously targeting the tumor and bone compartment yields superior outcomes than targeting either alone. We previously determined the dose and schedule of co-administering Ra-223 + docetaxel in a randomized phase I/IIa trial. The combination appeared to have improved declines in prostate specific antigen (PSA) and bone markers, delayed PSA progression, and was better tolerated (with adjusted dose/schedule) relative to standard docetaxel alone. We are now conducting a phase III study to determine the clinical benefit of the regimen. Methods: Randomization (1:1) of 738 men with mCRPC to docetaxel or docetaxel + Ra-223 is planned with a projected hazard ratio for treatment effect (15 vs 20 months median survival) of 0.75. Pts with ≥2 bone lesions and progression by Prostate Cancer Working Group 3 criteria are eligible. Other key inclusion criteria are an Eastern Cooperative Oncology Group performance status of 0–1 and normal organ function. Key exclusion criteria are: use of anticancer therapy ≤4 weeks (wks) before randomization and use of bone-seeking radiopharmaceuticals or chemotherapy in the castration-resistant setting, and bulky visceral metastases (≥3 lung and/or liver or a lesion ≥2 cm in the previous 8 wks). Subjects receive docetaxel 75 mg/m2 IV q3w for 10 doses or docetaxel 60 mg/m2 IV q3w for 10 doses + Ra-223 55 kBq/kg IV q6w for 6 doses. The primary endpoint is overall survival. Secondary and exploratory endpoints include: radiographic progression-free survival, symptomatic skeletal event-free survival, safety, markers of bone metabolism, alterations in circulating tumor cells and DNA, detection of androgen-receptor splice variant 7, changes in automated bone scan index (aBSI), and assessment of patient-reported outcome instruments (FACT-P, Brief Pain Inventory, Brief Fatigue Inventory). The study is open at 32 sites in the US and Netherlands and has 170 subjects enrolled. The study is sponsored by Memorial Sloan Kettering Cancer Center, and managed by the Prostate Cancer Clinical Trials Consortium. Clinical trial information: NCT03574571.

A phase III trial of docetaxel versus docetaxel and radium-223 (Ra-223) in patients with metastatic castration-resistant prostate cancer (mCRPC): DORA.

TPS5594 Background: Ra-223, a bone-targeted alpha therapy, prolongs survival in patients (pts) with symptomatic mCRPC to bone. Docetaxel targets microtubule trafficking improving survival in the mCRPC and metastatic hormone-sensitive settings. We hypothesized that simultaneously targeting the tumor and bone compartment yields superior outcomes than targeting either alone. We previously determined the dose and schedule of co-administering Ra-223 + docetaxel in a randomized phase I/IIa trial. The combination appeared to have improved declines in prostate specific antigen (PSA) and bone markers, delayed PSA progression, and was better tolerated (with adjusted dose/schedule) relative to standard docetaxel alone. We are now conducting a phase III study to determine the clinical benefit of the regimen. Methods: Randomization (1:1) of 738 men with mCRPC to docetaxel or docetaxel + Ra-223 is planned with a projected hazard ratio for treatment effect (15 vs 20 months median survival) of 0.75. Pts with ≥2 bone lesions and progression by Prostate Cancer Working Group 3 criteria are eligible. Other key inclusion criteria are an Eastern Cooperative Oncology Group performance status of 0-1 and normal organ function. Key exclusion criteria are: use of anticancer therapy ≤4 weeks (wks) before randomization and use of bone-seeking radiopharmaceuticals or chemotherapy in the castration-resistant setting, and bulky visceral metastases (≥3 lung and/or liver or a lesion ≥2 cm in the previous 8 wks). Subjects receive docetaxel 75 mg/m2 IV q3w for 10 doses or docetaxel 60 mg/m2 IV q3w for 10 doses + Ra-223 55 kBq/kg IV q6w for 6 doses. The primary endpoint is overall survival. Secondary and exploratory endpoints include: radiographic progression-free survival, symptomatic skeletal event-free survival, safety, markers of bone metabolism, alterations in circulating tumor cells and DNA, detection of androgen-receptor splice variant 7, changes in automated bone scan index (aBSI), and assessment of patient-reported outcome instruments (FACT-P, Brief Pain Inventory, Brief Fatigue Inventory). The study is open at 25 sites in the US and Netherlands, sponsored by Memorial Sloan Kettering Cancer Center, and managed by the Prostate Cancer Clinical Trials Consortium. Clinical trial information: NCT03574571 .

Using In Situ Padlock Probe Technology to Detect mRNA Splice Variants in Tumor Cells.

Advanced prostate cancer (PC) patients commonly receive anti-hormonal drugs targeting the androgen receptor (AR) signaling pathways. However, almost all patients acquire therapy resistance that can be caused by AR amplification or expression of AR splice variant 7 (AR-V7). Therefore, AR-V7 and AR expression are potential biomarkers for early detection of therapy resistance. Here, we present our padlock probe (PLP)-based approach for the in situ detection of AR full length, AR-V7, and prostate-specific transcripts in PC cell lines, which is applicable for circulating tumor cells (CTCs) isolated from cancer patients. First, PC cell lines are seeded on glass slides. Then, cDNA is created using target-specific reverse transcription primers. PLPs are hybridized to the cDNA and ligated to form circular single-stranded DNA molecules. The PLP sequence is ligated and amplified by rolling circle amplification and the resulting rolling circle products can be detected using fluorescently labeled probes. Quantification can be automated using the image analysis software CellProfiler.

Abstract 1335: Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer

Abstract Background: The androgen receptor (AR) is a clinically important driver in prostate cancer. In metastatic castration resistant prostate cancer (mCRPC), increased expression of the ligand-independent AR variant 7 (AR-v7) is a biomarker of hormonal therapy resistance. However, the prevalence and clinical importance of AR-v7 in non-metastatic CRPC (nmCRPC) is not yet known. Low circulating tumor cell (CTC) frequencies in these patients make emergence of AR-v7 during anti-androgen therapy difficult to study. We report blood-based detection of AR-v7 using digital droplet PCR (ddPCR) in nmCRPC patients enrolled in the SPARTAN trial, a randomized phase 3 study testing ADT vs apalutamide (APA)+ADT. Method: To investigate simultaneous and quantitative expression of AR-v7 and AR, we utilized ddPCR to measure individual mRNA transcripts in blood samples collected in PAXgene tubes. AR-v7 positivity was calculated as the normalized fraction of AR-v7 vs total AR transcripts (AR-v7/AR). Normalized AR-v7/AR frequency in healthy volunteers (HV) and mCRPC was measured to determine an expression cutoff to separate normal and prostate cancer blood samples. The ddPCR AR-v7 biomarker assay was then used to measure AR-v7 expression in ADT (N=47) and APA+ADT (N=53) SPARTAN samples taken at time of study initiation and correlated with clinical outcome. Results: By setting a cutoff at 0.3 AR-v7/AR normalized fraction we could differentiate HV from mCRPC patients. In nmCRPC, mean AR-v7 and AR-FL expression were calculated as 1.2 and 349.3 transcripts, respectively. The 0.3 AR-v7/AR normalized fraction cutoff could not differentiate AR-v7 expression between HV and nmCRPC. Using this assay, we detected AR-v7 transcripts in 47% of nmCRPC SPARTAN patients analyzed. However, results of AR-v7 expression as a continuous and discretized variable were inconclusive when correlated with clinical outcome. Conclusion: This study reports ddPCR-based detection of whole blood mRNA as a sensitive assay to detect simultaneously low and high expressing AR transcripts in nmCRPC. Our technical analysis demonstrates that unlike in mCRPC, low level transcript counts of AR-v7 in nmCRPC may not distinguish expression from baseline in healthy patients. Data from this limited cohort suggest that while AR-v7 is detected in 47% of patients, a higher threshold of expression may be biologically important for driving treatment resistance. Further analysis of this assay in mCRPC and APA refractory samples sequenced with other therapies are needed to confirm the clinical and biological utility of AR-v7 detection by ddPCR assay and inform disease continuum management. Citation Format: Mel Pilar Espaillat, Yashoda Rajpurohit, Mike Gormley, Denis Smirnov, Ian McCaffery, Angela Lopez-Gitlitz, Deborah Ricci, Shibu Thomas. Digital droplet PCR (ddPCR)-based detection of androgen receptor splice variant 7 (AR-v7) in non-metastatic castration resistant prostate cancer [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 1335.

A phase III trial of docetaxel versus docetaxel and radium-223 (Ra-223) in patients with metastatic castration-resistant prostate cancer (mCRPC): DORA.

TPS348 Background: Ra-223, a bone-targeted alpha therapy, is a well-tolerated treatment option that prolongs survival in patients (pts) with symptomatic mCRPC to bone. Docetaxel targets microtubule trafficking improving survival in the mCRPC and metastatic hormone-sensitive settings. We hypothesized that simultaneously targeting the tumor and bone compartment yields superior outcomes than targeting either alone. We previously determined the dose and schedule of co-administering Ra-223 + docetaxel in a randomized phase I/IIa trial. The combination appeared to have superior declines in prostate specific antigen (PSA) and bone markers, delayed PSA progression, and was better tolerated (with adjusted dose/schedule) relative to standard docetaxel alone. We are now conducting a phase III study. Methods: DORA is an open-label investigator-initiated phase III study sponsored by Memorial Sloan Kettering Cancer Center and managed by the Prostate Cancer Clinical Trials Consortium. Pts with mCRPC are randomized (1:1) to docetaxel or docetaxel + Ra-223. Pts with ≥2 bone lesions and progression by Prostate Cancer Working Group 3 criteria are eligible. Other key inclusion criteria are an Eastern Cooperative Oncology Group performance status of 0–1 and normal organ function. Key exclusion criteria are: use of anticancer therapy ≤4 weeks (wks) before randomization and use of bone-seeking radiopharmaceuticals or chemotherapy in the castration-resistant setting, and bulky visceral metastases (≥3 lung and/or liver or a lesion ≥2 cm in the previous 8 wks). Pts will receive docetaxel 75 mg/m2 IV q3w for 10 doses with oral prednisone 5 mg b.i.d. or docetaxel 60 mg/m2 IV q3w for 10 doses with oral prednisone 5 mg b.i.d. + Ra-223 55 kBq/kg IV q6w for 6 injections. The primary endpoint is overall survival. Secondary and exploratory endpoints include: radiographic progression-free survival, symptomatic skeletal event-free survival, safety, markers of bone metabolism, alterations in circulating tumor cells and DNA, detection of androgen-receptor splice variant 7 and changes in automated bone scan index. Enrollment of 738 pts is expected. The study is conducted in the US and Netherlands. Clinical trial information: NCT03574571.

Detection of androgen receptor (AR) and AR-V7 in small cell prostate carcinoma: Diagnostic and therapeutic implications

ObjectiveSmall cell prostate carcinoma (SCPC) is a rare and highly malignant subtype of prostate cancer. SCPC frequently lacks androgen receptor (AR) and prostate-specific antigen (PSA) expression, and often responds poorly to androgen deprivation therapy (ADT). AR splice variant-7 (AR-V7) is a truncated AR protein implicated in resistance to AR-targeting therapies. AR-V7 expression in castration-resistant prostate cancers has been evaluated extensively, and blood-based detection of AR-V7 has been associated with lack of response to abiraterone and enzalutamide. However, whether AR-V7 is expressed in SCPC is not known. MethodsUsing validated antibodies, we performed immunohistochemistry (IHC) assay for the full-length AR (AR-FL) and (AR-V7) on post-ADT surgical SCPC specimens. ResultsSeventy-five percent (9/12) of the specimens showed positive staining for the AR-FL 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-FL/AR-V7 staining. All specimens positive for AR-V7 were also positive for AR-FL. ConclusionThe study findings support the existence of measurable AR-FL and AR-V7 proteins in SCPC specimens. The results also have implications in detection of AR-V7 in specimens obtained through systemic sampling approaches such as circulating tumor cells. A positive AR-V7 finding by blood-based tests is not impossible in patients with SCPC who often demonstrate low PSA values.

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 909: Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9

Abstract Background: Androgen receptor (AR) is the key drug target in prostate cancer. AR splice variant-7 (AR-V7) is the most relevant AR variants implicated in castration-resistant prostate cancer (CRPC). Detection of AR-V7 in both liquid biopsies and tissue biopsies predicts poorer response to novel AR-targeting drugs. However, because AR-V7 always coexists with the full-length AR (AR-FL), the functional importance of AR-V7 needs to be dissected in the context of AR-FL. Lack of relevant cell line models presented a hurdle for detailed dissection of the role for AR-V7. It is essential to develop cell lines that express endogenous AR-V7 at a level comparable to clinical specimens, with or without AR-FL, to facilitate functional studies. The goal of this study is to develop and characterize LNCaP95 lines following specific and complete knock-out of AR molecules by CRISPR-Cas9 system. Methods: Different AR isoforms were knocked-out in parental LNCaP95 cells by CRISPR-Cas9. AR-FL was knocked-out through sgRNAs targeting AR exon5 or exon6. AR isoforms with functional N-terminal domain (total AR) were knocked-out through sgRNA targeting AR exon1. Complete knockout of AR-FL and total AR was achieved in LNCaP95 cells. The on-target editing of AR genomic regions were verified by Sanger Sequencing. Results: Complete knockout of AR-FL protein and total AR protein was confirmed in two clones carrying homologous indels, ARfl-KO#1/2 and AR-KO#1/2. Antibodies against different AR domains were used to differentiate expression of different AR isoforms, by Western blot and immunohistochemistry (IHC). Cell proliferation was not affected by androgen in AR-FL or total AR knockout cells, as shown in cell growth and clonogenic assays. The expression of AR-target genes such as prostate-specific antigen (PSA) were analyzed by realtime-PCR. PSA was expressed at similar level compared to parental cells in AR-FL knockout cells, which was totally lost in total AR knockout cells. Interestingly, total AR knockout cells are more resistant to ionizing radiation and PARP inhibitor olaparib compared to parental cell line. Conclusion: We have successfully developed the first prostate cancer cell line expressing endogenous AR-V7 at a level comparable to clinical specimens without AR-FL, allowing dissection of the functions mediated by AR-FL and AR-V7. Initial functional characterization suggested AR-V7 maintained the baseline level of AR signaling activity, which was not dependent on androgen. The chromatin-binding pattern of AR-V7 in the absence of AR-FL is under investigation. We have also developed a novel total AR knockout cell model from the AR-positive parental cell line. This model suggested prostate cancer cells were able to survive in an AR-null manner, although loss of AR significantly inhibited cell proliferation. Preliminary studies have found loss of total AR confers resistance to ionizing radiation and PARP inhibition. Citation Format: Yezi Zhu, Pei Zhao, Changxue Lu, Shihua Chen, Jun Luo. Development and characterization of androgen receptor knockout prostate cancer cell lines by CRISPR-Cas9 [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 909.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

AR-V7 in Asian patients with metastatic CRPC: A multinational, multicentre study.

TPS401 Background: The detection of androgen-receptor splice variant 7 (AR-V7) mRNA transcript in circulating tumor cells (CTCs) of progressive metastatic castration-resistant prostate cancer (mCRPC) patients is associated with resistance to abiraterone and enzalutamide. This finding was validated by other studies that found no association between the presence of AR-V7 transcripts and response to both docetaxel and cabazitaxel. The results suggest that AR-V7 could represent a biomarker to guide treatment selection in mCRPC. However, data in Asian population is limited. This multinational study aims to prospectively investigate the incidence and prevalence of ARV7 in Asian mCRPC patients. This is also the first study to evaluate 3 different detection platforms and help provide guidance on which will be the best platform to carry forward in terms of clinical utility. Methods: 101 Asian subjects with histologically confirmed mCRPC whom are going to be treated with AR-directed therapies (abiraterone, enzalutamide) or taxane chemotherapy (docetaxel) will be enrolled from Singapore, Malaysia, Thailand, Hong Kong and Japan. Subjects will be excluded if they plan to receive additional concurrent anticancer therapies. Peripheral-blood samples will be obtained at baseline and at the time of progression. We will use 3 methods to analyse the samples: (i) CTC enrichment platform followed by automated immunofluorescent staining for DNA, cytokeratins, CD45 (lymphocyte common antigen), and AR-V7; (ii) CTC enrichment platform followed by reverse-transcription polymerase chain reaction analysis; and (iii) the AdnaTest platform for molecular characterization of CTCs. Subjects will be followed up to 2 years. The clinical outcomes of AR-V7-positive and AR-V7-negative patients will be compared. PSA response rates will be compared using the Fisher exact test. Time-to-event outcomes (PFS, OS) will be evaluated using Kaplan-Meier analysis, and survival time differences will be compared using log-rank test. Cox regression analyses will be used to assess the effect of AR-V7 status in predicting clinical outcomes. The study is in progress; 32 of 101 planned patients have been enrolled at the end of September 2017.

AR-V7 detection by a novel junction-specific RNA in situ hybridization (RISH) test.

191 Background: Previous studies have reported an association between blood-based detection of androgen receptor splice variant-7 (AR-V7) and resistance to abiraterone/enzalutamide in men with metastatic castration-resistant prostate cancer (mCRPC). Further characterization of AR-V7 and its clinical correlates are necessary but depend on the development of methods that allow for reliable and specific measurement of AR-V7. In this study, we developed and evaluated a novel junction-specific AR-V7 RNA in situ hybridization (RISH) test. Methods: We developed a novel RISH assay targeting single exon-exon junction, allowing for highly-specific detection of mature cytoplasmic AR-FL and AR-V7 mRNA in native cell and tissue environment. We compared quantitative RISH AR-V7 results generated from an automated scoring system with AR-V7 levels quantified by RT-PCR in a panel of cell lines. Using the validated assay, we quantified AR-V7 signals in 58 prostate tumor biopsies. We conducted preliminary evaluation of the clinical correlates of AR-V7 detected. Results: The novel AR-V7 RISH test specifically detected mature cytoplasmic AR-V7 mRNA and did not detect nuclear pre-mRNA. AR-V7 RISH results were highly concordant with RT-PCR results. We generated quantitative AR-FL and AR-V7 data from 93.1% of the clinical specimens examined (54 of 58). Positive AR-V7 signals were detected in 23 specimens, all of which also co-expressed AR-FL. The median AR-V7/AR-FL ratio was 4.6%. Positive detection of AR-V7 was significantly correlated with prior treatment with second-line AR targeting agents and serum PSA ( p <0.05), but not with any other baseline clinical variables. Notably, all castration-sensitive prostate cancer specimens (CSPC) (n = 9) were AR-V7 negative by this method. Association between AR-V7 and treatment outcome has yet to be fully evaluated. Conclusions: We demonstrate for the first time a highly specific and quantifiable AR-V7 RISH test for detection of clinically significant levels of AR-V7 mRNA in prostate tissue specimens. This test may be applied to liquid biopsy specimens to expand the utility. Prospective studies can be designed to evaluate the potential utility of this novel test in drug development and patient management.

Development of AR-V7 as a putative treatment selection marker for metastatic castration-resistant prostate cancer.

Prostate cancer cells demonstrate a remarkable “addiction” to androgen receptor (AR) signaling in all stages of disease progression. As such, suppression of AR signaling remains the therapeutic goal in systemic treatment of prostate cancer. A number of molecular alterations arise in patients treated with AR-directed therapies. These molecular alterations may indicate the emergence of treatment resistance and may be targeted for the development of novel agents for prostate cancer. The presence of functional androgen receptor splice variants may represent a potential explanation for resistance to abiraterone and enzalutamide, newer AR-directed agents developed to treat metastatic castration-resistant prostate cancer (mCRPC). In the last 8 years, many androgen receptor splice variants have been identified and characterized. Among these, androgen receptor splice variant-7 (AR-V7) has been investigated extensively. In AR-V7, the entire COOH-terminal ligand-binding domain of the canonical AR is truncated and replaced with a variant-specific peptide of 16 amino acids. Functionally, AR-V7 is capable of mediating constitutive nuclear localization and androgen receptor signaling in the absence of androgens, or in the presence of enzalutamide. In this review, we will focus on clinical translational studies involving detection/measurement of AR-V7. Methods have been developed to detect AR-V7 in clinical mCRPC specimens. AR-V7 can be reliably measured in both tissue and circulating tumor cells derived from mCRPC patients, making it possible to conduct both cross-sectional and longitudinal clinical correlative studies. Current evidence derived from studies focusing on detection of AR-V7 in mCRPC support its potential clinical utility as a treatment selection marker.

Label-Free Detection of AR-V7 mRNA in Prostate Cancer Using Yb2Ti2O7 –Based Electrolyte-Insulator-Semiconductor Biosensors

We developed an Yb2Ti2O7-electrolyte-insulator-semiconductor (EIS) biosensor that can rapidly discriminate clinical statuses of prostate cancer (PCa) by detecting the existence of androgen-receptor splice variant 7 (AR-V7) messenger RNA. The structural feature of YbTixOy sensing membranes anneal to Si substrates through reactive cosputtering with different annealing temperatures investigated. Results showed annealed at 900°C exhibited higher sensitivity of 61.11 mV/pH, lower hysteresis voltage of 1.6 mV, and smaller drift rate of 0.16 mV/hr. This attributed to the well-crystallized Yb2Ti2O7 structure, detected using X-ray diffraction and X-ray photoelectron spectroscopy. We then compared two different methods, encapsulating or covalently bonding of the probe, to detect existence of AR-V7 in PCa. After hybridization of the probe with the target cDNA sequence, the covalent binding of AR-V7 probe method exhibited a larger VREF shift in the capacitance-voltage curves for the detection of AR-V7. Furthermore, highly reliable and stable results (>95% repetition rate) also could be observed when compared to that encapsulated probe on the EIS. Our data demonstrated this AR-V7 probe-based Yb2Ti2O7-EIS biosensor is a rapid, simple, label-free, economical, and accurate diagnostic device, and provide fast identification of resistant statuses in circulating tumor cells of PCa, essential for clinicians to make defining decisions.

AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer

BackgroundThe androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone.MethodsWe used a quantitative reverse-transcriptase–polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression–free survival), clinical or radiographic progression–free survival, and overall survival.ResultsA total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 53%, P=0.004) and shorter PSA progression–free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression–free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7–positive patients had lower PSA response rates than AR-V7–negative patients (0% vs. 68%, P=0.004) and shorter PSA progression–free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression–free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA.ConclusionsDetection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.)

Ar-V7 Splice Variant and Resistance to Enzalutamide and Abiraterone in Men with Metastatic Castration-Resistant Prostate Cancer (Mcrpc): Overall Survival Results

ABSTRACT Aim: Androgen receptor splice variant-7 (AR-V7) is a truncated form of the AR that lacks the ligand-binding domain, the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We previously reported (ASCO 2014; abstract 5001) that detection of AR-V7 in circulating tumor cells (CTCs) from men with mCRPC was associated with resistance to enzalutamide and abiraterone: PSA responses and progression-free survival (PFS) were inferior in AR-V7–positive men treated with both agents. Here, we present overall survival (OS) data from this study. Methods: We used qRT-PCR to interrogate CTCs for the presence or absence of AR-V7 from prospectively enrolled patients with mCRPC starting treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status and OS. Multivariable Cox regression analyses were conducted to determine the independent effect of AR-V7 status on OS. We also performed a combined analysis of OS using data from all patients. Results: 31 enzalutamide-treated men and 31 abiraterone-treated men were enrolled, of which 12/31 (38.7%) and 6/31 (19.4%) had detectable AR-V7 in pretreatment CTC samples, respectively. In men receiving enzalutamide [n = 31], AR-V7–positive patients demonstrated inferior OS compared to AR-V7–negative patients (HR 6.9, 95% CI 1.7–28.1, log-rank P = 0.002). Similarly, in men receiving abiraterone [n = 31], AR-V7–positive patients had inferior OS (HR 12.7, 95% CI 1.3–125.3, log-rank P = 0.006). In the combined analysis [n = 62], the negative prognostic impact of AR-V7 was maintained (HR 8.3, 95% CI 2.5–27.4, log-rank P Conclusions: Detection of AR-V7 in CTCs from men with mCRPC is associated with resistance to both enzalutamide and abiraterone, as evidenced by inferior PSA responses, PFS and OS. AR-V7 status may be used as a non-invasive biomarker to predict resistance to AR-targeting agents, facilitate treatment selection, and fuel the development AR N-terminal–domain inhibitors. Disclosure: E.S. Antonarakis is a paid consultant/advisor for Janssen, Sanofi and Dendreon. He receives research funding from Janssen, Johnson & Johnson, Sanofi, Dendreon, Aragon, Exelixis, Millennium, Genentech and Novartis. All other authors have declared no conflicts of interest.