Loss Of Rb Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

In This Issue

In This Issue

MP33-01 METASTATIC CASTRATION-RESISTANT PROSTATE CANCER REMAINS DEPENDENT ON ONCOGENIC DRIVERS IN PRIMARY TUMORS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I (MP33)1 Sep 2021MP33-01 METASTATIC CASTRATION-RESISTANT PROSTATE CANCER REMAINS DEPENDENT ON ONCOGENIC DRIVERS IN PRIMARY TUMORS Seiji Arai, David Einstein, Kazuhiro Suzuki, Steven Balk, Adam Sowalsky, and Joshua Russo Seiji AraiSeiji Arai More articles by this author , David EinsteinDavid Einstein More articles by this author , Kazuhiro SuzukiKazuhiro Suzuki More articles by this author , Steven BalkSteven Balk More articles by this author , Adam SowalskyAdam Sowalsky More articles by this author , and Joshua RussoJoshua Russo More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002042.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Localized prostate cancer (PC) can be curable, but metastatic recurrence is unfortunately common. Metastatic PC is initially sensitive to androgen receptor inhibition, but eventually becomes castration-resistant (mCRPC). Early use of more intensive therapies targeting androgen receptor and other oncogenic drivers may be more effective than in advanced mCRPC. However, as oncogenic alterations found in mCRPC are less frequent in treatment-naïve primary PC, analysis of primary tumors may not reveal targetable metastatic drivers that are subclonal in the primary tumor or acquired at metastatic sites. METHODS: We analyzed PC samples spanning one patient’s clinical course: diagnostic biopsies, pre-/post-enzalutamide metastatic biopsies, and rapid autopsy samples including a patient-derived xenograft (PDX). RESULTS: Consistent with mCRPC being driven by non-truncal alterations, we identified RB1 loss throughout mCRPC samples, but not in the primary tumor. However, we identified a truncal EGFR-activating mutation in the primary that was maintained in mCRPC samples and the PDX, and showed that the PDX responded to the EGFR inhibitor. CONCLUSIONS: Truncal alterations can drive advanced mCRPC, and suggest that earlier detection and targeting of these truncal alterations may be effective at halting disease progression. Source of Funding: Congressionally Directed Medical Research Program (CDMRP), Prostate Cancer Research Program (Physician Research Award W81XWH-17-1-0350, Early Investigator Award W81XWH-18-1-0531, Idea Development Awards W81XWH-18-1-0379 and W81XWH-20-1-0925), the Prostate Cancer Foundation (Young Investigator Award 18YOUN24), the Intramural Research Program of the NIH, National Cancer Institute (NIH P01 - CA163227, SPORE in Prostate Cancer P50 – CA090281, and NIH P20 – CA233255) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e606-e606 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Seiji Arai More articles by this author David Einstein More articles by this author Kazuhiro Suzuki More articles by this author Steven Balk More articles by this author Adam Sowalsky More articles by this author Joshua Russo More articles by this author Expand All Advertisement Loading ...

Circulating Cell-Free DNA-Based Detection of Tumor Suppressor Gene Copy Number Loss and Its Clinical Implication in Metastatic Prostate Cancer.

Current liquid biopsy assays lack sufficient sensitivity to detect copy number loss, which limits the interrogation of critical tumor suppressor gene deletions during cancer progression and treatment. Here we describe a liquid biopsy assay with improved sensitivity for detection of copy number loss in blood samples with low levels of circulating tumor DNA, and demonstrate its utility by profiling PTEN, RB1, and TP53 genetic loss in metastatic prostate cancer patients.

Neuroendocrine Neoplasms of the Larynx: A Clinicopathologic Analysis of 27 Neuroendocrine Tumors and Neuroendocrine Carcinomas.

Laryngeal neuroendocrine neoplasms (NENs) are rare and heterogeneous, encompassing well-differentiated neuroendocrine tumors (NETs; grade 1, 2, and 3), neuroendocrine carcinomas (NECs, small cell and large cell types), and mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN). We aimed to study the clinicopathologic spectrum of these neoplasms. A retrospective review of all primary laryngeal NENs diagnosed from 2005 to 2017 was undertaken. Mitotic index was divided into < 2, ≥ 2-10, and > 10 mitoses/2 mm2, with a Ki-67 labelling index of < 2%, ≥ 2-20%, and > 20% for the NET grade 1, 2 and 3 categories, respectively. A total of 27 patients were included. The median age at presentation was 60years; the male-to-female ratio was 8:1. Supraglottis (n = 22) was the most frequently affected subsite. There were 9 NETs grade 2 (G2), and 18 NECs cases. There were no NET grade 1 or 3 cases in our cohort. Among the NETs G2, the morphology was epithelioid (2), plasmacytoid (3), clear (2), oncocytic (1), and rhabdoid (1). Unique 'glomeruloid structures' (n = 5), calcification (n = 3), lymphoid aggregates (n = 5), intranuclear inclusions (n = 2), hyaline globules (n = 3), and Leisegang rings (n = 2) were identified. NECs comprised 16 small cell neuroendocrine carcinoma and 2 large cell neuroendocrine carcinoma. On immunohistochemistry, tumor cells expressed AE1/AE3 (86%), synaptophysin (100%), chromogranin (100%), INSM1 (100%), calcitonin (33.3%). In the NEC group, p53 aberrant expression (87.5%), Retinoblastoma (Rb) loss (88.2%), and diffuse p16 immunoreactivity (66.7%) were additionally observed. Lymph-node metastasis was detected in 62.5% and 85.7%, while distant metastasis in 55.6% and 76.9%, respectively in NET G2 and NEC. Laryngeal NENs are aggressive neoplasms with a high rate of nodal and distant metastasis. Awareness of the wide pathologic spectrum of laryngeal NENs and appropriate use of IHC is needed to render an accurate diagnosis. Ki67 assessment is strongly recommended for laryngeal NEN prognostication.

Circulating tumor cell heterogeneity in neuroendocrine prostate cancer by single cell copy number analysis

Neuroendocrine prostate cancer is an aggressive variant of prostate cancer that may arise de novo or develop from pre-existing prostate adenocarcinoma as a mechanism of treatment resistance. The combined loss of tumor suppressors RB1, TP53, and PTEN are frequent in NEPC but also present in a subset of prostate adenocarcinomas. Most clinical and preclinical studies support a trans-differentiation process, whereby NEPC arises clonally from a prostate adenocarcinoma precursor during the course of treatment resistance. Here we highlight a case of NEPC with significant intra-patient heterogeneity observed across metastases. We further demonstrate how single-cell genomic analysis of circulating tumor cells combined with a phenotypic evaluation of cellular diversity can be considered as a window into tumor heterogeneity in patients with advanced prostate cancer.

Abstract 2509: RB1-loss in castration-resistant prostate cancer reprograms androgen receptor signaling and confers vulnerability to LSD1 inhibition

Abstract Background: Retinoblastoma protein (Rb) functions to repress the transcription activity of E2Fs by forming an Rb-E2F repressor complex and the hyperphosphorylation of Rb during G1/S cell cycle transition results in its disassociation with E2Fs, thus inducing E2F-dependent transcription of DNA replication and cell cycle. Genomic RB1-loss is frequently occurred in castration-resistant prostate cancer (CRPC) (~10-15%) and is significantly associated with poor clinical outcomes. RB1-loss is also more frequently found in CRPC with neuroendocrine features (CRPC-NE) (~45-50%). However, it remains unclear on the global activities of Rb-E2F repressor complex functions in modulating transcriptional networks in CRPC. In this study, we performed a comprehensive analysis to functionally characterize the activities of Rb-E2F in multiple CRPC models and aimed to identify vulnerabilities in CRPC with RB1-loss. Methods: C4-2 cells (PTEN-loss, p53 positive) stably infected with doxycycline-inducible lentiviral shRNA against RB1 were generated. VCaP cells (PTEN positive, p53 R248W mut) that transfected with siRNAs against RB1 (siRB) were also examined. The chromatin landscape of Rb-E2F complex was determined by Rb ChIP-seq. Rb-E2F transcriptome was obtained from RNA-seq analysis in the C4-2 stable line treated with/out doxycycline and VCaP cells with siRB. An integrated analysis combines the ChIP-seq and RNA-seq data was also performed to characterize the direct targets of Rb-E2F. Furthermore, we have also assessed the effect of RB1 loss on AR transcriptional program using AR ChIP-seq and RNA-seq in cells treated with/out DHT. Results: Using an integrated cistromic and transcriptomic analysis, we have characterized Rb-E2F activities in multiple CRPC models by identifying the Rb-E2F directly regulated genes. Our results revealed that Rb-E2F has distinct binding sites and targets in TP53-mutated CRPC, and that the expression of Rb-E2F directly repressed genes is increased in the neuroendocrine (NE) subtype of CRPC. Furthermore, we have found that loss of Rb expression redistributes the chromatin binding of androgen receptor (AR), leading to the transcriptional activation of a subset of genes that are increased in CRPC-NE. Remarkably, we have found that the chromatin binding of E2F1 is regulated by an epigenetic factor LSD1/KDM1A (a lysine-specific demethylase that demethylates histone and non-histone proteins) and loss of Rb sensitizes the E2F signaling to the regulation by LSD1. Importantly, LSD1 inhibitor treatment was more effective in CRPC xenograft tumors with RB silencing. Conclusion: Taken together, these results characterized the direct transcriptomic changes in RB1-loss CRPC, indicated a reprogramming of AR signaling by loss of RB1, and suggested LSD1 as a potential therapeutic target in RB1-loss CRPC or CRPC-NE by modulating chromatin binding of E2Fs. Citation Format: Wanting Han, Mingyu Liu, Dong Han, Muqing Li, Anthia A. Toure, Susan C. Patalano-Salsman, Jill A. Macoska, Shuai Gao, Changmeng Cai. RB1-loss in castration-resistant prostate cancer reprograms androgen receptor signaling and confers vulnerability to LSD1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2509.

Abstract 1326: Second generation androgen receptor signaling inhibitor resistant neuroendocrine like prostate cancer: A mechanistic study

Abstract Background:Failure of castration resistant prostate cancer patients to Androgen Receptor Signaling Inhibitor (ARSI) poses a major challenge in terms of therapeutic development. The heterogeneous nature of prostate cancer makes it difficult to stratify the treatment modalities. A multitude of variable pathological and genetic alterations induced by systemic treatment adds further to the therapeutic complexity. Upon continued exposure to enzalutamide (ARSI), nearly 15-20% of castration resistant patients advance towards a highly aggressive neuroendocrine like phenotype with concomitant functional loss of RB1 and TP53 and persistent AR expression in some. Elucidation of the underlying mechanism of resistance among this aggressive subset of patients is of utmost importance to clinically stratify the treatment options and target these advanced neuroendocrine like patients, who currently lack specific treatment approaches. The following study aims to identify the molecular signature responsible for driving this enzalutamide resistant neuroendocrine like development. Objective:The primary objective of the study is to identify the molecular mechanism behind ARSI induced neuroendocrine like Prostate cancer development. Methods:We have developed two cell line model systems. C4-2 has been genetically modified to develop a simultaneous genetic loss of RB1 and TP53 (DKD). C4-2B has been kept on continued exposure to Enzalutamide to develop Enzalutamide Resistant (C4-2BER). C4-2BER maintains AR expression while DKD loses AR expression, resembling different stages in neuroendocrine like development. Using Western Blot and RT PCR analysis the neuroendocrine like background of these resistant cell line models has been characterized. We performed an RNA-Seq with C4-2B and its resistant model C4-2BER and further downstream analysis through gene ontology and GSEA were performed. Results:Model system characterization study revealed neuroendocrine like characteristics and gene expression. Genetic analysis revealed an upregulation of multiple genes in C4-2BER in comparison to its parental C4-2B, which is comparable to patient dataset available online. GSEA analysis revealed several neuronal biology related pathways to be prominently upregulated among C4-2BER. Further analysis revealed that expression of WT1, which binds near most of these upregulated genes, is downregulated in C4-2BER. Conclusions:The downregulation of WT1 is noted during progression from CRPC towards enzalutamide resistant neuroendocrine development in C4-2BER, which is in turn accompanied with various epigenetic changes. Understanding such pattern of changes and identifying the underlying molecular signature would help elucidate the targets behind treatment induced neuroendocrine characteristics from a therapeutic point of view. Citation Format: Sreyashi Bhattacharya, Ridwan Islam, Sanika Bodas, Navatha Polavaram, Kaustubh Datta, Samikshan Dutta. Second generation androgen receptor signaling inhibitor resistant neuroendocrine like prostate cancer: A mechanistic study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1326.

Abstract 358: A prospective study of prostate cancer metastases identifies an androgen receptor activity-low, stemness program associated with resistance to androgen receptor axis inhibitors and unveils mechanisms of clonal evolution

Abstract Background: The androgen receptor axis inhibitors (ARi) (e.g, enzalutamide, abiraterone acetate) are administered in daily practice for men with metastatic castration-resistant prostate cancer (mCRPC). However, not all patients respond, and mechanisms of both primary and acquired resistance remain largely unknown. Methods: In a prospective trial MATCH-R (NCT02517892), 55 mCRPC patients underwent whole exome sequencing (WES) (n=45) and RNA-sequencing (RNA-seq) (n=52) of metastatic biopsies before starting ARi. Also, 16 mCRPC patients underwent biopsy at time of resistance (WES=14, RNA-seq = 14). The objectives were to identify genomic alterations associated with resistance to ARi as well as to describe clonal evolution. Primary resistance was determined at 4 months of treatment using composite criteria for progression that included serum prostate specific antigen measurements, bone scan, CT imaging and symptom assessments. Acquired resistance was defined by occurrence of progressive disease after initial response or stable disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using Wilcoxon and Fisher's exact tests. Results: At 4 months, 22/55 patients in the cohort had disease progression (primary resistance). No genomic alterations from WES analysis were significantly associated with primary resistance. Analysis of sequential biopsies suggests that mCRPC follows mainly a parallel evolution model and involve DNA-repair related mutational processes. At time of acquired resistance to ARi, most tumors acquired new drivers affecting AR pathway (e.g, AR, NCOR1/2) or lineage switching (e.g, RB1, PTEN, TP53). Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis to identify pathways whose activity state correlated with resistance. AR gene alterations and AR expression were similar between responding and non-responding patients. Transcriptional analysis demonstrated that multiple specific gene sets — including those linked to low AR transcriptional activity, stemness program, RB loss and homologous repair deficiency — were activated in both primary and acquired resistance. Conclusion: Resistance to AR axis inhibitors results from multiple transcriptional programs already activated in pre-treatment samples. Clonal evolution analysis along with RNA-seq data indicate the role of genomic instability and lineage switching in driving acquired resistance Citation Format: Naoual Menssouri, Loic Poiraudeau, Carole Helissey, Ludovic Bigot, Jonathan Sabio, Tony Ibrahim, Claudio Nicotra, Maud Ngocamus, Lambros Tselikas, Thierry De Baere, Etienne Rouleau, Ludovic Lacroix, Anne Chaucherau, Luc Friboulet, Ronan Flippot, Giulia Baciarello, Laurence Albiges, Emeline Colomba, Pernelle Lavaud, Stefan Michiels, Aline Maillard, Antoine Italiano, Fabrice Barlesi, Jean-Charles Soria, Jean-Yves Scoazec, christophe Massard, Benjamin Besse, Fabrice André, Karim Fizazi, Daniel Gautheret, Yohann Loriot. A prospective study of prostate cancer metastases identifies an androgen receptor activity-low, stemness program associated with resistance to androgen receptor axis inhibitors and unveils mechanisms of clonal evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 358.

Abstract 1422: Neuropilin-2, a potential target against neuroendocrine like therapy resistant prostate cancer

Abstract Background: Prostate cancer (PCa) patient management is becoming increasingly challenging owing to the emergence of advanced therapy-resistant PCa. Published reports from our laboratory demonstrated that neuropilin-2 (NRP2), a non-tyrosine kinase transmembrane receptor, is associated with therapy resistance in aggressive PCa. Such advanced PCa is often associated with diverse genetic changes that complicate the therapeutic interventions. One example is the loss of tumor suppressor gene TP53 along with RB1 to develop therapy resistant neuroendocrine (NE)-like PCa that evades the standard androgen depletion therapy (ADT) and is resistant to first-line chemotherapy. Our results indicate that loss of TP53 and RB1 is associated with significantly increased expression of NRP2. Significance of the problem: Switch to NE-like lineage has been found to be associated with resistance to androgen receptor (AR) targeted therapy as well as chemotherapy. Patients with NE-like PCa show poor prognosis owing to the heterogeneous feature of the tumors and unavailability of effective therapy against this type of cancer. Therefore, identification of potential molecular target(s) is necessary to combat this disease. Objective: The objective of this study is to comprehend the underlying molecular mechanism/s of increased NRP2 expression in PCa, and whether targeting NRP2 would be effective against therapy resistant NE-like PCa, especially in combination with first-line chemotherapeutic agents. Study design: Current study was performed to test the NRP2 status in NE-like PCa cells and to understand if NRP2 is associated with the therapy resistance in this type of aggressive cancer. Moreover, efficacy of NRP2 depletion to sensitize the cancer cells to the chemotherapeutic agents used clinically was evaluated. A stable cell line model of NE-like PCa was developed and characterized by knocking down TP53 and RB1 genes in castration resistant prostate cancer (CRPC) cells. The resistance of the cells against the clinically used chemotherapy was investigated. In addition, efficacy of the chemotherapeutic agents following NRP2 knockdown was evaluated in in-vitro and in-vivo models. For in-vivo studies, PCa bone metastatic mouse model was developed in our laboratory and the effect of the depletion of NRP2 in combination with chemotherapeutic agent was evaluated. Results: NRP2 was significantly expressed in the NE-like PCa cells and knockdown of NRP2 sensitized the originally resistant NE-like cells to chemotherapeutic drugs. Tumor burden was also reduced in the bone metastatic mouse model upon NRP2 depletion when given in combination with chemotherapy. Conclusion: Therefore, targeting NRP2 can be an effective therapeutic strategy to combat highly aggressive and therapy resistant NE-like PCa. Citation Format: Ridwan Islam, Navatha Polavaram, Zhengdong Hong, Sreyashi Bhattacharya, Sanika Bodas, Benjamin Teply, Michael Muders, Kaustubh Datta, Samikshan Dutta. Neuropilin-2, a potential target against neuroendocrine like therapy resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1422.

Abstract 2714: Development of a phenotypically diverse model of sarcoma formation to identify key drivers of tumorigenesis across subtypes

Abstract Approximately 13,000 people this year in the United States alone will be diagnosed with sarcoma and nearly 5,000 will lose their lives to this devastating disease. Soft tissue sarcomas are a clinically and biologically heterogeneous group of tumors made up of approximately fifty subtypes that are classified by their presumed mesenchymal origin. The development of novel therapies for sarcoma has been hampered by the considerable heterogeneity of the disease and the limited understanding of the molecular mechanisms driving sarcoma formation and evolution. Due to this, clinically, the majority of patients with aggressive soft tissue sarcoma subtypes are treated similarly despite evidence these subtypes are unique. Our lab has focused on the development of a new model for the study of sarcoma subtypes using a primary human transformation system. Based on TCGA data, one characteristic that is shared between sarcoma subtypes is the loss or silencing of p53 and Rb1. We have started with immortalized mesenchymal stem cells (MSC), the presumed cell of origin for sarcoma and introduced lentiviral constructs targeting p53 and Rb1 by CRISPR technology to delete these genes. Following this, we have added a barcoded oncogene library with genes implicated in sarcoma specific biology or across cancer subtypes. Using this system, we have successfully transformed these MSCs into high grade soft tissue sarcomas. The current goal is to continue to manipulate this system to develop multiple soft tissue sarcoma subtypes. By starting with the same cell of origin and arriving at different sarcoma subtypes, we can begin to understand the relationship between these subtypes which will inform clinical trial design. Using this system, we can investigate the development of sarcoma, understand subtype heterogeneity, and utilize this tool to develop novel therapies for our patients. Citation Format: Janai Carr-Ascher. Development of a phenotypically diverse model of sarcoma formation to identify key drivers of tumorigenesis across subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2714.

Abstract 3027: Role of BCOR in retinoblastoma

Abstract Retinoblastoma, which is usually initiated by the biallelic mutational inactivation of RB1, is the most common primary eye cancer in children and is fatal if left untreated. While retinoblastoma is initially sensitive to chemotherapy, it demonstrates a strong propensity for therapeutic escape, which is a leading cause of ocular morbidity, eye loss, and reduced survival. This problem is exacerbated by a poor understanding of how retinoblastoma progresses after the initial loss of RB1. Genomic analysis of primary retinoblastoma tumors has revealed the corepressor BCOR as the second most common target of mutations in retinoblastoma. Here, we explored the role of BCOR in normal retinal development to provide insights into its role in retinoblastoma. Mass spectrometry following immunoprecipitation of wildtype BCOR in primary retinoblastoma cells revealed novel BCOR interacting proteins in the Polycomb and MiDAC epigenetic complexes. Subsequent studies revealed potential functional consequences of BCOR mutation that may promote retinoblastoma progression. This work reveals new insights the role of epigenetic deregulation in retinoblastoma progression, and they nominate new candidates for therapeutic intervention. Citation Format: Michelle Garying Zhang, Dawn Owens, Stefan Kurtenbach, Jeffim Kuznetsov, Matthew Fields, Daniel Pelaez, James W. Harbour. Role of BCOR in retinoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3027.

Abstract 591: Developing an immunofluorescence assay for detecting Rb and phospho-Rb on circulating tumor cells in breast cancer

Abstract Endocrine therapies (ET) such as tamoxifen, fulvestrant, and aromatase inhibitors (AIs) are the standard-of-care first-line treatment in majority of estrogen receptor (ER)-positive breast cancers (BC). Recent clinical studies using cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) plus AIs or fulvestrant have shown significant improvement in survival outcomes in patients with ER+ metastatic BC compared to standalone ETs. CDK4/6i exert their action by inhibiting the phosphorylation of retinoblastoma (Rb) protein and consequently inducing cell cycle arrest. However, not all patients respond to this combination therapy and those, who initially respond, eventually develop resistance. Emerging studies suggest that the intrinsic resistance to CDK4/6i could be due to the loss of Rb or its mutations. Therefore, CDK4/6i resistance can be evaluated by measuring expression of total and phospho-Rb and Rb mutations. However, repeated biopsies to evaluate biomarkers in tumors is not feasible in patients. In this research, we aimed to develop an immunofluorescence assay to evaluate the expression of Rb and phospho-Rb using circulating tumor cells (CTCs) which can help predict response and resistance to CDK4/6i. CTCs serve as representative of the tumor bulk in patients and animal models and allow for less-invasive, frequent blood collection and real-time monitoring of treatment response. MCF7 cells treated with vehicle or abemaciclib (500 nM) for 48 hours were spiked into blood from non-tumor bearing mice. The MCF7 cells-spiked blood was processed using ScreenCell® device and the cells were transferred to slides and stained with DAPI, pan-cytokeratin, CD45, estrogen receptor, Rb and phospho-Rb. Tumor cells were defined as pan-cytokeratin-positive, CD45-negative, and nuclear stain-positive cells. Various antibody combinations were examined to increase the sensitivity of the IF assay for individual markers as well as the multiplexed assay. We also developed quantitative assessment approach to detect per-cell intensity of various markers. Treatment with abemaciclib reduced the intensity of phospho/Total Rb from 2.6 +/- 0.6 to 0.8 +/- 0.2 units (p &amp;lt; 0.05, t-test, n=8-9) in the vehicle-treated samples. There was no significant difference in the Rb intensity between the treatment groups. Ongoing studies focus on validation of the assay using preclinical models and clinical samples. Citation Format: Tanya Kumar, Hariprasad Thangavel, Noor Mazin Abdulkareem, Raksha Bhat, Meghana V. Trivedi. Developing an immunofluorescence assay for detecting Rb and phospho-Rb on circulating tumor cells in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 591.

Abstract 2418: PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression

Abstract Background: The loss of PTEN function, one of the most frequent mutations in prostate cancer (CaP), is presumed to drive disease progression through AKT activation. However, two transgenic mouse CaP models, Pten/RbPE:-/- and Akap12-/-;RbPE:-/-, exhibited different CaP and metastasis development even though both shared Rb loss plus Akt activation, based on increased relative AKTpoS473 levels. Experimental Procedures: We analyzed whether PI3K-p110 and/or AKT isoform reliance, using isoform-specific RNAi or small molecule inhibitors, played a role in the different CaP outcomes. Findings: In comparison to Akap12/Rb-null high-grade prostatic intraepithelial neoplasias, Pten/Rb-null adenocarcinomas had increased protein and activation levels of AKT2 and decreased relative levels of Smad4, a known CaP metastasis suppressor. Comparison of isogenic PTEN +/- pairs of human or mouse CaP cell lines indicated that PTEN-deficiency correlated with dependence on both p110β and AKT2 for survival or oncogenic growth/invasiveness, whereas PTEN expression correlated with greater dependence on AKT1 plus p110α. Inhibition of AKT2 in PTEN-null CaP cells induced higher SMAD4 levels and decreased chemotaxis and Matrigel invasiveness. Given that PTEN likely regulates non-redundant roles of the AKT isoforms during CaP progression, we sought to characterize AKT isoform substrates by phosphoproteomics analysis. Specifically, after endogenous kinases were inactivated in mouse Pten/Rb-null adenocarcinoma cell lysates using 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA), dialyzed samples were subjected to in vitro kinase reactions using enzymatically-active, full-length baculovirus-expressed AKT1, AKT2 or AKT3, or as a negative control, a heat-inactivated mixture of all three isoforms. Tryptic peptides isolated on TiO2 beads were subjected to LC-MS/MS. Data will be shown on the role of AKT isoform-shared vs. -preferred substrates in specific parameters of CaP progression controlled by PTEN. Conclusion: Our data suggest that in the context of RB1 loss, PTEN controls a signaling plasticity and drug sensitivity through specific pairings of PI3K-p110 and AKT isoforms. Citation Format: Karina Miller, Seamus Degan, Yanqing Wang, Sheng-Yu Ku, David W. Goodrich, Irwin H. Gelman. PTEN regulated PI3K-p110 and AKT isoform plasticity controls metastatic prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2418.

Molecular Profiling of Relapsed and Refractory Myeloma Identifies Frequent Bi-Allelic Driver Events — Interim Results of the ‘Myeloma UK Seven’ Biomarker Trial

IntroductionPomalidomide and low-dose dexamethasone (PomDex) are approved for relapsed and refractory myeloma (RRMM) with Overall Response Rates of about 30%, including durable remissions. Combining PomDex with biologic agents can improve response, but there are currently no markers to predict which patients benefit from the addition of drugs to PomDex alone. Myeloma UK (MUK) seven is a multi-center UK phase II trial that is designed to generate evidence for improved outcomes and treatment stratification in RRMM by: 1) building a bio-repository for molecular characterization of RRMM and discovery of predictive markers for response 2) patient randomisation to Cyclophosphamide-PomDex (CyPomDex) or PomDex to investigate if the cost-effective CyPomDex combination increases progression free survival compared to PomDex 3) a flexible design that allows addition of trial arms with other promising PomDex combinations. We report high patient and investigator bio-sampling adherence and first comprehensive genetic profiling results in RRMM using a novel molecular assay platform.MethodsMUKseven (NCT02406222) will in total recruit 250 RRMM patients who are currently randomised 1:1 to CyPomDex or PomDex therapy. Registered patients undergo bone marrow sampling at screening, Cycle 1 Day 14, Cycle 4 Day 14 and at disease progression, and peripheral blood sampling on Cycle 1 Day 1. Patient consent includes mandatory bio-sampling at screening. Material is shipped to central laboratories for processing and storage of CD138-purified (>95%) MM tumor and non-tumor bone marrow microenvironment cells. Participants are currently recruited from over 30 NHS Hospitals in the MUK Early Phase Clinical Network.Digital multiplex ligation-dependent probe amplification (dMLPA®; MRC Holland) is a novel assay for high-throughput genome-wide copy number aberration (CNA) profiling. The MM specific D006 assay contains 160 probes that interrogate regions frequently affected by CNA in MM, including hotspots CDKN2C, CKS1B, MYC and regions encoding for therapeutic targets such as MCL1 or SLAMF7. In addition, 219 probes are distributed over all chromosomal arms and inform a virtual karyogram. The targeted design allows for analysis of over 80 patient samples in a single MiSeq (Illumina) run, using 10-20 ng DNA input material.ResultsBetween March 2016 and July 2017 a total of 83 patients were randomised to MUKseven with a median follow up of 6 cycles (1-13). Bone marrow material was received by central laboratories for all patients (100%) at screening, 69/79 (87.3%) at Cycle 1 day 14, 47/54 (87.0%) at Cycle 4 day 14 and 13/29 (44.8%) at disease progression. Peripheral blood was received for 73/79 (92.4%) on Cycle 1 Day 1. These return rates are higher than those reported for recent commercial trials. Of importance, for 52% of patients, matched bone marrow samples from earlier disease stages collected as part of UK academic research studies were available.We profiled the first 81 RRMM samples from trial entry with dMLPA and compared CNA frequency to a recently published newly diagnosed MM (NDMM) cohort which was assessed with MLPA (Shah V, Leukemia 2017). We found significantly more focal bi-allelic CNA of driver lesions in RRMM than in NDMM (P = 0.002). These included homozygous deletions of CDKN2C (6.2 vs 1.9%) and RB1 (3.7 vs 0.8%), amplifications of MYC (8.6 vs 2.3%), and heterozygous losses of TP53 (16.0 vs 9.6%). There were significantly more RRMM patients with one or more of the following bi-allelic changes: CDKN2C, RB1 or CYLD homozygous loss, or 1q amplification (18.5 vs 8.2%; P = 0.007), and 3.7% of patients in the RRMM cohort showed more than one bi-allelic change. We identified frequent homozygous deletions in NFκB pathway genes such as CYLD (4.9%), and recurrent amplifications of therapeutic targets like MCL1 and SLAMF7 on chromosome 1 (8.6% amplifications). The trial is recruiting and profiling of additional cases as well as longitudinal profiling of cases with material from earlier disease stages is ongoing.ConclusionWe demonstrate highly successful conduct of a multi-center biomarker discovery trial by the Myeloma UK Clinical Trial Network and the UK Myeloma Research Alliance. Our results demonstrate that bi-allelic CNA increase significantly in RRMM and some of these may help guide therapy. Recruitment and analyses of additional cases enrolled into MUKseven are ongoing and updated data will be presented at the meeting. DisclosuresPawlyn:Takeda: Honoraria, Other: Travel support; Janssen: Other: Travel support; Celgene: Honoraria, Other: Travel support. Atanesyan:MRC-Holland: Employment. Savola:MRC-Holland: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership. Garg:Janssen: Other: travel support, Research Funding, Speakers Bureau; Takeda: Other: travel support; Novartis: Other: travel support, Research Funding. Cook:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetcs: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau. Boyd:Janssen: Honoraria, Other: Travel support; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Kaiser:Takeda: Consultancy; BMS: Consultancy, Other: Travel expenses; Janssen: Honoraria; Chugai: Consultancy; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

A Subset of Localized Prostate Cancer Displays an Immunogenic Phenotype Associated with Losses of Key Tumor Suppressor Genes.

A subset of primary prostate cancer expresses programmed death-ligand 1 (PD-L1), but whether they have a unique tumor immune microenvironment or genomic features is unclear. We selected PD-L1-positive high-grade and/or high-risk primary prostate cancer, characterized tumor-infiltrating lymphocytes with multiplex immunofluorescence, and identified genomic alterations in immunogenic and nonimmunogenic tumor foci. One quarter of aggressive localized prostate cancer cases (29/115) had tumor PD-L1 expression more than 5%. This correlated with increased density of CD8+ T cells, a large fraction coexpressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8+PD-1+ cells did not express terminal exhaustion markers (TIM3 or LAG3), while a subset expressed TCF1. Consistent with these CD8+PD-1+TCF1+ cells being progenitors, they were found in antigen-presenting cell niches in close proximity to MHC-II+ cells. CD8 T-cell density in immunogenic prostate cancer and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent, the latter being strongly associated with a dendritic cell gene set in The Cancer Genome Atlas. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present. A subset of localized prostate cancer is immunogenic, manifested by PD-L1 expression and CD8+ T-cell content comparable with RCC. The CD8+ T cells include effector cells and exhausted progenitor cells, which may be expanded by immune checkpoint inhibitors (ICI). Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of patients with localized prostate cancer.

Retinoblastoma: from discovery to clinical management.

The retinoblastoma gene (RB1) was the first tumour suppressor cloned; the role of its protein product (RB) as the principal driver of the G1 checkpoint in cell cycle control has been extensively studied. However, many other RB functions are continuously reported. Its role in senescence, DNA repair and apoptosis, among others, is indications of the significance of RB in a vast network of cellular interactions, explaining why RB loss or its malfunction is one of the leading causes of a large number of paediatric and adult cancers. RB was first reported in retinoblastoma, a common intraocular malignancy in the paediatric population worldwide. Currently, its diagnosis is clinical, and in nondeveloped countries, where the incidence is higher, it is performed in advanced stages of the disease, compromising the integrity of the eye and the patient's life. Even though new treatments are being continuously developed, enucleation is still a major choice due to the late disease stage diagnosis and treatments costs. Research into biomarkers is our best option to improve the chances of good results in the treatment and hopes of patients' good quality of life. Here, we recapitulated the history of the disease and the first treatments to put the advances in its clinical management into perspective. We also review the different functions of the protein and the progress in the search for biomarkers. It is clear that there is still a long way to go, but we should offer these children and their families a better way to deal with the disease with the community's effort.

Temporal evolution of cellular heterogeneity during the progression to advanced AR-negative prostate cancer

Despite advances in the development of highly effective androgen receptor (AR)-directed therapies for the treatment of men with advanced prostate cancer, acquired resistance to such therapies frequently ensues. A significant subset of patients with resistant disease develop AR-negative tumors that lose their luminal identity and display neuroendocrine features (neuroendocrine prostate cancer (NEPC)). The cellular heterogeneity and the molecular evolution during the progression from AR-positive adenocarcinoma to AR-negative NEPC has yet to be characterized. Utilizing a new genetically engineered mouse model, we have characterized the synergy between Rb1 loss and MYCN (encodes N-Myc) overexpression which results in the formation of AR-negative, poorly differentiated tumors with high metastatic potential. Single-cell-based approaches revealed striking temporal changes to the transcriptome and chromatin accessibility which have identified the emergence of distinct cell populations, marked by differential expression of Ascl1 and Pou2f3, during the transition to NEPC. Moreover, global DNA methylation and the N-Myc cistrome are redirected following Rb1 loss. Altogether, our data provide insight into the progression of prostate adenocarcinoma to NEPC.

Establishment and characterization of a novel treatment-related neuroendocrine prostate cancer cell line KUCaP13.

The prevalence of neuroendocrine prostate cancer (NEPC) arising from adenocarcinoma (AC) upon potent androgen receptor (AR) pathway inhibition is increasing. Deeper understanding of NEPC biology and development of novel therapeutic agents are needed. However, research is hindered by the paucity of research models, especially cell lines developed from NEPC patients. We established a novel NEPC cell line, KUCaP13, from tissue of a patient initially diagnosed with AC which later recurred as NEPC. The cell line has been maintained permanently in vitro under regular cell culture conditions and is amenable to gene engineering with lentivirus. KUCaP13 cells lack the expression of AR and overexpress NEPC‐associated genes, including SOX2, EZH2, AURKA, PEG10, POU3F2, ENO2, and FOXA2. Importantly, the cell line maintains the homozygous deletion of CHD1, which was confirmed in the primary AC of the index patient. Loss of heterozygosity of TP53 and PTEN, and an allelic loss of RB1 with a transcriptomic signature compatible with Rb pathway aberration were revealed. Knockdown of PEG10 using shRNA significantly suppressed growth in vivo. Introduction of luciferase allowed serial monitoring of cells implanted orthotopically or in the renal subcapsule. Although H3K27me was reduced by EZH2 inhibition, reversion to AC was not observed. KUCaP13 is the first patient‐derived, treatment‐related NEPC cell line with triple loss of tumor suppressors critical for NEPC development through lineage plasticity. It could be valuable in research to deepen the understanding of NEPC.

Malignant glioma subset from Actuate 1801: A phase 1/2 study of 9-ING-41, a glycogen synthase kinase 3 beta (GSK-3β) inhibitor, as a single agent and combined with chemotherapy, in patients with refractory hematologic malignancies or solid tumors.

2051 Background: GSK-3β, a serine/threonine kinase, is a key regulator of metabolism and glycogen biosynthesis. GSK-3β aberrant overexpression promotes tumor progression and chemotherapy resistance through NF-κB and p53-mediated apoptotic pathways. GSK-3β inhibition impacts immunomodulation through downregulation of checkpoints, such as PD-L1 and LAG-3, and increasing NK and T-cell mediated killing of tumor cells. 9-ING-41 is a small-molecule potent selective GSK-3β inhibitor with preclinical antitumor activity against several tumor types. In chemoresistant PDX models of glioblastoma (GBM), 9-ING-41 enhanced the antitumor effect of CCNU and CPT-11. Methods: In the first-in-human study (NCT03678883), patients (pts) with refractory malignancies received 9-ING-41 monotherapy (n = 65) or in combination with one of 8 cytotoxic regimens after prior treatment with the same chemotherapy (n = 162). We report the subset of pts with recurrent gliomas treated with 9-ING-41 monotherapy IV twice a week in 21-day cycles at different dose levels (3.3, 5, 9.3, 15mg/kg), or in combination with lomustine 30 mg/m² orally once weekly in 84-day cycles. Primary objective was safety and tolerability. Results: An RP2D of 15mg/kg IV was confirmed across all 9 regimens, no accentuation of chemotherapy-related toxicity noted. Of 18 glioma patients enrolled, 13 were GBM, 2 anaplastic astrocytomas, 1 diffuse astrocytoma, and 1 anaplastic oligodendroglioma. Four patients received single agent 9-ING-41, 14 treated concurrently with lomustine. Demographics: 6 female, 12 male; median age 52 (30-69) years; median ECOG was 1 (0-2). All received first-line radiation and temozolomide (18/18), prior therapies for recurrences included nitrosoureas (15/18), bevacizumab (8/18), TTFields (6/18), immune checkpoint inhibitor (4/18). Median recurrences 3 (1-6). Genomic alterations from available NGS reports included: IDH WT (11), IDH mutation (3), MGMT promoter unmethylated (11), MGMT promoter methylated (1), 1p19q co-deletion (10), EGFR amplification (6), EGFR v3 mutation (3), TERT promoter mutation (6), PTEN loss (3), NF1 rearrangement (2), ATRX loss (2), TP53 mutated (4), CDKN2A deletion (2), RB1 loss (1), PALB-2 mutation (10). No SAEs or grade 3/4 AEs attributed to 9-ING-41 were noted; AEs included G1/2 transient vision changes (9/18, 50%), infusion reactions (4/18, 22%). Side effects from lomustine included: G3/4 thrombocytopenia (3/14, 21%), and G1/2 fatigue (4/14, 28%). Best overall response: 1 minimal response (-43%) after 2 cycles of 9-ING-41 and lomustine. Median days on therapy was 55 (4-305), 4/18 (22%) were stable for 20 weeks or longer. Conclusions: These results show 9-ING-41 alone or in combination is safe and warrants further study in glioma patients. Clinical trial information: NCT03678883.