PTEN Loss Research Articles

'High proliferative cribriform prostate cancer' defines a patient subgroup with an inferior prognosis.

A cribriform pattern, reactive stroma (RS), PTEN, Ki67 and ERG are promising prognostic biomarkers in primary prostate cancer (PCa). We aim to determine the relative contribution of these factors and the Cancer of the Prostate Risk Assessment Postsurgical (CAPRA-S) score in predicting PCa prognosis. We included 475 patients who underwent radical prostatectomy (2010-12, median follow-up = 8.7 years). Cribriform pattern was identified in 57% of patients, PTEN loss in 55%, ERG expression in 51%, RS in 39% and high Ki67 in 9%. In patients with multiple samples from the same malignant focus and either PTEN loss or high Ki67, intrafocal heterogeneity for PTEN and Ki67 expression was detected in 55% and 89%, respectively. In patients with samples from two or more foci, interfocal heterogeneity was detected in 46% for PTEN and 6% for Ki67. A cribriform pattern and Ki67 were independent predictors of biochemical recurrence (BCR) and clinical recurrence (CR), whereas ERG expression was an independent predictor of CR. Besides CAPRA-S, a cribriform pattern provided the highest relative proportion of explained variation for predicting BCR (11%), and Ki67 provided the highest relative proportion of explained variation for CR (21%). In patients with a cribriform pattern, high Ki67 was associated with a higher risk of BCR [hazard ratio (HR) = 2.83, P < 0.001] and CR (HR = 4.35, P < 0.001). High Ki67 in patients with a cribriform pattern identifies a patient subgroup with particularly poor prognosis, which we termed 'high proliferative cribriform prostate cancer'. These results support reporting a cribriform pattern in pathology reports, and advocate implementing Ki67.

Gender Differences in a Mouse Model of Hepatocellular Carcinoma Revealed Using Multi-Modal Imaging.

The worldwide incidence of hepatocellular carcinoma (HCC) continues to rise, in part due to poor diet, limited exercise, and alcohol abuse. Numerous studies have suggested that the loss or mutation of PTEN plays a critical role in HCC tumorigenesis through the activation of the PI3K/Akt signaling axis. The homozygous knockout of PTEN in the livers of mice results in the accumulation of fat (steatosis), inflammation, fibrosis, and eventually progression to HCC. This phenotype bears a striking similarity to non-alcoholic steatohepatitis (NASH) which is thought to occupy an intermediate stage between non-alcoholic fatty liver disease (NAFLD), fibrosis, and HCC. The molecular and physiological phenotypes that manifest during the transition to HCC suggest that molecular imaging could provide a non-invasive screening platform to identify the hallmarks of HCC initiation prior to the presentation of clinical disease. We have carried out longitudinal imaging studies on the liver-specific PTEN knockout mouse model using CT, MRI, and multi-tracer PET to interrogate liver size, steatosis, inflammation, and apoptosis. In male PTEN knockout mice, significant steatosis was observed as early as 3 months using both magnetic resonance spectroscopy (MRS) and computed tomography (CT). Enhanced uptake of the apoptosis tracer 18F-TBD was also observed in the livers of male PTEN homozygous knockout mice between 3 and 4 months of age relative to heterozygous knockout controls. Liver uptake of the inflammation tracer [18F]4FN remained relatively low and constant over 7 months in male PTEN homozygous knockout mice, suggesting the suppression of high-energy ROS/RNS with PTEN deletion relative to heterozygous males where the [18F]4FN liver uptake was elevated at early and late time points. All male PTEN homozygous mice developed HCC lesions by month 10. In contrast to the male cohort, only 20% (2 out of 10) of female PTEN homozygous knockout mice developed HCC lesions by month 10. Steatosis was significantly less pronounced in the female PTEN homozygous knockout mice relative to males and could not accurately predict the eventual occurrence of HCC. As with the males, the [18F]4FN uptake in female PTEN homozygous knockout mice was low and constant throughout the time course. The liver uptake of 18F-TBD at 3 and 4.5 months was higher in the two female PTEN knockout mice that would eventually develop HCC and was the most predictive imaging biomarker for HCC in the female cohort. These studies demonstrate the diagnostic and prognostic role of multi-modal imaging in HCC mouse models and provide compelling evidence that disease progression in the PTEN knockout model is highly dependent on gender.

An updated literature on BRAF inhibitors (2018-2023).

BRAF is the most common serine-threonine protein kinase and regulates signal transduction from RAS to MEK inside the cell. The BRAF is a highly active isoform of RAF kinase. BRAF has two domains such as regulatory and kinase domains. The BRAF inhibitors bind in the c-terminus of the kinase domain and inhibit the downstream pathways. The mutation occurs mainly in the A-loop of the kinase domain. The mutation occurs due to a conversion of valine to glutamate/lysine/arginine/aspartic acid at 600th position. Among the diverse mutations, BRAFV600E is the most common and responsible for numerous cancer such as melanoma, colorectal, ovarian, and thyroid cancer. Due to mutations in RAC1, loss of PTEN, NF1, CCND1, USP28-FBW7 complex, COT overexpression, and CCND1 amplification, the BRAF kinase enzyme developed resistance over the commercially available BRAF inhibitors. There is still unmute urgence for the development of BRAF inhibitors to overcome the persistent limitation such as resistance, mutation, and adverse effects of drugs. In the current study, we described the structure, activation, downstream signaling pathway, and mutation of BRAF. Our group also provided a detailed review of BRAF inhibitors from the last five years (2018-2023) highlighting the structure-activity relationship, mechanistic study, and molecular docking studies. We hope that the current analysis will be a useful resource for researchers and provide chemists a glimpse into the future as design and development of more effective and secure BRAF kinase inhibitors.

PTEN Expression and Specific Clinicopathological Associations in Colorectal Carcinomas

Introduction: Colorectal carcinoma (CRC) refers to any cancerthat affects the colon and the rectum and it is a significantpublic health issue.1 It is the third most common cancer to bediagnosed globally and the fourth most frequent reason forcancer-related mortality. The diagnosis of CRCs occurs inplaces with affluent economies about 60% of the time. Thepresent study assessed phosphatase and tensin homolog(PTEN) expression in colorectal cancer and its associationwith tumor characteristics.Materials and Methods: The study was carried out over thecourse of 18 months, from 1 February 2021 to 31 July 2022, atthe SRMS IMS Bareilly Department of Pathology. The analysisincluded surgically removed specimens of colorectal cancerthat had been obtained at the SRMS, IMS Department ofPathology. The patient’s medical records provided the clinicalinformation.Results: Loss of PTEN expression was seen in 76.9% of CRCcases, while 23.1% of cases revealed positive expression.Maximum cases were of age&gt;60 years and male predominancewas noted. In 62.5% of cases with tumor size ≥5 cm displayedloss of PTEN expression. In 52.5% loss of PTEN tumor caseswere histologically moderately differentiated, 37.5% were welldifferentiated, and 10% were poorly differentiated. 50% of caseswith pathologic tumor stage (pT3) and 65% left-sided tumorsshowed loss of PTEN expression.Conclusion: Loss of PTEN expression was observed in themajority of colorectal tumors along with its negative associationwith tumor size and pathologic tumor staging.

Clinical, pathological, and comprehensive molecular analysis of the uterine clear cell carcinoma: a retrospective national study from TMRG and GINECO network

BackgroundUterine clear cell carcinomas (CCC) represent less than 5% of uterine cancers. Their biological characteristics and clinical management remain uncertain. A multicenter study to explore both clinical and molecular features of these rare tumors was conducted.MethodsThis multicenter retrospective national study was performed within the French TMRG (Rare Gynecologic Malignant Tumors) network. Clinical data and, when available, FFPE blocks were collected. Clinical features, treatments, and outcome (progression-free survival (PFS) and overall survival (OS)) were analyzed and correlated to the protein (tissue micro-array), RNA (Nanostring nCounter® technology), and DNA (array-Comparative Genomic hybridization and target-next generation sequencing) levels using the tumor samples available.ResultsSixty-eight patients with uterine CCC were enrolled, 61 from endometrial localization and 5 with cervix localization. Median age at diagnosis was 68.9 years old (range 19–89.7). Most tumors were diagnosed at an early stage (78% FIGO stage I–II). Hysterectomy (performed in 90%) and lymph node dissection (80%) were the most frequent surgical treatment. More than 70% of patients received external beam radiotherapy and 57% received brachytherapy. Nearly half (46%) of the patients received chemotherapy. After a median follow-up of 24.7 months, median PFS was 64.8 months (95 CI [5.3–124.4]) and median OS was 79.7 (IC95 [31.0–128.4]). Low hormone receptor expression (13% estrogen-receptor positive), frequent PI3K pathway alterations (58% PTEN loss, 50% PIK3CA mutations), and P53 abnormalities (41%) were observed. Mismatch repair deficiency was identified in 20%. P16 expression was associated with shorter PFS (HR = 5.88, 95 CI [1.56–25], p = 0.009). Transcriptomic analyzes revealed a specific transcriptomic profile notably with a high expression of immune response-associated genes in uterine CCC displaying a very good overall prognosis.ConclusionsUterine CCC reported to be potentially MSI high, hormone receptors negative, and sometimes TP53 mutated. However, some patients with immune response-associated features and better prognosis may be candidate to treatment de-escalation and immunotherapy.

A New Type of Endometrial Cancer Models in Mice Revealing the Functional Roles of Genetic Drivers and Exploring their Susceptibilities.

Endometrial cancer (EC) is the most common female reproductive tract cancer and its incidence has been continuously increasing in recent years. The underlying mechanisms of EC tumorigenesis remain unclear, and efficient target therapies are lacking, for both of which feasible endometrial cancer animal models are essential but currently limited. Here, an organoid and genome editing-based strategy to generate primary, orthotopic, and driver-defined ECs in mice is reported. These models faithfully recapitulate the molecular and pathohistological characteristics of human diseases. The authors names these models and similar models for other cancers as organoid-initiated precision cancer models (OPCMs). Importantly, this approach can conveniently introduce any driver mutation or a combination of driver mutations. Using these models,it is shown that the mutations in Pik3ca and Pik3r1 cooperate with Pten loss to promote endometrial adenocarcinoma in mice. In contrast, the Kras G12D mutati led to endometrial squamous cell carcinoma. Then, tumor organoids are derived from these mouse EC models and performed high-throughput drug screening and validation. The results reveal distinct vulnerabilities of ECs with different mutations. Taken together, this study develops a multiplexing approach to model EC in mice and demonstrates its value for understanding the pathology of and exploring the potential treatments for this malignancy.

Artificial Intelligence-Based PTEN Loss Assessment as an Early Predictor of Prostate Cancer Metastasis After Surgery: A Multicenter Retrospective Study

Phosphatase and tensin homolog (PTEN) loss is associated with adverse outcomes in prostate cancer and can be measured via immunohistochemistry. The purpose of the study was to establish the clinical application of an in-house developed artificial intelligence (AI) image analysis workflow for automated detection of PTEN loss on digital images for identifying patients at risk of early recurrence and metastasis. Postsurgical tissue microarray sections from the Canary Foundation (n = 1264) stained with anti-PTEN antibody were evaluated independently by pathologist conventional visual scoring (cPTEN) and an automated AI-based image analysis pipeline (AI-PTEN). The relationship of PTEN evaluation methods with cancer recurrence and metastasis was analyzed using multivariable Cox proportional hazard and decision curve models. Both cPTEN scoring by the pathologist and quantification of PTEN loss by AI (high-risk AI-qPTEN) were significantly associated with shorter metastasis-free survival (MFS) in univariable analysis (cPTEN hazard ratio [HR], 1.54; CI, 1.07-2.21; P = .019; AI-qPTEN HR, 2.55; CI, 1.83-3.56; P < .001). In multivariable analyses, AI-qPTEN showed a statistically significant association with shorter MFS (HR, 2.17; CI, 1.49-3.17; P < .001) and recurrence-free survival (HR, 1.36; CI, 1.06-1.75; P = .016) when adjusting for relevant postsurgical clinical nomogram (Cancer of the Prostate Risk Assessment [CAPRA] postsurgical score [CAPRA-S]), whereas cPTEN does not show a statistically significant association (HR, 1.33; CI, 0.89-2; P = .2 and HR, 1.26; CI, 0.99-1.62; P = .063, respectively) when adjusting for CAPRA-S risk stratification. More importantly, AI-qPTEN was associated with shorter MFS in patients with favorable pathological stage and negative surgical margins (HR, 2.72; CI, 1.46-5.06; P = .002). Workflow also demonstrated enhanced clinical utility in decision curve analysis, more accurately identifying men who might benefit from adjuvant therapy postsurgery. This study demonstrates the clinical value of an affordable and fully automated AI-powered PTEN assessment for evaluating the risk of developing metastasis or disease recurrence after radical prostatectomy. Adding the AI-qPTEN assessment workflow to clinical variables may affect postoperative surveillance or management options, particularly in low-risk patients.

PTEN loss confers resistance to anti-PD-1 therapy in non-small cell lung cancer by increasing tumor infiltration of regulatory T cells.

Immunotherapy resistance in non-small cell lung cancer (NSCLC) may be mediated by an immunosuppressive microenvironment, which can be shaped by the mutational landscape of the tumor. Here, we observed genetic alterations in the PTEN/PI3K/AKT/mTOR pathway and/or loss of PTEN expression in >25% NSCLC patients, with higher frequency in lung squamous carcinomas (LUSCs). Patients with PTEN-low tumors had higher levels of PD-L1 and PD-L2 and showed worse progression-free survival when treated with immunotherapy. Development of a Pten-null LUSC mouse model revealed that tumors with PTEN loss were refractory to anti-PD-1, highly metastatic and fibrotic, and secreted TGF-β/CXCL10 to promote conversion of CD4+ lymphocytes into regulatory T cells (Tregs). Human and mouse PTEN-low tumors were enriched in Tregs and expressed higher levels of immunosuppressive genes. Importantly, treatment of mice bearing Pten-null tumors with TLR agonists and anti-TGF-β antibody aimed to alter this immunosuppressive microenvironment led to tumor rejection and immunological memory in 100% of mice. These results demonstrate that lack of PTEN causes immunotherapy resistance in LUSC by establishing an immunosuppressive tumor microenvironment that can be reversed therapeutically.

Nomogram for predicting the biochemical recurrence of prostate cancer after neoadjuvant androgen deprivation therapy

BackgroundA predictive model for biochemical recurrence (BCR) of prostate cancer (PCa) after neoadjuvant androgen deprivation therapy (nADT) has not been established. This study was aimed at determining multiparameter variables that could be used to construct a nomogram to predict the post-nADT BCR of PCa.MethodsOverall, 43 radical prostatectomy specimens from PCa patients who had undergone nADT were collected. Multiparameter variables were analyzed by univariate and then multivariate logistic analyses to identify the independent prognostic factors for predicting BCR. The predictive model was established using Lasso regression analysis.ResultsUnivariate logistic analysis revealed six variables, pathology stage; margins; categorization as group A, B, or C; nucleolus grading; percentage of tumor involvement (PTI); and PTEN status were significantly associated with the BCR of PCa (all p < 0.05). Multivariate logistic regression analysis suggested that categorization as group C, severe nucleolus grading, PTI less than or equal to 5%, and PTEN loss were positively correlated with BCR (all p < 0.05). A nomogram comprising the four variables predicting BCR was constructed, and it exhibited good discrimination (AUC: 0.985; specificity: 86.2%; sensitivity: 100%). Calibration plots for the probability of freedom from BCR at 1 and 2 years showed a good match between the prediction by the nomogram.ConclusionsWe constructed and validated a nomogram to predict the risk of BCR in PCa patients after nADT. This nomogram is a complement to the existing risk stratification systems for PCa, which could have marked implications for clinical decision-making for PCa patients after nADT.

BIOL-23. LOSS OF MICRORNAS COOPERATES WITH TUMOR SUPPRESSORS TO INITIATE MYC-DEPENDENT METASTATIC MEDULLOBLASTOMA

Abstract To determine the role of microRNA regulation in brain tumor development, we incorporated a conditional allele of the microRNA processing enzyme Dicer to a glioma mouse model based on inactivation of the tumor suppressors Nf1, Trp53, and Pten using the Nestin-creERT2 transgene. Loss of Dicer and tumor suppressors at adult ages led to glioma development; however, mutant mice tamoxifen induced at early postnatal ages developed medulloblastoma (MB) instead of glioma. The switch in tumor spectrum occurred with 100% penetrance and the cerebellar tumors were histologically indistinguishable from human MB. The minimum genetic mutations required for MB formation were Dicer1 and Trp53, while additional loss of Pten produced more invasive tumors with leptomeningeal metastasis. Analysis of tumor transcriptome and MB subtype-specific markers show that Dicer tumors most resemble human Sonic Hedgehog (SHH) MB. The presence of leptomeningeal metastasis, Trp53 loss and upregulation of Mycn and Gli2 in the setting of activated Shh signaling suggests that this model phenocopies extremely high risk p53-mutated SHH MB, which carries poor prognosis and is associated with the majority of treatment failures in SHH MB. Analysis of pre-symptomatic mutant mice showed proliferative defects and retained cells in the external granule cell layer (EGL), suggesting that tumors may arise from cerebellar granule neuron precursors (CGNPs). However, targeting CGNPs using Math1-creERT2 did not lead to MB development, suggesting that an earlier EGL precursor may be required for tumorigenesis. Dicer tumors exhibit reduced microRNAs and RNA-induced silencing complexes consistent with loss of Dicer. One of these downregulated microRNAs, miR-101, is a regulator of Mycn and its overexpression leads to Mycn downregulation and inhibition of MB growth. Likewise, genetic and pharmacologic inhibition of Myc leads to inhibition of MB growth. These studies demonstrate a role for microRNAs in Myc-dependent MB development and show an alternative pathway for SHH MB tumorigenesis.

Predicting Prostate Cancer Molecular Subtype With Deep Learning on Histopathologic Images

Microscopic examination of prostate cancer has failed to reveal a reproducible association between molecular and morphologic features. However, deep-learning algorithms trained on hematoxylin and eosin (H&E)-stained whole slide images (WSI) may outperform the human eye and help to screen for clinically-relevant genomic alterations. We created deep-learning algorithms to identify prostate tumors with underlying ETS-related gene (ERG) fusions or PTEN deletions using the following 4 stages: (1) automated tumor identification, (2) feature representation learning, (3) classification, and (4) explainability map generation. A novel transformer-based hierarchical architecture was trained on a single representative WSI of the dominant tumor nodule from a radical prostatectomy (RP) cohort with known ERG/PTEN status (n = 224 and n = 205, respectively). Two distinct vision transformer-based networks were used for feature extraction, and a distinct transformer-based model was used for classification. The ERG algorithm performance was validated across 3 RP cohorts, including 64 WSI from the pretraining cohort (AUC, 0.91) and 248 and 375 WSI from 2 independent RP cohorts (AUC, 0.86 and 0.89, respectively). In addition, we tested the ERG algorithm performance in 2 needle biopsy cohorts comprised of 179 and 148 WSI (AUC, 0.78 and 0.80, respectively). Focusing on cases with homogeneous (clonal) PTEN status, PTEN algorithm performance was assessed using 50 WSI reserved from the pretraining cohort (AUC, 0.81), 201 and 337 WSI from 2 independent RP cohorts (AUC, 0.72 and 0.80, respectively), and 151 WSI from a needle biopsy cohort (AUC, 0.75). For explainability, the PTEN algorithm was also applied to 19 WSI with heterogeneous (subclonal) PTEN loss, where the percentage tumor area with predicted PTEN loss correlated with that based on immunohistochemistry (r = 0.58, P = .0097). These deep-learning algorithms to predict ERG/PTEN status prove that H&E images can be used to screen for underlying genomic alterations in prostate cancer.

Abstract A049: Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer

Abstract Introduction: ETS gene rearrangements involving ERG, ETV1 and ETV4, as well as PTEN deletions are among the most common genomic alterations in prostate cancer. Although these events are found to be mutually exclusive, the multifocality of prostate cancer suggests that they may co-occur in some tumors. The immunohistochemistry (IHC) detection of both ERG overexpression and PTEN protein loss are reliable substitutes for fluorescence in situ hybridization (FISH) assay to probe genomic alterations. The lack of specific antibodies that can discriminate between multiple ETS factors, however, has limited the evaluation for the co-occurrence of these genomic alterations in prostate cancer. Improving our ability to identify the multifocality or inter-focal heterogeneity of tumors can enhance our understanding of biological mechanisms of tumor initiation and progression, and help stratify patients for treatment. Methods: Using monoclonal antibodies (mAbs) that we developed against ETV1 and ETV4, we examined their expression in relation to ERG and PTEN expression by IHC using two TMAs constructed from primary prostate cancer specimens of independent patient cohorts: one was constructed from multiple 1 mm cores representing distinct tumor focus from multifocal tumors from 50 African American (AA) and 50 Caucasian American (CA) men; the other, from a single 2 mm core of individual tumors from a cohort of 152 AA and 304 CA men. Positive expression for each ETS factor and PTEN were evaluated for association with race and clinico-pathologic features that include PSA at diagnosis, Gleason score, pathologic T stage, biochemical recurrence, and metastasis. Univariate analysis of BCR free survival as a function of ERG, ETV1 and ETV4 and PTEN expression was performed for both patient cohorts. Results: ERG(+) expression alone or in combination with PTEN loss were significantly higher in CA men in both patient cohorts. When expression of ETS factors were evaluated together with PTEN status, positive expression of ETS factors and PTEN loss were significantly more prevalent among CA men in both patient cohorts ETV1 and ETV4 expression were detected at 5% or less in both patient cohorts. Cases harboring tumor foci that were positive for more than one marker evaluated frequently showed either intra-tumor or inter-tumor heterogeneity. Univariate analysis of PTEN-ETS protein expression status and BCR free survival showed that patients with loss of PTEN expression trends towards poorer BCR free survival regardless of ETS expression status. Conclusion: We showed that the IHC detection of multiple aberrantly expressed proteins such as ERG, ETV1, ETV4 and PTEN in prostate tumor specimens using highly specific mAbs can reveal the heterogeneity and multi-focality in prostate cancer, which could be used to enhance the stratification of patients for novel treatment strategies. Citation Format: Shyh-Han Tan, Cara Schafer, Denise Young, Jiji Jiang, Yingjie Song, Albert Dobi, Gyorgy Petrovics, Bettina F. Drake, Gregory T. Chesnut, Isabell A. Sesterhenn. Immunohistochemical detection of ERG, ETV1, ETV4, and PTEN in prostate tumor specimens reveals the heterogeneity of multi-focal prostate cancer [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 A049.

Abstract B012: Genomic and epigenomic drivers of double-negative metastatic prostate cancer

Abstract Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen receptor (AR) signaling. Androgen deprivation therapy and second-generation AR-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by whether the tumor expresses either AR or neuroendocrine (NE) markers. Among the subtypes of mCRPC, the molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterize genomic and epigenomic features of treatment-emergent mCRPC subtypes in 210 tumors by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing. We show that AR-/NE- tumors exhibit a clinically and molecularly distinct phenotype. Patients with AR-/NE- mCRPC tumors have the shortest survival, and these tumors preferentially harbor amplification of the chromatin remodeler CHD7 and loss of PTEN. We demonstrate that methylation changes in CHD7 candidate enhancers are linked to elevated CHD7 expression in AR-/NE+ tumors. Moreover, we use genome-wide methylation analysis to nominate the Krüppel-like factor gene KLF5 as a driver of the AR-/NE- phenotype and link its activity to loss of the tumor suppressor RB1. These observations reveal the aggressiveness of the AR-/NE- tumors and elucidate genomic and epigenomic drivers of mCRPC subtypes, which may facilitate the identification of novel therapeutic targets in this highly aggressive disease. Citation Format: Arian Lundberg, Meng Zhang, Rahul R. Aggarwal, Haolong Li, Li Zhang, Adam Foye, Martin Sjöström, Jonathan Chou, Kevin Chang, Thaidy Moreno-Rodriguez, Raunak Shrestha, Avi Baskin, Xiaolin Zhu, Alana S. Weinstein, Noah Younger, Joshi J. Alumkal, Tomasz M. Beer, Kim N. Chi, Christopher P. Evans, Martin Gleave, Primo N. Lara, Robert E. Reiter, Matthew B. Rettig, Owen N. Witte, Alexander W. Wyatt, Felix Y. Feng, Eric J. Small, David A. Quigley. Genomic and epigenomic drivers of double-negative metastatic prostate cancer [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 B012.

Abstract A018: Increased RAD21 promotes prostate cancer development

Abstract Prostate cancer (PCa) is one of the most common cancers among men, leading to the second cause of death for men with cancers in the US. Aneuploidy, featured by imbalanced chromosome numbers, is a hallmark of cancer. Epidemiology studies on primary PCa cohorts of Physicians' Health Study and Health Professionals Follow-up Study (PHS and HPFS) have shown that high levels of whole-genome aneuploidy correlate with lethal progression in PCa. However, the detail mechanisms of how aneuploidy drives aggressiveness of PCa are still unclear. Here, we used the case of chromosome 8q (chr 8q, the long arm of chr 8) gain to study aneuploidy-associated prostatic malignancies. Chr 8q gains are the most frequently gain events that occur in around 23% of PCa cases. By using the PHS and HPFS cohorts, we modeled the increased expression of each gene located on chr 8q, for predicting the risks for lethal progression, and obtained the odds ratio (OR) for each. Then, we ranked the ORs for lethal progression and identified several important genes highly associated with lethality when overexpressed. Among them, the cohesin subunit gene, RAD21, is one of the top associators. Increased RAD21 mRNA level, per se, is highly correlated with lethality in all PCa cases, and the lethality is synergistically aggravated in the cases with both increased RAD21 expression and chr 8q gains. indicating that RAD21 cooperates with other chr 8q genes to drive cancer progression and chr 8q gains. To determine how RAD21 overexpression promotes PCa, we studied the effect of overexpression of RAD21 in early prostatic oncogenic events. We utilized the isogenic mouse prostate organoid models carrying an inducible the fusion-oncogene, TMPRSS2-ERG (T-ERG), which 50% of PCa cases harbor. We found that induction of T-ERG leads to a strong oncogenic replication stress at an early stage. Such stress results in an increase in apoptosis and growth impairment in these primary organoids. Overexpression of RAD21, mimicking the chr 8q gain situation, mitigates such replication stress and rescues the growth defect caused by T-ERG. These data suggest the role of increased RAD21 in promoting oncogenic growth of PCa cells by reducing oncogenic toxicity at an early stage. In addition, we showed that such role of RAD21 overexpression sustains in the more advanced cancerous organoid (ERG positive and PTEN loss) and promotes the growth of the cancer organoids. Consistently, increased RAD21 expression correlates with increase proliferative markers in human prostate cancer cases. In conclusion, we identified that overexpression of multiple chr 8q genes are correlated with lethal progression in primary PCa, and RAD21 is one of such genes. Increased RAD21 plays a fundamental role in reducing toxic DNA damage caused by prostatic oncogenesis. Citation Format: Xiaofeng A. Su, Konrad H. Stopsack, Daniel R. Schmidt, Duanduan Ma, Zhe Li, Matthew G. Vander Heiden, Angelika Amon, Lorelei A. Mucci. Increased RAD21 promotes prostate cancer development [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 A018.

Abstract B065: Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer

Abstract The phosphoinositide 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is dysregulated in the majority of advanced prostate cancer patients, often linked to phosphate and tensin homolog (PTEN) inactivation. An increasing number of studies demonstrates the reciprocal crosstalk between the PI3K/AKT/mTOR and the androgen receptor (AR) pathways with the inhibition of one of them leading to the compensatory activation of the other one. We compared the effects of different PI3K/AKT/mTOR pathway inhibitors as single agents or in combination with the AR inhibitor darolutamide.In vitro impact of the compounds was evaluated in prostate cancer cell lines by proliferation and apoptosis assays, and by RNA-seq analysis. In vivo effects were determined in a patient-derived (PDX) prostate cancer model in an efficacy study followed by immunohistochemistry analysis. Analysis of VCaP cells treated with the androgen R1881 showed that beside androgen response, the PI3K/AKT/mTOR pathway was an essential hallmark characteristic of androgen action. Additional darolutamide treatment opposed androgen effects on both pathways. We therefore evaluated the impact of selective inhibitors of PI3K or AKT and saw strong in vitro antiproliferative effects in the prostate cancer cell lines tested. We then focused on the pan-PI3K inhibitor copanlisib and observed pronounced synergistic effects with darolutamide in the VCaP model. This was accompanied by induction of PARP cleavage and activation of caspase 3/7. A detailed transcriptomic analysis revealed that the combination of both inhibitors resulted in more pronounced transcript changes, compared to individual treatments. Principal component analysis furthermore showed the combination to be closer to the DMSO control along the axis that represents androgen regulation. We observed a pronounced downregulation of cell proliferation and cell division genes following combination treatment compared to individual treatments, a prominent example being the proliferation marker KI67. In vivo studies performed with the PDX LuCaP 35 model revealed a superior inhibitory effect of the combination treatment with darolutamide and copanlisib when compared to single agents. Immunohistochemistry analysis revealed a significant upregulation of a pro-apoptotic pathway in tumors treated with darolutamide and copanlisib which was not observed in single treatment arms, when compared to vehicle control. In summary, we found that different PI3K and AKT inhibitors impaired the in vitro proliferation of prostate cancer cell lines. Combining the AR inhibitor darolutamide with the pan-PI3K inhibitor copanlisib led to increased apoptosis and down-regulation of cell division and proliferation genes. Importantly, the improved tumor inhibition by the combination was also observed in vivo. These results warrant further analysis of the impact of combined AR and PI3K pathway inhibition in prostate cancer, especially when aberrations in the PI3K/AKT/mTOR pathway or PTEN loss are observed. Citation Format: Simon Heller, Tatsuo Sugawara, Ekaterina Nevedomskaya, Simon J. Baumgart, Holly Nguyen, Eva Corey, Annika Böhme, Oliver von Ahsen, Oliver Politz, Bernard Haendler. Combining the androgen receptor inhibitor darolutamide with PI3K/AKT/mTOR pathway inhibitors has superior efficacy in preclinical models of prostate cancer [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 B065.

Abstract PR007: ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells

Abstract TMPRSS2-ERG translocations are initiating lesions in nearly 50% of human prostate adenocarcinomas, often in conjunction with PTEN loss. In mice, prostate-specific expression of ERG using a Probasin-Cre driver (Pb-Cre4) is sufficient to induce invasive Ck8+ luminal adenocarcinoma with high penetrance in the context of Pten loss. Because TMPRSS2 expression in the human prostate is androgen-regulated and thereby largely restricted to luminal epithelial cells, it has been assumed that luminal cells are the likely cell of origin for TMPRSS2-ERG positive prostate cancers. To gain further support for this hypothesis, we examined the early stages of luminal adenocarcinoma initiation in the Pb-Cre ERG/Pten model at a single cell level. To our surprise, we found a highly proliferative population of ERG+ Ck5+ (a putative basal marker) cells that are enriched at the invasive front in a background of more canonical ERG+/Ck8+/Ck5- luminal tumor cells. We next performed lineage tracing experiments using Ck5-, Ck8- as well as Nkx3.1-Cre drivers, introduced via viral injection or tamoxifen induction, which collectively support the hypothesis that ERG-driven tumors originate in a subset of basal cells defined by Ck5+/Nkx3.1+. This conclusion is further supported by orthotopic transplantation of freshly isolated primary mouse prostate basal (Cd49f high) or luminal (Cd24 high) cells showing that ERG+/Ck8+ tumors arise preferentially from basal cells following ex vivo ERG/Pten activation. Time course experiments in organoids and in vivo show that ERG promotes Ck8+ luminal differentiation from highly proliferative Ck5+ cells, progressing through a Ck5+/Ck8+ intermediate cell state. Collectively, these data implicate a tumor initiating cell population defined by a mix of basal (Ck5) and luminal (Nkx3.1) markers that, under the influence of ERG expression, can expand and invade while retaining full luminal differentiation potential. Our results also suggest that widely held assumptions regarding luminal-specific expression of canonical markers such as Nkx3.1 and Probasin may need revision. Citation Format: Weiran Feng, Erik Ladewig, Nazifa Salsabeel, Huiyong Zhao, Young Sun Lee, Anuradha Gopalan, Hanzhi Luo, Wenfei Kang, Ning Fan, Eric Rosiek, Ignas Masilionis, Katia Manova-Todorova, Ronan Chaligné, Elisa de Stanchina, Brett S. Carver, Yu Chen, Dong Gao, Christina S. Leslie, Charles L. Sawyers. ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells [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 PR007.

Abstract B049: Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence

Abstract Background: In patients who receive definitive therapy for locally advanced prostate cancer, biochemical recurrence is due to local recurrence and/or the presence of undetected metastatic disease at the time of initial diagnosis and treatment. Approximately 25-40% of patients who receive radiotherapy plus androgen deprivation therapy (ADT) with curative intent are expected to recur within 5-10 years. We hypothesized that applying multiparametric (mp) MRI, gene expression, and genomic analyses to prostate tumors prior to EBRT+ADT would identify biomarkers predictive of recurrence (NCT01834001). Methods: At baseline and 6 months after completion of radiotherapy, mpMRI was performed and lesions were manually contoured. MR/ultrasound-fusion biopsies acquired at baseline targeted to up to three distinct MR lesions per patient. 29 patients with intermediate and high risk localized prostate cancer treated with EBRT+ADT with sufficient baseline biopsy tissue were selected for this analysis (median follow-up: 91 months). Tumor tissues were macrodissected to obtain DNA and RNA from 60 distinct biopsy regions (1-4 per patient). DNA was used for generating whole-exome sequencing libraries (92.5 × median coverage); RNA was converted into cDNA and hybridized to Affymetrix Human Exon 1.0 microarrays. Secondary gene expression cohorts with clinical annotation were obtained from the University of Miami and Queen’s University Belfast. Gene expression signatures were processed using Ingenuity Pathway Analysis (IPA) and the Decipher GRID. Results: Five of the 29 patients experienced biochemical failure by the time of data analysis; four of these patients were identified to have distant metastases with no evidence of local recurrence at the time of biochemical failure. Baseline and posttreatment lesion volumes by MRI and posttreatment ADCmax (apparent diffusion coefficient) were positively associated with recurrence (p&amp;lt;0.05). The Decipher signature positively correlated with these three MR features. Comparison of gene expression using a linear mixed-effect model identified 1,120 differentially expressed transcripts. IPA nominated TGF-beta signaling as the topmost enriched upstream regulator (z=3.32). This result was validated in the Miami (z=2.54) and Belfast (z=3.67) cohorts. By network analysis, genes that independently tracked with the baseline and posttreatment volumes and posttreatment ADCmax MR features similarly converged to the TGF-beta pathway. Co-occurrence of biallelic TP53 alterations with 1- or 2-copy PTEN losses was observed exclusively in patients who recurred. Conclusions: Genomic, transcriptomic and radiomic analyses have nominated predictive biomarkers that identify the subset of patients with locally advanced prostate cancer destined to recur after definitive EBRT+ADT. Identification of molecular features predictive of failure suggests that aggressive pre-existing subclones harboring these alterations have metastatic potential. Emerging systemic therapies including TGF-beta ligand traps targeted to these tumors may improve overall outcomes. Citation Format: Adam G. Sowalsky, Anson T. Ku, Uma Shankavaram, Shana Y. Trostel, Houssein A. Sater, Stephanie A. Harmon, Nicole V. Carrabba, Yang Liu, Radka Stoyanova, Elai Davicioni, Alan Pollack, Baris Turkbey, Deborah E. Citrin. Radiogenomic profiling of prostate tumors prior to external beam radiotherapy (EBRT) converges on a transcriptomic signature of TGF-beta activity driving tumor recurrence [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 B049.

Dual targeting of melanoma translation by MNK/eIF4E and PI3K/mTOR inhibitors

Melanoma is relatively resistant to chemotherapy, and no targeted therapies are fully effective. The most common mutations in melanoma result in hyperactivation of the mitogen-activated protein kinase (MAPK) and PI3K/AKT/ mTOR pathways responsible for initiating and controlling oncogenic protein translation. This makes both the signaling pathways potentially important therapeutic targets in melanoma. Our studies were carried out on human melanoma cell lines WM793 and 1205 LU with similar genomic alteration (BRAFV600E and PTEN loss). We used a highly specific PI3K/mTOR inhibitor, dactolisib (NVP-BEZ235), and Mnk inhibitor - CGP57380 alone and in combination. Here, we explore the mechanism of action of these drugs alone and in combination, as well as their effect on the viability and invasiveness of melanoma cells. Although when used independently, both drugs suppressed cell proliferation and migration, their combination has additional antitumor effects. We demonstrate that simultaneous inhibition of both pathways may prevent possible drug resistance.

PTEN potentiation of oncolytic HSV therapy for glioblastoma

PTEN potentiation of oncolytic HSV therapy for glioblastoma

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Background: Emerging evidence suggests the prognostic impact of the tumor microenvironment (TME) in peripheral T-cell lymphomas (PTCLs). To better understand PTCL pathobiology, we performed an integrative multi-omics study to explore the genetic subtypes and the TME signatures of PTCLs, especially nodal T-follicular helper T-cell lymphomas (nTFHLs) and PTCL, not otherwise specified (PTCL-NOS). Method: We performed whole-exome sequencing of 92 nTFHLs and 37 PTCL-NOSs, a total of 129 cases. Ninety-two nTFHLs included 85 angioimmunoblastic T-cell lymphomas (AITLs), four nPTCL with TFH phenotypes, two follicular T-cell lymphomas, and one unclassifiable nTFHL. Genetic drivers were integrated using non-negative matrix factorization consensus clustering (NMF). We estimated the proportion of immune cell fractions from bulk RNA sequencing data (n = 57) using CIBERSORTx and classified the TME signatures using hierarchical clustering. Results: NMF clustering analysis revealed three genetic subtypes within our cohort, except for 10 cases lacking recurrent genetic abnormalities (denoted as cluster 0). C2 was characterized by TP53 mutation, CDKN2A loss, PTEN loss, and chromosomal instability, corresponding to the previously described GATA3-PTCL subtype. C1 and C3 shared TFH-related genomic alterations, but C3 had significantly higher frequencies of chromosome (Chr) 5 gain, Chr 21 gain, RHOA, IDH2, and CD28 mutations. A higher frequency of rash and higher C-reactive protein levels than the other AITLs characterized C3-AITL. Pairwise association analysis of genomic alterations revealed that multiple TET2 mutations, rather than a single TET2 mutation, significantly co-occurred with IDH2 and RHOA mutations. A Bradley-Terry model estimated that the first TET2 and DNMT3A mutations occurred earlier, and the second or later TET2, RHOA, and IDH2 mutations occurred later. Compared to C1, significantly inferior survival was observed in C2 (hazard ratio (HR), 2.52; 95% CI, 1.37–4.63) and C3(HR, 2.14; 95% CI, 1.17–3.89; Figure A). The TME signatures were divided into TME1 (B-cells and follicular helper T-cells), TME2 (activated memory CD4 T-cells, M2 macrophages, and CD8 T-cells), and TME3 (naïve CD4 T-cells and activated mast cells). mTORC1 signaling was enriched in the TME2 signature compared to TME1 and TME3 signatures. The TME2 signature was significantly associated with shorter survival than the TME3 signature (HR: 3.4, 95% CI 1.6–7.5; Figure B). C2 had significantly more TME2 signature than C1 (64.3% vs. 7.7%), while C1 had significantly more TME3 signature than C2 (80.8% vs. 28.6%, p = 0.006; Figure C). Keywords: genomics, epigenomics, and other -omics, microenvironment Encore Abstract—previously submitted to EHA 2023 The research was funded by: Japan Society for the Promotion of Science KAKENHI grants (grant numbers: 19K23879, 22K19451, and 21H02945) and Japan Agency for Medical Research and Development (AMED) grants (20ck0106544 and 21ck0106644)