Clear Cell Renal Carcinoma Tumors Research Articles

Novel Peptide-Based 68Ga-Labeled Radiotracer for Preclinical Studies of TIM3 Expression.

T-cell immunoglobulin and mucin domain-3 (TIM3) is an immune checkpoint that plays a negative regulatory role in the immune response. TIM3-targeted drugs inhibit this negative regulation, thereby modulating the level of immune response activation. In the previous investigation, several peptides targeting TIM3 were identified through screening from a phage peptide library. In this research, three peptides were selected to construct the radioactive molecular probes according to the characteristic that targeting TIM3 drugs would lead to the increase of interferon-γ (IFN-γ) secretion. Molecular docking was performed to assess the binding properties of the selected peptides with the TIM3 protein. To further enhance the targeting properties, one of the peptides with a higher-affinity peptide was structurally modified. Then, 68Ga was used to construct the peptide probe 68Ga-DOTA-peptide by labeling the six peptides with 68Ga riboprobes, and the binding affinity and specificity were assessed using TIM3 overexpressing cell line A549TIM3 and the parental A549 cells. In addition, in Micro-PET/CT imaging, transfected model mice were dynamically imaged for 30 min after injection of 3.7-7.4 MBq 68Ga-DOTA-peptides via the tail vein. Meanwhile, the same dose of molecular probes was injected in the MC38 model (colorectal cancer in mice) and the CCRCC (clear cell renal cell carcinoma) xenografted model, followed by static scans at 15, 30, and 60 min postinjection. Finally, immunohistochemical (IHC) staining was performed to assess TIM3 expression in the dissected tumor tissues. The molecular docking results showed that the binding energy of P26 to TIM3 protein was -6.5 kcal/mol, which was lower than that of P24 to TIM3 protein, -3.6 kcal/mol, indicating that the affinity of P26 peptide to TIM3 protein was higher than that of P24 and P20 peptide. After structural modification of the P26 peptide, P26NH2, r-NH2, and P26X2 were obtained, and the above peptides were successfully constructed into six targeting TIM3 peptide probes by 68Ga labeling. Cellular uptake experiments demonstrated that 68Ga-DOTA-P26, 68Ga-DOTA-P26NH2, and 68Ga-DOTA-r-NH2 showed significantly higher uptake in A549TIM3 cells than in A549 cells and could be blocked by the unlabeled peptide. Micro-PET imaging experiments showed that the uptake of each probe in the A549TIM3 model tumor tissue was significantly higher than that in the A549 model tumor tissue, and a comparison of the tumor-to-cardiac uptake ratios of each group showed that the 68Ga-DOTA-P26 had a better tumor-to-cardiac uptake ratio in the A549TIM3 model than several other molecular probes, and in the MC38 model, similar results were obtained, with the difference that the 68Ga-DOTA-P26NH2 had the highest tumor-to-cardiac uptake ratio in the CCRCC model. Finally, validation by IHC showed that A549TIM3, MC38, and CCRCC tumor tissues had varying degrees of TIM3 expression. Upon comparison of ex vivo and in vivo studies, one of them, the 68Ga-DOTA-P26 probe, demonstrated significant target specificity for TIM3. These results suggest that studying peptide probes targeting TIM3 will promote the process of TIM3-targeted drug research and is expected to guide the application of TIM3 immune checkpoint drugs in immunotherapy.

Renal cancer cells acquire immune surface protein through trogocytosis and horizontal gene transfer.

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9 (CAIX), a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells in tumor cells following trogocytosis. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells during trogocytosis. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expressed both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other types of cancers.

Knockdown of PIK3R6 impedes the onset and advancement of clear cell renal cell carcinoma

ABSTRACT In this research, we investigated the role of PIK3R6, a regulatory subunit of PI3Kγ, known for its tumor-promoting properties, in clear cell renal cell carcinoma (CCRCC). Utilizing the UALCAN website, we found PIK3R6 upregulated in CCRCC, correlating with lower survival rates. We compared PIK3R6 expression in CCRCC tumor tissues and adjacent normal tissues using immunohistochemistry. Post RNA interference-induced knockdown of PIK3R6 in 786-O and ACHN cell lines, we performed CCK-8, colony formation, Edu staining, flow cytometry, wound healing, and transwell assays. Results showed that PIK3R6 silencing reduced cell proliferation, migration, and invasion, and induced G0/G1 phase arrest and apoptosis. Molecular analysis revealed decreased CDK4, Cyclin D1, N-cadherin, Vimentin, Bcl-2, p-PI3K and p-AKT, with increased cleaved caspase-3, Bax, and E-cadherin levels in CCRCC cells. Moreover, inhibiting PIK3R6 hindered tumor growth. These findings suggest a significant role for PIK3R6 in CCRCC cell proliferation and metastasis, presenting it as a potential therapeutic target.

Abstract 4357: Examining the role of end-stage kidney as a potential intermediate stage in the development of renal cell carcinomas; understanding the pathogenesis allowing for early detection

Abstract Introduction: Clear cell renal cell carcinoma (CCRCC) and papillary renal cell carcinoma (PRCC) are both thought to arise from the renal proximal tubules. End stage renal disease (ESRD) patients have an increased incidence of developing PRCC and CCRCC. Some studies showed a progenitor cell population that is inherently present in renal tubules, and is significantly increased after renal injury. The studies have also illustrated similarities between these progenitor cells and PRCC & CCRCC; hypothesizing that they are the cell of origin of these tumors. This trend was not observed in other renal cell carcinomas such as chromophobe renal cell carcinoma (chRCC). This study aims to further examine the possible role of ESRD in the development of PRCC and CCRCC to understand this initial step of renal cell carcinoma pathogenesis. Methods: A cohort of n = 39 PRCC, 25 CCRCC, 63 normal, 10 end-stage and 18 chRCC cases were selected. RNA sequencing was performed and differential-gene-expression analysis was conducted between normal, ESRD and tumor using DESeq2 software. Pathway analysis was performed using GSEA. Genes from oncogenic pathways that have been studied to be involved in renal cancer development were selected to perform consensus clustering analysis on Genepattern. Results: Oncogenic and developmental pathways enriched in ESRD are consistently overlapping with that of CCRCC and PRCC tumors. 50% of end-stage developmental pathways are found in both CCRCC and PRCC but only 4.5% in chRCC. 62.4% and 46.4% of end-stage oncogenic pathways are found in CCRCC and PRCC respectively but only 16% in chRCC. Clustering analysis showed ESRD cases clustered together with CCRCC and PRCC groups and not with chRCC. Overall, the ESRD cases exhibited a gene expression profile more closely resembling that of CCRCC and PRCC than chRCC. Conclusion: Our findings support the hypothesis that ESRD provides a favorable environment for tumor growth promoting pathogenesis of both PRCC and CCRCC. The enrichment of the same developmental pathways in both ESRD and the tumors suggests that the progenitor cell population in the renal tubules, mentioned above, could be the origin of both PRCC and CCRCC lesions. Further analysis would allow the uncovering of the pathways that lead to tumor progression from these cells of origin, and can be used to determine biomarkers for early detection of CCRCC and PRCC. This is of particular benefit for those with increased risk as the ESRD patients. Citation Format: Mingyuan Wan, Vincent Castillo, Rola Saleeb. Examining the role of end-stage kidney as a potential intermediate stage in the development of renal cell carcinomas; understanding the pathogenesis allowing for early detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4357.

High TEAD4 Expression is Associated With Aggressive Clear Cell Renal Cell Carcinoma, Regardless of YAP1 Expression.

Yes-associated protein 1 (YAP1) and transcriptional coactivator TEA domain transcription factor 4 (TEAD4) are the main effectors of the Hippo signaling pathway. Deregulation of the Hippo signaling pathway significantly impacts tumorigenesis and tumor progression. We evaluated the mRNA expression level of YAP1 and TEAD4 using the Gene Expression Profiling Interactive Analysis database and investigated the roles of YAP1 and TEAD4 in 349 surgically resected clear cell renal cell carcinoma (CCRCC) samples through immunohistochemical analysis. High YAP1 and TEAD4 expression were observed in 57 (16.3%) and 131 (37.5%) cases, respectively. High YAP1 expression was associated with a low nuclear grade only. High TEAD4 expression was significantly associated with large tumor size, high nuclear grade, lymphovascular invasion, advanced pT classification, advanced clinical stage, sarcomatous differentiation, and metastasis. CCRCC with YAP1-low/TEAD4-high expression was significantly associated with aggressive clinicopathological variables and poor outcomes. For CCRCC, higher tumor stage, sarcomatous differentiation, and metastasis were the independent prognostic factors for overall survival (OS) and disease-free survival (DFS). High TEAD4 expression was significantly associated with short OS and DFS but was not an independent prognostic factor. High TEAD4 and YAP1-low/TEAD4-high expression significantly correlated with adverse clinicopathological factors and worse OS and DFS in patients with CCRCC. YAP1 expression was not significantly associated with clinicopathological factors or patient survival. Therefore, TEAD4 plays a critical role in CCRCC tumor progression independent of YAP1 and may be a potential biomarker and therapeutic target for CCRCC.

Abstract A012: Investigating the heterogeneity and clinical significance of tumor-associated macrophages in renal cell carcinoma milieu

Abstract Background: Tumor-associated macrophages (TAMs) exert a range of immunomodulatory functions in the tumor microenvironment (TME) and have been considered key regulators of tumor development. The interactions of TAMs with cancer and stromal cells in the TME have been shown to promote and sustain tumor progression; however, TAMs are largely phenotypically and functionally heterogeneous. Studies at a single-cell level are therefore necessary to understand biological and clinical relevance of heterogeneity of TAMs. Objective: To investigate the heterogeneity of TAM populations in renal cell carcinoma (RCC), the most common form of Kidney cancer, and to determine their connections to patient outcome. Methods and Results: We leverage single-cell RNA sequencing analysis of six primary RCC tumors and three Lung metastatic clear cell RCC (ccRCC) tumors, from which we identified eight TAM and two monocyte clusters shared across all nine patient tissue samples. Differential gene expression and pathway analyses revealed distinct subsets of upregulated genes and cellular pathways activated in each subpopulation. Survival analysis using gene signatures characteristic of each TAM population in datasets of ccRCC tumors (n=533) from TCGA revealed two subpopulations associated with poor patient outcome in ccRCC. Of these, one subpopulation, which we labelled as TAM-8, was characterized by upregulation of pro-inflammatory cytokines, chemokines, and growth factors such as CCL20, CXCL8, and IL1A/B. To our knowledge, this TAM population has not been described in RCC previously. We performed additional in silico functional analyses to further characterize the function of TAM-8 subpopulation, which indicated an active role in promoting immune cell infiltration and angiogenesis via various pro-inflammatory signaling pathways. Conclusion: This study provides new insight into the heterogeneity of TAM populations in RCC and defines CCL20/CXCL8/IL1High TAM-8 population as a potential driver of poor outcome and a candidate therapeutic target. Citation Format: Evelyn M. Zavacky, Minjun Kim, Ariel Madrigal, Zohreh Mehrjoo, Jonathon Spicer, Simon Tanguay, Fadi Brimo, Hamed Najafabadi, Yasser Riazalhosseini. Investigating the heterogeneity and clinical significance of tumor-associated macrophages in renal cell carcinoma milieu [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A012.

Deciphering intratumor heterogeneity in clear cell renal cell carcinoma utilizing clinicopathologic and molecular platforms

Clear cell renal cell carcinoma (CCRCC) is a common renal malignancy known for its lethality and chromosome 3p aberrancies associated with loss of VHL. It has been shown that additional prognostic molecular markers exist in other transcriptional modifiers such as BAP1 and SETD2. Molecular heterogeneity has been described between primary and metastatic sites as well as genetic diversity in spatial tumor analysis; however, morphologic and proteogenomic heterogeneity information is lacking. We assessed 77 nephrectomy specimens with a diagnosis of CCRCC for morphologic architectural patterns including nodular growth patterns and variations in WHO/ISUP grade. Evaluation of highly heterogeneous areas with immunohistochemical (IHC) staining for BAP1, UCHL1, SETD2, and CAIX was performed and correlated with morphologic and histology data. Ultimately, high variability in the morphologic and histological findings matched the complexity of the IHC findings. Alterations in expression of CAIX and UCHL1 correlated with alterations in transcriptional regulators BAP1 and SETD2 within the tumor. High-grade morphology, such as eosinophilia, were areas enriched for alteration of biomarker expression. This highly complex data set of morphologic and biomarker characteristics highlights the heterogeneity of morphology amongst high-grade CCRCC tumors.

Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis.

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

A Novel Biomarker, FKBP10, for Poor Prognosis Prediction in Patients with Clear Cell Renal Cell Carcinoma.

Objective To screen genes associated with poor prognosis of clear cell renal cell carcinoma (CcRCC) from the public databases HPA (Human Protein Atlas), UALCAN, and GEPIA (Gene Expression Profiling Interactive Analysis) and to investigate the expression of FKBP10 in CcRCC and the effect on prognosis of the patients and the biological behavior of CcRCC cells. Methods The tumor tissues and adjacent noncancerous tissues of 42 patients with CcRCC diagnosed and treated in our hospital were collected, and the general information of the patients was recorded. FKBP10 expression in the tissues was determined by qRT-PCR and western blot, and its relationship with general information and prognosis of patients was analyzed. Knockdown or overexpression experiments were carried out with the human proximal tubule epithelial cell line HK-2 and CcRCC cell lines Caki-1, 786-O, ACHN, and A498 to verify the relationship between FKBP10 expression and cell proliferation and adhesion ability using MTT assay and fibronectin adhesion assay, respectively. Western blot was utilized to examine the protein expression level of c-Myc, cyclin D1, and Bcl-2 in the cells. Results FKBP10 was highly expressed in CcRCC tissues and cells and was correlated with poor prognosis. In addition, FKBP10 expression was positively correlated with CcRCC tumor size and staging and negatively correlated with tumor differentiation. Moreover, knockdown of FKBP10 significantly inhibited the proliferation of CcRCC cells, notably declined the protein expression of c-Myc, cyclin D1, and Bcl-2, and promoted cell adhesion. Conclusion FKBP10 is highly expressed in CcRCC tissues and cells and is associated with poor prognosis in patients. FKBP10 participated in the occurrence and development of CcRCC by promoting cell proliferation and inhibiting apoptosis and adhesion.

Identification of FDFT1 as a potential biomarker associated with ferroptosis in ccRCC.

Renal cell carcinoma (RCC) seriously threatens people's lives and health. The identification of some precise biomarkers during the process of RCC progression and the pathophysiologic procedure is critical for improving the diagnosis and management of RCC. Evidence suggests that ferroptosis may play a pivotal role in eradicating clear cell RCC (ccRCC, KIRC) tumor cells. We screened out the target prognostic ferroptosis-associated genes and examined the functions of farnesyl-diphosphate farnesyltransferase (FDFT1) in 786-O cells by plasmid transfection. In our study, we identified FDFT1 as a potential marker correlating with ferroptosis in KIRC. Upregulated FDFT1 inhibited cell proliferation, migration, and invasion, and the underlying antitumor effects may occur via the AKT signaling pathway. Our study provides helpful evidence to study the complex physiopathology of KIRC.

T and NK cell abundance defines two distinct subgroups of renal cell carcinoma

ABSTRACT Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3high, 25/52) and NK cells (NKhigh, 27/52). CD3high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.

Expression of transient receptor potential melastatin 4 in differential diagnosis of eosinophilic renal tumors.

Immunohistochemical and molecular studies to differentiate eosinophilic kidney tumors are gradually increasing. The present study investigated the role of transient receptor potential cation channel subfamily M member 4 (TRPM4), a non-selective cation channel associated with migration, proliferation and invasion in cancer cells, in this differentiation. The aim was to investigate the effectiveness of TRPM4 in differentiation of eosinophilic kidney tumors. The study included a total of 112 patients, including 97 eosinophilic kidney tumors with the diagnoses of 33 eosinophilic clear cell renal cell carcinoma (CCRCC), 35 eosinophilic chromophobe renal cell carcinoma (ChRCC), 8 papillary renal cell carcinoma type 2 (P2RCC), 21 renal oncocytoma (RO), as well as 15 papillary renal cell carcinoma type 1 to differentiate from P2RCC. For TRPM4, diffuse staining (>10%) was observed in 2 CCRCC, 15 ChRCC, 20 RO and 4 P2RCC cases. There was a significant difference between eosinophilic CCRCC and other eosinophilic tumors (P<0.05). While basolateral staining was observed in papillary tumors, membrane staining was observed in other stained cases. It was hypothesized that the use of TRPM4 along with morphological findings, cytokeratin 7 and other markers may be useful for the differentiation of eosinophilic kidney tumors.

Sequencing of Renal Mass Biopsy and Ablation: Results from the National Cancer Database.

We assessed current utilization and sequencing of renal mass biopsy (RMB) and thermal ablation for renal cell carcinoma (RCC) patients in the United States. The 2004-2014 National Cancer Database was queried for adult patients with histopathologically diagnosed American Joint Committee on Cancer stage I RCC ≤5 cm undergoing RMB and thermal ablation. RMB sequencing was stratified into "staged RMB" (separate sessions for RMB/ablation) or "concomitant RMB" (same date RMB/ablation). Demographics, cancer variables and time trends were compared. Univariate and multivariable logistic regression identified predictors of concomitant RMB, and evaluated their impact on inpatient hospital stay and 30-day unplanned readmissions. A total of 6,323 patients were included, of whom 2,913 (46.1%) underwent staged RMB and 3,410 (53.9%) underwent concomitant RMB. Concomitant RMB was more frequently performed until 2012, and staged RMB thereafter (trend p <0.001). In multivariable logistic regression models, private insurance, clear cell RCC, right-sided disease and smaller tumor diameter demonstrated higher probability of concomitant RMB. Furthermore, geographical differences across the United States were evident (concomitant RMB West/South Central region 64.6% versus New England 29.3%, multivariable p <0.05). Concomitant RMB was associated with greater odds of inpatient hospital stay (multivariable OR=1.42, 95% CI=1. 27-1. 60; p <0.001) and 30-day unplanned hospital readmissions (OR=1.55, 95% CI=1.07-2.27; p=0.022). Staged RMB is more frequently performed in more recent years and is associated with lower odds of inpatient hospital stay and unplanned readmissions within 30 days. These potential benefits, combined with the potential to limit ablation of benign tumors, need to be weighed against additional health care costs and inconvenience associated with 2 interventions.

Rac Signaling Drives Clear Cell Renal Carcinoma Tumor Growth by Priming the Tumor Microenvironment for an Angiogenic Switch.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.

Characterization of clear cell renal cell carcinoma and other renal tumors: evaluation of dual-energy CT using material-specific iodine and fat imaging.

This study aimed to assess material-specific iodine and fat images for diagnosis of clear cell renal cell carcinoma (cc-RCC) compared to papillary RCC (p-RCC) and other renal masses. With IRB approval, we identified histologically confirmed solid renal masses that underwent rapid-kVp-switch DECT between 2016 and 2018: 25 cc-RCC (7 low grade versus 18 high grade), 11 p-RCC, and 6 other tumors (2 clear cell papillary RCC, 2 chromophobe RCC, 1 oncocytoma, 1 renal angiomyomatous tumor). A blinded radiologist measured iodine and fat concentration on material-specific iodine-water and fat-water basis pair images. Comparisons were performed between groups using univariate analysis and diagnostic accuracy calculated by ROC. Iodine concentration was higher in cc-RCC (6.14 ± 1.79mg/mL) compared to p-RCC (1.40 ± 0.54mg/mL, p < 0.001), but not compared to other tumors (5.0 ± 2.2mg/mL, p = 0.370). Intratumoral fat was seen in 36.0% (9/25) cc-RCC (309.6 ± 234.3mg/mL [71.1-762.3ng/mL]), 9.1% (1/11) papillary RCC (97.11mg/mL), and no other tumors (p = 0.036). Iodine concentration ≥ 3.99mg/mL achieved AUC and sensitivity/specificity of 0.88 (CI 0.76-1.00) and 92.31%/82.40% to diagnose cc-RCC. To diagnose p-RCC, iodine concentration ≤ 2.5mg/mL achieved AUC and sensitivity/specificity of 0.99 (0.98-1.00) and 100%/100%. The presence of intratumoral fat had AUC 0.64 (CI 0.53-0.75) and sensitivity/specificity of 34.6%/93.8% to diagnose cc-RCC. A logistic regression model combining iodine concentration and presence of fat increased AUC to 0.91 (CI 0.81-1.0) with sensitivity/specificity of 80.8%/93.8% to diagnose cc-RCC. Iodine concentration values are highly accurate to differentiate clear cell RCC from papillary RCC; however, they overlap with other tumors. Fat-specific images may improve differentiation of clear cell RCC from other avidly enhancing tumors. • Clear cell renal cell carcinoma (RCC) has significantly higher iodine concentration than papillary RCC, but there is an overlap in values comparing clear cell RCC to other renal tumors. • Iodine concentration ≤ 2.5mg/mL is highly accurate to differentiate papillary RCC from clear cell RCC and other renal tumors. • The presence of microscopic fat on material-specific fat images was specific for clear cell RCC, helping to differentiate clear cell RCC from other avidly enhancing renal tumors.

Abstract 1086: Immune checkpoint inhibitors CD300A and LAIR1 are associated with poor prognosis in clear cell renal cell carcinoma

Abstract Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) have been shown to improve the survival of patients with clear cell renal cell carcinoma (CCRCC). However, a benefit for CCCRC patients was observed with the FDA-approved PD-1 inhibitor Nivolumab irrespective of PD-1 ligand (PD-L1) expression status indicating there are potentially additional CCRCC checkpoint molecules worth exploring for targeted therapy. We therefore analyzed RNAseq data (n=534, CCRCCs; n=72, normal kidney specimens) from the TCGA project, and found that PD-L1 was not upregulated in CCRCC when compared to normal tissue whereas other checkpoint members i.e. CD300A and LAIR1 were among the most significantly upregulated genes in CCRCCs. In terms of overall survival, high expression of PD-L1 was associated with better outcome in CCRCCs while high expression of CD300A and LAIR1 were significantly associated with worse outcomes. We confirmed these findings in two independent cohorts of CCRCC patients. Furthermore, we performed cell type enrichment analysis, which showed that CD300A and LAIR1 are most likely expressed by tumor-associated macrophages in the CCRCC tumor microenvironment. Our results suggest that PD1/PDL1 might be suboptimal targets in CCRCC and that there are additional unexplored immune checkpoint molecules that warrant our attention. Note: This abstract was not presented at the meeting. Citation Format: Jonas Sjolund, David Lindgren, Helén Nilsson, Martin E. Johansson, Hakan Axelson, Kristian Pietras. Immune checkpoint inhibitors CD300A and LAIR1 are associated with poor prognosis in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1086.

Current Landscape and the Potential Role of Hypoxia-Inducible Factors and Selenium in Clear Cell Renal Cell Carcinoma Treatment.

In the last two decades, the discovery of various pathways involved in renal cell carcinoma (RCC) has led to the development of biologically-driven targeted therapies. Hypoxia-inducible factors (HIFs), angiogenic growth factors, von Hippel–Lindau (VHL) gene mutations, and oncogenic microRNAs (miRNAs) play essential roles in the pathogenesis and drug resistance of clear cell renal cell carcinoma. These insights have led to the development of vascular endothelial growth factor (VEGF) inhibitors, Mechanistic target of rapamycin (mTOR) inhibitors, and immunotherapeutic agents, which have significantly improved the outcomes of patients with advanced RCC. HIF inhibitors will be a valuable asset in the growing therapeutic armamentarium of RCC. Various histone deacetylase (HDAC) inhibitors, selenium, and agents like PT2385 and PT2977 are being explored in various clinical trials as potential HIF inhibitors, to ameliorate the outcomes of RCC patients. In this article, we will review the current treatment options and highlight the potential role of selenium in the modulation of drug resistance biomarkers expressed in clear cell RCC (ccRCC) tumors.

Multiregion Quantification of Extracellular Signal-regulated Kinase Activity in Renal Cell Carcinoma

To personalize treatment for renal cell carcinoma (RCC), it would be ideal to confirm the activity of druggable protein pathways within individual tumors. We have developed a high-resolution nanoimmunoassay (NIA) to measure protein activity with high precision in scant specimens (eg, fine needle aspirates [FNAs]). Here, we used NIA to determine whether protein activation varied in different regions of RCC tumors. Since most RCC therapies target angiogenesis by inhibiting the vascular endothelial growth factor (VEGF) receptor, we quantified phosphorylation of extracellular signal-regulated kinase (ERK), a downstream effector of the VEGF signaling pathway. In 90 ex vivo FNA biopsies sampled from multiple regions of 38 primary clear cell RCC tumors, ERK phosphorylation differed among patients. In contrast, within individual patients, we found limited intratumoral heterogeneity of ERK phosphorylation. Our results suggest that measuring ERK in a single FNA may be representative of ERK activity in different regions of the same tumor. As diagnostic and therapeutic protein biomarkers are being sought, NIA measurements of protein signaling may increase the clinical utility of renal mass biopsy and allow for the application of precision oncology for patients with localized and advanced RCC. Patient summaryIn this report, we applied a new approach to measure the activity of extracellular signal-regulated kinase (ERK), a key cancer signaling protein, in different areas within kidney cancers. We found that ERK activity varied between patients, but that different regions within individual kidney tumors showed similar ERK activity. This suggests that a single biopsy of renal cell carcinoma may be sufficient to measure protein signaling activity to aid in precision oncology approaches.

Targeting the Mevalonate Pathway Suppresses VHL-Deficient CC-RCC through an HIF-Dependent Mechanism.

Clear cell renal cell carcinoma (CC-RCC) is a devastating disease with limited therapeutic options available for advanced stages. The objective of this study was to investigate HMG-CoA reductase inhibitors, also known as statins, as potential therapeutics for CC-RCC. Importantly, treatment with statins was found to be synthetically lethal with the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in 90% of CC-RCC driving the disease. This effect has been confirmed in three different CC-RCC cell lines with three different lipophilic statins. Inhibition of mevalonate synthesis by statins causes a profound cytostatic effect at nanomolar concentrations and becomes cytotoxic at low micromolar concentrations in VHL-deficient CC-RCC. The synthetic lethal effect can be fully rescued by both mevalonate and geranylgeranylpyrophosphate, but not by squalene, indicating that the effect is due to disruption of small GTPase isoprenylation and not the inhibition of cholesterol synthesis. Inhibition of Rho and Rho kinase (ROCK) signaling contributes to the synthetic lethality effect, and overactivation of hypoxia-inducible factor signaling resulting from VHL loss is required. Finally, statin treatment is able to inhibit both tumor initiation and progression of subcutaneous 786-OT1-based CC-RCC tumors in mice. Thus, statins represent potential therapeutics for the treatment of VHL-deficient CC-RCC. Mol Cancer Ther; 17(8); 1781-92. ©2018 AACR.

Abstract B059: SETD2-PI3Kβ synthetic lethality

Abstract Introduction: Renal cell carcinoma (RCC) is a high-risk and high-mortality cancer for which several new targeted therapies exist. However, responses of primary clear cell RCC (ccRCC) tumors and metastases to these targeted agents are variable, and complete responses are relatively uncommon, underscoring the need to identify reliable biomarkers to predict tumor behavior. Mutations in the tumor suppressor gene SETD2, encoding a lysine methyltransferase, occur in 3-16% of ccRCC tumors while the SETD2 protein deficiency rate has been reported to be as high as 34%. The phosphatidylinositide 3-kinases (PI3Ks) have emerged as important therapeutic targets for the treatment of solid and hematologic malignancies, including ccRCC. In this study, we determine the extent to which small-molecule inhibitors against the p110α, p110β, and p110δ isoforms of PI3K inhibit growth of RCC with SETD2 loss. Methods: RNAi oligonucleotides and small-molecule inhibitors targeting the p110α (BYL719), p110β (TGX221, AZD8186), and p110δ (idelalisib) isoforms of PI3K were tested against ccRCC cell lines with SETD2 loss. The effect of drugs on cell growth and survival was determined by (1) proliferation in monolayer, (2) 3D growth in Matrigel, and (3) cell migration in wound healing assay. Downstream signaling pathways were evaluated by immunoblot analysis. Results: Genetic and pharmacologic inhibition of p110β resulted in inhibition of ccRCC cell lines with SETD2 loss. ccRCC-derived SETD2 deficient (-/-) 786-O and A498 cells exhibited increased sensitivity to TGX221 (IC50 = 2.3 - 4.5 µM, respectively) and AZD8186 (IC50 = 1.79 - 0.24 µM, respectively) as compared to SETD2 proficient (+/+) 786-O cells (IC50 = &amp;gt;10 µM). Inhibition of the p110δ isoform alone had a modest effect on ccRCC cell growth and migration. Importantly, genetic (with siRNA) and pharmacologic (with BYL719) inhibition of p110α had no effect on the ccRCC cell lines. Inhibition of AKT with the small-molecule inhibitor MK2206 recapitulated the effects seen with AZD8186 in ccRCC cell lines with SETD2 loss, suggesting PI3Kβ mediates the synthetic lethality via AKT signaling in the context of SETD2 loss in ccRCC. Conclusions: Our work strongly suggests a synthetic lethal-type of interaction between PI3Kβ inhibition and SETD2 loss and offers a rationale for further translational and clinical investigation of small-molecule inhibitors of PI3Kβ in RCC. Xenograft studies are ongoing to investigate the in vivo efficacy of AZD8186. Citation Format: Esteban Terzo, Valerie M. Jansen, Yun-Chen Chiang, Kimryn W. Rathmell. SETD2-PI3Kβ synthetic lethality [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B059.