SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-ß-independent manner.

Histone-lysine N-methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial-to-mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor-beta-independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA-seq and assay for transposase-accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild-type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell-intrinsic and cell-extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

Cancer associated macrophage-like cells in metastatic renal cell carcinoma predicts for poor prognosis and tracks treatment response in real time.

Renal Cell Carcinoma (RCC) is a fatal urological cancer, with one third of patients diagnosed with metastasis, resulting in a 5-year survival of only 12%. Recent advancements in therapies have increased survival in mRCC, but lack efficacy in subtypes, due to treatment resistance and toxic side effects. Currently, white blood cells, hemoglobin, and platelets are limitedly used as blood based biomarkers to help determine RCC prognosis. Cancer associated macrophage-like cells (CAMLs) are a potential mRCC biomarker which have been identified in peripheral blood of patients with malignant tumors and have been shown to predict poor clinical patient outcomes based on their number and size. In this study, blood samples from 40 RCC patients were obtained to evaluate the clinical utility of CAMLs. CAML changes were monitored during treatment regimens to evaluate their ability to predict treatment efficacy. It was observed that patients with smaller CAMLs had better progression free survival (HR = 2.84, 95% CI 1.22-6.60, p = 0.0273) and overall survival (HR = 3.95, 95% CI 1.45-10.78, p = 0.0154) versus patients with larger CAMLs. These findings suggest that CAMLs can be used as a diagnostic, prognostic, and predictive biomarker for patients with RCC which may help improve management of advanced RCC.

Generation and characterization of a monoclonal antibody that binds to Galectin-1.

Galectin-1 is a ß-galactoside-binding lectin that has been implicated as a suppressive molecule in cancer and autoimmune diseases. Gal-1 has known immunomodulatory activity and was found to be expressed on regulatory T cells, leading to the potential for targeted immunotherapies. Anti-Gal-1 monoclonal antibodies were generated in this study using classical hybridoma techniques. MAb 6F3 was found to bind to Gal-1 by Western blot and ELISA. Flow cytometry was used to determine cell surface and intracellular binding of mAb 6F3 to Gal-1 in PBMC-derived Tregs and tumor cells, including Treg-like cell lines. These results suggest mAb 6F3 may be used to further study Gal-1 protein expression and function.

Extensive intratumor regional epigenetic heterogeneity in clear cell renal cell carcinoma targets kidney enhancers and is associated with poor outcome.

BACKGROUND: Clear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations. RESULTS: A multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential. CONCLUSION: Taken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers.

Performing Automatic Identification and Staging of Urothelial Carcinoma in Bladder Cancer Patients Using a Hybrid Deep-Machine Learning Approach.

Accurate clinical staging of bladder cancer aids in optimizing the process of clinical decision-making, thereby tailoring the effective treatment and management of patients. While several radiomics approaches have been developed to facilitate the process of clinical diagnosis and staging of bladder cancer using grayscale computed tomography (CT) scans, the performances of these models have been low, with little validation and no clear consensus on specific imaging signatures. We propose a hybrid framework comprising pre-trained deep neural networks for feature extraction, in combination with statistical machine learning techniques for classification, which is capable of performing the following classification tasks: (1) bladder cancer tissue vs. normal tissue, (2) muscle-invasive bladder cancer (MIBC) vs. non-muscle-invasive bladder cancer (NMIBC), and (3) post-treatment changes (PTC) vs. MIBC.

The impact of genetic aberrations on response to radium-223 treatment for castration-resistant prostate cancer with bone metastases.

BACKGROUND: Radium (Ra)-223 is an established treatment option for patients with metastatic castrate-resistant prostate cancer (mCRPC) who have symptomatic bone metastases without soft tissue disease. Studies have indicated genetic aberrations that regulate DNA damage response (DDR) in prostate cancer can increase susceptibility to treatments such as poly ADP-ribose polymerase inhibitors and platinum-based therapies. This study aims to evaluate mCRPC response to Ra-223 stratified by tumor genomics. METHODS: This is a retrospective study of mCRPC patients who received Ra-223 and genetic testing within the Mayo Clinic database (Arizona, Florida, and Minnesota) and Tulane Cancer Center. Patient demographics, genetic aberrations, treatment responses in terms of alkaline phosphatase (ALP) and prostate-specific antigen (PSA), and survival were assessed. Primary end points were ALP and PSA response. Secondary end points were progression-free survival (PFS) and overall survival (OS) from time of first radium treatment. RESULTS: One hundred and twenty-seven mCRPC patients treated with Ra-223 had germline and/or somatic genetic sequencing. The median age at time of diagnosis and Ra-223 treatment was 61.0 and 68.6 years, respectively. Seventy-nine (62.2%) had Gleason score ≥ 8 at time of diagnosis. 50.4% received prior docetaxel, and 12.6% received prior cabazitaxel. Notable alterations include TP53 (51.7%), BRCA 1/2 (15.0%), PTEN (13.4%), ATM (11.7%), TMPRSS2-ERG (8.2%), RB deletion (3.4%), and CDK12 (1.9%). There was no significant difference in ALP or PSA response among the different genetic aberrations. Patients with a TMPRSS2-ERG mutation exhibited a trend toward lower OS 15.4 months (95% confidence interval [CI] 10.0-NR) versus 26.8 months (95% CI 20.9-35.1). Patients with an RB deletion had a lower PFS 6.0 months (95% CI 1.28-NR) versus 9.0 months (95% CI 7.3-11.1) and a lower OS 13.9 months (95% CI 5.2-NR) versus 26.5 months (95% CI 19.8-33.8). CONCLUSIONS: Among mCRPC patients treated with Ra-223 at Mayo Clinic and Tulane Cancer Center, we did not find any clear negative predictors of biochemical response or survival to treatment. TMPRSS2-ERG and RB mutations were associated with a worse OS. Prospective studies and larger sample sizes are needed to determine the impact of genetic aberrations in response to Ra-223.

Intracellular Signaling Pathways Mediating Tyrosine Kinase Inhibitor Cardiotoxicity.

Tyrosine kinase inhibitors (TKIs) are used to treat several cancers; however, a myriad of adverse cardiotoxic effects remain a primary concern. Although hypertension (HTN) is the most common adverse effect reported with TKI therapy, incidents of arrhythmias (eg, QT prolongation, atrial fibrillation) and heart failure are also prevalent. These complications warrant further research toward understanding the mechanisms of TKI-induced cardiotoxicity. Recent literature has given some insight into the intracellular signaling pathways that may mediate TKI-induced cardiac dysfunction. In this article, we discuss the cardiotoxic effects of TKIs on cardiomyocyte function, signaling, and possible treatments.

MTAP deficiency creates an exploitable target for antifolate therapy in 9p21-loss cancers.

Methylthioadenosine phosphorylase, an essential enzyme for the adenine salvage pathway, is often deficient (MTAPdef) in tumors with 9p21 loss and hypothetically renders tumors susceptible to synthetic lethality by antifolates targeting de novo purine synthesis. Here we report our single arm phase II trial (NCT02693717) that assesses pemetrexed in MTAPdef urothelial carcinoma (UC) with the primary endpoint of overall response rate (ORR). Three of 7 enrolled MTAPdef patients show response to pemetrexed (ORR 43%). Furthermore, a historic cohort shows 4 of 4 MTAPdef patients respond to pemetrexed as compared to 1 of 10 MTAP-proficient patients. In vitro and in vivo preclinical data using UC cell lines demonstrate increased sensitivity to pemetrexed by inducing DNA damage, and distorting nucleotide pools. In addition, MTAP-knockdown increases sensitivity to pemetrexed. Furthermore, in a lung adenocarcinoma retrospective cohort (N = 72) from the published BATTLE2 clinical trial (NCT01248247), MTAPdef associates with an improved response rate to pemetrexed. Our data demonstrate a synthetic lethal interaction between MTAPdef and de novo purine inhibition, which represents a promising therapeutic strategy for larger prospective trials.

Identification of a CD4+ T cell line with Treg-like activity.

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4+ CD25high Foxp3+ Tregs and demonstrate they suppress stimulated CD4+ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3+ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4+PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4+PBMC proliferation by MoT cells and Tregs, suggesting cell-cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity.We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4+PBMCs in a cell contact-dependent manner, suggesting that a Foxp3+Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.

Differential impact of tumor suppressor gene (TP53, PTEN, RB1) alterations and treatment outcomes in metastatic, hormone-sensitive prostate cancer.

BACKGROUND: Altered tumor suppressor genes (TSG-alt) in prostate cancer are associated with worse outcomes. The prognostic value of TSG-alt in metastatic, hormone-sensitive prostate cancer (M1-HSPC) is unknown. We evaluated the effects of TSG-alt on outcomes in M1-HSPC and their prognostic impact by first-line treatment. METHODS: We retrospectively identified patients with M1-HSPC at our institution treated with first-line androgen deprivation therapy plus docetaxel (ADT + D) or abiraterone acetate (ADT + A). TSG-alt was defined as any alteration in one or more TSG. The main outcomes were Kaplan-Meier-estimated progression-free survival (PFS) and overall survival, analyzed with the log-rank test. Clinical characteristics were compared with the χ2 test and Kruskal-Wallis rank sum test. Cox regression was used for univariate and multivariable analyses. RESULTS: We identified 97 patients with M1-HSPC: 48 (49%) with ADT + A and 49 (51%) with ADT + D. Of 96 patients with data available, 33 (34%) had 1 TSG-alt, 16 (17%) had 2 TSG-alt, and 2 (2%) had 3 TSG-alt. The most common alterations were in TP53 (36%) and PTEN (31%); 6% had RB1 alterations. Median PFS was 13.1 (95% CI, 10.3-26.0) months for patients with normal TSGs (TSG-normal) vs. 7.8 (95% CI, 5.8-10.5) months for TSG-alt (P = 0.005). Median PFS was lower for patients with TSG-alt vs TSG-normal for those with ADT + A (TSG-alt: 8.0 [95% CI, 5.8-13.8] months vs. TSG-normal: 23.2 [95% CI, 13.1-not estimated] months), but not with ADT + D (TSG-alt: 7.8 [95% CI, 5.7-12.9] months vs. TSG-normal: 9.5 [95% CI, 4.8-24.7] months). On multivariable analysis, only TSG-alt predicted worse PFS (hazard ratio, 2.37; 95% CI, 1.42-3.96; P < 0.001). CONCLUSIONS: The presence of TSG-alt outperforms clinical criteria for predicting early progression during first-line treatment of M1-HSPC. ADT + A was less effective in patients with than without TSG-alt. Confirmation of these findings may establish the need for inclusion of molecular stratification in treatment algorithms.

Phase Ib Study of Atezolizumab Plus Interferon-α with or without Bevacizumab in Patients with Metastatic Renal Cell Carcinoma and Other Solid Tumors.

This Phase Ib study combined programmed death-ligand 1 inhibitor, atezolizumab, with other immunomodulatory agents in locally advanced and metastatic solid tumors. Arms B-D evaluated atezolizumab plus interferon-α, with/without vascular endothelial growth factor inhibitor, bevacizumab, in renal cell carcinoma (RCC) and other solid tumors. Arm B predominantly recruited patients with previously treated RCC or melanoma to receive atezolizumab plus interferon α-2b. Arm C investigated atezolizumab plus polyethylene glycol (PEG)-interferon α-2a in previously treated RCC. Arm D evaluated atezolizumab plus PEG-interferon α-2a and bevacizumab. Primary objectives were safety and tolerability; secondary objectives included clinical activity. Combination therapy was well tolerated, with safety profiles consistent with known risks of individual agents. The most frequent treatment-related toxicities were fatigue, chills, and pyrexia. The objective response rate (ORR) in arm B was 20.0% overall and 17.8% in patients with previously treated checkpoint inhibitor-naive RCC (n = 45). No responses were reported in arm C. The highest ORR in arm D was 46.7% in patients with treatment-naive RCC (n = 15). Data showed preliminary clinical activity and acceptable tolerability of atezolizumab plus interferon α-2b in patients with previously treated checkpoint inhibitor-naive RCC and of atezolizumab plus PEG-interferon α-2a and bevacizumab in patients with treatment-naive RCC.

Clinical Results and Biomarker Analyses of Axitinib and TRC105 versus Axitinib Alone in Patients with Advanced or Metastatic Renal Cell Carcinoma (TRAXAR).

LESSONS LEARNED: The combination of carotuximab with axitinib did not provide a benefit over axitinib monotherapy in patients with metastatic clear cell renal cell carcinoma who had previously progressed on one or more vascular endothelial growth factor (VEGF)-targeted therapies. Exploratory evaluation of pretreatment circulating biomarkers suggested the combination might benefit patients who have low baseline VEGF levels. BACKGROUND: Endoglin is an angiogenic receptor expressed on proliferating tumor vessels and renal cell carcinoma (RCC) stem cells that is implicated as a mechanism of resistance to vascular endothelial growth factor receptor (VEGFR) inhibitors. This study evaluated an antiendoglin monoclonal antibody (carotuximab, TRC105) combined with axitinib in patients with advanced or metastatic clear cell renal cell carcinoma (mccRCC) who had progressed following one or more prior VEGF inhibitors. METHODS: TRAXAR was a multicenter, international randomized 1:1 (stratified by ECOG, 0 vs. 1), phase II study of carotuximab combined with axitinib versus axitinib alone in mccRCC patients who had progressed following one or more prior VEGF inhibitors. The primary endpoint was progression-free survival (PFS) assessed by independent central review (ICR) per RECIST 1.1 RESULTS: A total of 150 patients were randomized. The combination therapy resulted in shorter median PFS by RECIST 1.1 than axitinib monotherapy (6.7 vs. 11.4 months). The combination was tolerated similarly to axitinib monotherapy, and there were no treatment related deaths. Exploratory evaluation of pretreatment circulating biomarkers suggested the combination might benefit patients who have low baseline VEGF levels. CONCLUSION: The combination of carotuximab with axitinib did not demonstrate additional efficacy over single agent axitinib in patients with mccRCC who progressed following one or more prior VEGF inhibitor treatment.

A Living, Interactive Systematic Review and Network Meta-analysis of First-line Treatment of Metastatic Renal Cell Carcinoma.

CONTEXT: Identifying the most effective first-line treatment for metastatic renal cell carcinoma (mRCC) is challenging as rapidly evolving data quickly outdate the existing body of evidence, and current approaches to presenting the evidence in user-friendly formats are fraught with limitations. OBJECTIVE: To maintain living evidence for contemporary first-line treatment for previously untreated mRCC. EVIDENCE ACQUISITION: We have created a living, interactive systematic review (LISR) and network meta-analysis for first-line treatment of mRCC using data from randomized controlled trials comparing contemporary treatment options with single-agent tyrosine kinase inhibitors. We applied an advanced programming and artificial intelligence-assisted framework for evidence synthesis to create a living search strategy, facilitate screening and data extraction using a graphical user interface, automate the frequentist network meta-analysis, and display results in an interactive manner. EVIDENCE SYNTHESIS: As of October 22, 2020, the LISR includes data from 14 clinical trials. Baseline characteristics are summarized in an interactive table. The cabozantinib + nivolumab combination (CaboNivo) is ranked the highest for the overall response rate, progression-free survival, and overall survival, whereas ipilimumab + nivolumab (NivoIpi) is ranked the highest for achieving a complete response (CR). NivoIpi, and atezolizumab + bevacizumab (AteBev) were ranked highest (lowest toxicity) and CaboNivo ranked lowest for treatment-related adverse events (AEs). Network meta-analysis results are summarized as interactive tables and plots, GRADE summary-of-findings tables, and evidence maps. CONCLUSIONS: This innovative living and interactive review provides the best current evidence on the comparative effectiveness of multiple treatment options for patients with untreated mRCC. Trial-level comparisons suggest that CaboNivo is likely to cause more AEs but is ranked best for all efficacy outcomes, except NivoIpi offers the best chance of CR. Pembrolizumab + axitinib and NivoIpi are acceptable alternatives, except NivoIpi may not be preferred for patients with favorable risk. Although network meta-analysis provides rankings with statistical adjustments, there are inherent biases in cross-trial comparisons with sparse direct evidence that does not replace randomized comparisons. PATIENT SUMMARY: It is challenging to decide the best option among the several treatment combinations of immunotherapy and targeted treatments for newly diagnosed metastatic kidney cancer. We have created interactive evidence summaries of multiple treatment options that present the benefits and harms and evidence certainty for patient-important outcomes. This evidence is updated as soon as new studies are published.

ZMYND8 preferentially binds phosphorylated EZH2 to promote a PRC2-dependent to -independent function switch in hypoxia-inducible factor-activated cancer.

Both gene repressor (Polycomb-dependent) and activator (Polycomb-independent) functions of the Polycomb protein enhancer of zeste homolog 2 (EZH2) are implicated in cancer progression. EZH2 protein can be phosphorylated at various residues, such as threonine 487 (T487), by CDK1 kinase, and such phosphorylation acts as a Polycomb repressive complex 2 (PRC2) suppression "code" to mediate the gene repressor-to-activator switch of EZH2 functions. Here we demonstrate that the histone reader protein ZMYND8 is overexpressed in human clear cell renal cell carcinoma (ccRCC). ZMYND8 binds to EZH2, and their interaction is largely enhanced by CDK1 phosphorylation of EZH2 at T487. ZMYND8 depletion not only enhances Polycomb-dependent function of EZH2 in hypoxia-exposed breast cancer cells or von Hippel-Lindau (VHL)-deficient ccRCC cells, but also suppresses the FOXM1 transcription program. We further show that ZMYND8 is required for EZH2-FOXM1 interaction and is important for FOXM1-dependent matrix metalloproteinase (MMP) gene expression and EZH2-mediated migration and invasion of VHL-deficient ccRCC cells. Our results identify a previously uncharacterized role of the chromatin reader ZMYND8 in recognizing the PRC2-inhibitory phosphorylation "code" essential for the Polycomb-dependent to -independent switch of EZH2 functions. They also reveal an oncogenic pathway driving cell migration and invasion in hypoxia-inducible factor-activated (hypoxia or VHL-deficient) cancer.

Integrative molecular characterization of sarcomatoid and rhabdoid renal cell carcinoma.

Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.

Identification of DNA methylation signatures associated with poor outcome in lower-risk Stage, Size, Grade and Necrosis (SSIGN) score clear cell renal cell cancer.

BACKGROUND: Despite using prognostic algorithms and standard surveillance guidelines, 17% of patients initially diagnosed with low risk clear cell renal cell carcinoma (ccRCC) ultimately relapse and die of recurrent disease, indicating additional molecular parameters are needed for improved prognosis. RESULTS: To address the gap in ccRCC prognostication in the lower risk population, we performed a genome-wide analysis for methylation signatures capable of distinguishing recurrent and non-recurrent ccRCCs within the subgroup classified as 'low risk' by the Mayo Clinic Stage, Size, Grade, and Necrosis score (SSIGN 0-3). This approach revealed that recurrent patients have globally hypermethylated tumors and differ in methylation significantly at 5929 CpGs. Differentially methylated CpGs (DMCpGs) were enriched in regulatory regions and genes modulating cell growth and invasion. A subset of DMCpGs stratified low SSIGN groups into high and low risk of recurrence in independent data sets, indicating that DNA methylation enhances the prognostic power of the SSIGN score. CONCLUSIONS: This study reports a global DNA hypermethylation in tumors of recurrent ccRCC patients. Furthermore, DMCpGs were capable of discriminating between aggressive and less aggressive tumors, in addition to SSIGN score. Therefore, DNA methylation presents itself as a potentially strong biomarker to further improve prognostic power in patients with low risk SSIGN score (0-3).

8q24 clear cell renal cell carcinoma germline variant is associated with VHL mutation status and clinical aggressiveness.

BACKGROUND: The four most commonly-mutated genes in clear cell renal cell carcinoma (ccRCC) tumors are BAP1, PBRM1, SETD2 and VHL. And, there are currently 14 known RCC germline variants that have been reproducibly shown to be associated with RCC risk. However, the association of germline genetics with tumor genetics and clinical aggressiveness are unknown. METHODS: We analyzed 420 ccRCC patients from The Cancer Genome Atlas. Molecular subtype was determined based on acquired mutations in BAP1, PBRM1, SETD2 and VHL. Aggressive subtype was defined clinically using Mayo SSIGN score and molecularly using the ccA/ccB gene expression subtype. Publically-available Hi-C data were used to link germline risk variants with candidate target genes. RESULTS: The 8q24 variant rs35252396 was significantly associated with VHL mutation status (OR = 1.6, p = 0.0037) and SSIGN score (OR = 1.9, p = 0.00094), after adjusting for multiple comparisons. We observed that, while some germline variants have interactions with nearby genes, some variants demonstrate long-range interactions with target genes. CONCLUSIONS: These data further demonstrate the link between rs35252396, HIF pathway and ccRCC clinical aggressiveness, providing a more comprehensive picture of how germline genetics and tumor genetics interact with respect to tumor development and progression.

Macrophage HIF-1α Is an Independent Prognostic Indicator in Kidney Cancer.

PURPOSE: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1α and HIF-2α. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2α, and a tumor suppressor role for HIF-1α. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC. EXPERIMENTAL DESIGN: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC. RESULTS: HIF-1α was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2α and HAF were expressed primarily in tumor cells. TAM-associated HIF-1α was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1α was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2α were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1α in M2-polarized CD163-expressing TAMs. CONCLUSIONS: These findings highlight a potential role of TAM HIF-1α in ccRCC progression and support the reevaluation of HIF-1α as a therapeutic target and marker of disease progression.