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Blockade of the PD1 pathway is a broadly effective cancer therapy, but additional immune-inhibitory pathways contribute to tumor immune evasion. HERV–H LTR-associating 2 (HHLA2; also known as B7H5 and B7H7) is a member of the B7 family of immunoregulatory ligands that mediates costimulatory effects through its interaction with the CD28 family member transmembrane and immunoglobulin domain containing 2 (TMIGD2). However, HHLA2 has also been known to have inhibitory effects on T cells. Here, we report that we have identified killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) as an inhibitory receptor for HHLA2 in T cells and natural killer (NK) cells and have generated HHLA2 and KIR3DL3 antibodies that block the immune-inhibitory activity of HHLA2, preserving the costimulatory signal. It is known that HHLA2 is frequently expressed in several tumor types, including clear cell renal cell carcinoma (ccRCC). We found that HHLA2 expression was nonoverlapping with PDL1 expression in ccRCC, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PDL1. Blockade of both the PD1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion.See related Spotlight on p. 128

Supplementary Figures 1-7 and Supplementary Tables 1-2

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

PD-L1 positivity in relation to tumor grade within individual lesions.

PD-L1 expression in primary clear-cell renal cell carcinoma (ccRCC) increases the likelihood of response to anti–PD-1 inhibition, but fails to identify all responders. We hypothesized that PD-L1 levels assessed in randomly selected areas of the primary tumors may not accurately reflect expression levels in metastatic lesions, which are the target of systemic therapy. Therefore, we compared PD-L1 expression in a series of primary ccRCC and their metastases. Tissue blocks from 53 primary ccRCCs and 76 corresponding metastases were retrieved. Areas with predominant and highest nuclear grade were selected. Slides were immunostained with a validated anti–PD-L1 antibody (405.9A11). Membranous expression in tumor cells was quantified using H-score. Expression in tumor-infiltrating mononuclear cells (TIMC) was quantified using a combined score. Discordant tumor cell PD-L1 staining between primary tumors and metastases was observed in 11 of 53 cases (20.8%). Overall, tumor cell PD-L1 levels were not different in primary tumors and metastases (P = 0.51). Tumor cell PD-L1 positivity was associated with higher T stage (P = 0.03) and higher Fuhrman nuclear grade (P < 0.01). Within individual lesions, PD-L1 positivity was heterogeneous and almost exclusively detected in high nuclear grade areas (P < 0.001). No difference was found in PD-L1 levels in TIMCs between primary tumors and metastases (P = 0.82). The heterogeneity of PD-L1 expression in ccRCC suggests that its assessment as a predictive biomarker for PD-1 blockade may require analysis of metastatic lesions. Notably, because PD-L1 expression was mostly detected in high nuclear grade areas, to avoid false-negative results, these areas should be specifically selected for assessment. Cancer Immunol Res; 3(10); 1158–64. ©2015 AACR.

Figure S1: Impedance barrier function assay: The effect of S1P and Ex82 was analyzed using a transendothelial electrical impedance assay. S1P treatment (10 nM) of an endothelial monolayer strongly increases electrical impedance (B) vs control (A) whereas Ex82 has the opposite effect and significantly decreases electrical impedance (D). These results are consistent with the known barrier function of S1P1. In addition, pretreatment with Ex82 blocked the S1P dependent increase in electrical impedance (C).

Figure S2: Co-targeting S1P1 and VEGFR2 pathways induced reduction in permeability of retinal vessels. Ter 119 staining (panels A) visualize red blood cells and show that the combination of DC101 and the S1P1 antagonist in the retina led to reduction of the areas of hemorrhage within the vascular plexus and restricted only to the angiogenic front indicating that the remaining vessels in the remodeled area are less permeable.

Time interval between the resection of the primary tumor and the resection of each metastatic lesion. The discordance in PD-L1 expression between the primary tumor and each metastasis is also noted.

Table S1: Plasma S1P levels in patients with RCC; Table S2: Table of ASL MRI values.

Purpose: Renal cell carcinoma (RCC) has the potential for cure with surgery when diagnosed at an early stage. Kidney injury molecule-1 (KIM-1) has been shown to be elevated in the plasma of RCC patients. We aimed to test whether plasma KIM-1 could represent a means of detecting RCC prior to clinical diagnosis.Experimental Design: KIM-1 concentrations were measured in prediagnostic plasma from 190 RCC cases and 190 controls nested within a population-based prospective cohort study. Cases had entered the cohort up to 5 years before diagnosis, and controls were matched on cases for date of birth, date at blood donation, sex, and country. We applied conditional logistic regression and flexible parametric survival models to evaluate the association between plasma KIM-1 concentrations and RCC risk and survival.Results: The incidence rate ratio (IRR) of RCC for a doubling in KIM-1 concentration was 1.71 [95% confidence interval (CI), 1.44–2.03, P = 4.1 × 10−23], corresponding to an IRR of 63.3 (95% CI, 16.2–246.9) comparing the 80th to the 20th percentiles of the KIM-1 distribution in this sample. Compared with a risk model including known risk factors of RCC (age, sex, country, body mass index, and tobacco smoking status), a risk model additionally including KIM-1 substantially improved discrimination between cases and controls (area under the receiver-operating characteristic curve of 0.8 compared with 0.7). High plasma KIM-1 concentrations were also associated with poorer survival (P = 0.0053).Conclusions: Plasma KIM-1 concentrations could predict RCC incidence up to 5 years prior to diagnosis and were associated with poorer survival. Clin Cancer Res; 24(22); 5594–601. ©2018 AACR.

Purpose: VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC.Experimental Design: In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK–S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC.Results: Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI.Conclusions: Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy. Clin Cancer Res; 21(8); 1925–34. ©2015 AACR.

Supplementary Figure from Renal Cancer Resistance to Antiangiogenic Therapy Is Delayed by Restoration of Angiostatic Signaling

Related Article from The Challenge of Herbal Therapies for Prostate Cancer

Purpose:The use of VEGFR TKIs for the adjuvant treatment of renal cell carcinoma (RCC) remains controversial. We investigated the effects of adjuvant VEGFR TKIs on circulating cytokines in the ECOG-ACRIN 2805 (ASSURE) trial.Experimental Design:Patients with resected high-risk RCC were randomized to sunitinib, sorafenib, or placebo. Plasma from 413 patients was analyzed from post-nephrectomy baseline, 4 weeks, and 6 weeks after treatment initiation. Mixed effects and Cox proportional hazards models were used to test for changes in circulating cytokines and associations between disease-free survival (DFS) and cytokine levels.Results:VEGF and PlGF increased after 4 weeks on sunitinib or sorafenib (P < 0.0001 for both) and returned to baseline at 6 weeks on sunitinib (corresponding to the break in the sunitinib schedule) but not sorafenib (which was administered continuously). sFLT-1 decreased after 4 weeks on sunitinib and 6 weeks on sorafenib (P < 0.0001). sVEGFR-2 decreased after both 4 and 6 weeks of treatment on sunitinib or sorafenib (P < 0.0001). Patients receiving placebo had no significant changes in cytokine levels. CXCL10 was elevated at 4 and 6 weeks on sunitinib and sorafenib but not on placebo. Higher baseline CXCL10 was associated with worse DFS (HR 1.41 per log increase in CXCL10, Bonferroni-adjusted P = 0.003). This remained significant after adjustment for T-stage, Fuhrman grade, and ECOG performance status.Conclusions:Among patients treated with adjuvant VEGFR TKIs for RCC, drug–host interactions mediate changes in circulating cytokines. Elevated baseline CXCL10 was associated with worse DFS. Studies to understand functional consequences of these changes are under way.

1. Supplementary table 1S describing details of biopsies undergoing WES. 2. Table 2S includes the primers used in the CTC gene expression analysis. 3. Table 3S includes summary of PSA response based on prior treatment. 4. Table 4S includes objective response in the overall cohort and based on patients with measurable and non-measurable disease. 5. Table 5S is the PSA progression and radiographic PFS based on prior lines of treatment. 6. Table 6S is the adverse events in >10% of patients. 7. Table 7S is a listing of the biopsy procedures and outcomes. 8. Table 8S is the frequency of genomic alterations in the baseline and progression samples and PSA response data. 9. Table 9S includes the details for the AR mutations observed in the baseline and progression samples. 10. Figure 1S includes the KM curves for PSA progression and radiographic PFS. 11. Figure 2S includes the Consort Diagram for the study. 12. Figure 3S includes the integrative data of paired biopsy and CTC samples. 13. Figure 4S includes the KM curves for time to PSA progression, radiographic PFS, and overall survival in the CTC biomarker groups.

Distribution of baseline KIM-1 values

Purpose:No circulating biomarkers are currently available to identify patients at highest risk of recurrence after nephrectomy for renal cell carcinoma (RCC). Kidney injury molecule-1 (KIM-1) is overexpressed in RCC and its ectodomain circulates in plasma. We investigated whether plasma KIM-1 is a prognostic biomarker in patients with localized RCC after nephrectomy.Experimental Design:The ECOG-ACRIN E2805 (ASSURE) trial evaluated adjuvant sunitinib, sorafenib, or placebo in resected high-risk RCC. KIM-1 levels were measured from banked plasma at trial enrollment 4–12 weeks after nephrectomy. Lognormal accelerated failure time models were used to test for association between KIM-1 and disease-free survival (DFS) as well as overall survival (OS).Results:Plasma from 418 patients was analyzed. Higher post-nephrectomy KIM-1 was associated with worse DFS across all study arms after adjustment for Fuhrman grade, T stage, N stage, and tumor histology [survival time ratio 0.56 for 75th vs. 25th percentile of KIM-1; 95% confidence interval (CI), 0.42–0.73; P < 0.001]. The association between KIM-1 and DFS was stronger among patients with pathologic nodal involvement (Pinteraction = 0.0086). The addition of post-nephrectomy KIM-1 improved the concordance of clinical prognostic models [Stage, Size, Grade, and Necrosis (SSIGN) concordance 0.57 vs. 0.43, P = 0.05; UCLA International Staging System (UISS) concordance 0.60 vs. 0.40, P = 0.0005]. Higher post-nephrectomy KIM-1 was also associated with worse OS after multivariable adjustment (survival time ratio 0.71 for 75th vs. 25th percentile of KIM-1; 95% CI, 0.56–0.91; P < 0.001).Conclusions:Post-nephrectomy plasma KIM-1 is associated with DFS and OS in RCC, and may be a biomarker for microscopic residual disease.

Supplementary Table from Renal Cancer Resistance to Antiangiogenic Therapy Is Delayed by Restoration of Angiostatic Signaling

Supplemental Table 1: Catalog numbers for MSD assays Supplemental Figure 1: Hazard ratio for CXCL10 and DFS by treatment arm Supplemental Figure 2: Assessment of log-linearity for relationship between IP-10 and recurrence risk Supplemental Table 2: Median changes in cytokines at weeks 4 and 6 Supplemental Table 3: Relationship between on-treatment cytokine levels and DFS Supplemental Table 4: Subset analysis for cytokine changes among patients who did not recur Supplemental Table 5: Evaluation for interaction effects between treatment arm and the association between baseline CXCL10 and DFS

Treatment of metastatic renal cell cancer (RCC) with antiangiogenic agents that block vascular endothelial growth factor (VEGF) receptor 2 signaling produces tumor regression in a substantial fraction of patients; however, resistance typically develops within 6 to 12 months. The purpose of this study was to identify molecular pathways involved in resistance. Treatment of mice bearing either 786-0 or A498 human RCC xenografts with sorafenib or sunitinib produced tumor growth stabilization followed by regrowth despite continued drug administration analogous to the clinical experience. Tumors and plasma were harvested at day 3 of therapy and at the time of resistance to assess pathways that may be involved in resistance. Serial perfusion imaging, and plasma and tumor collections were obtained in mice treated with either placebo or sunitinib alone or in combination with intratumoral injections of the angiostatic chemokine CXCL9. Sunitinib administration led to an early downmodulation of IFNγ levels as well as reduction of IFNγ receptor and downstream angiostatic chemokines CXCL9 to 11 within the tumor. Intratumoral injection of CXCL9, although producing minimal effects by itself, when combined with sunitinib resulted in delayed resistance in vivo accompanied by a prolonged reduction of microvascular density and tumor perfusion as measured by perfusion imaging relative to sunitinib alone. These results provide evidence that resistance to VEGF receptor therapy is due at least in part to resumption of angiogenesis in association with reduction of IFNγ-related angiostatic chemokines, and that this resistance can be delayed by concomitant administration of CXCL9. Mol Cancer Ther; 9(10); 2793–802. ©2010 AACR.

Supplemental data for DART Part 1 manuscript. Contains 1 figure and 3 tables. Supplemental Figure 1: Percent change from baseline in serum receptor expression. Samples were taken at cycle 1 day 1 (baseline), cycle 2 day 1, cycle 2 day 8, cycle 3 day 1, and the last visit. Percent change from baseline was calculated for each patient and averaged across all patients. (A) Serum VEGFR3 receptor expression (B) Serum VEGFR2 receptor expression. Supplemental Table 1: Management of Adverse Events Related to Dalantercept/Placebo Supplemental Table 2: Summary Pharmacokinetic Parameters for Dalantercept plus Axitinib Supplemental Table 3: Biomarker Expression Levels in Biopsied Tissue Samples

Purpose:Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance.Patients and Methods:Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing.Results:A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression.Conclusions:Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

Sensitivity analysis AFT model for DFS with imputation of KIM-1 levels below lower limit of detection (9.1 pg/mL)

Purpose: Activin receptor-like kinase 1 (ALK1) is a novel target in angiogenesis. Concurrent targeting of ALK1 and VEGF signaling results in augmented inhibition of tumor growth in renal cell carcinoma (RCC) xenograft models. Dalantercept is an ALK1-receptor fusion protein that acts as a ligand trap for bone morphogenetic proteins 9 and 10. The DART Study evaluated the safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity of dalantercept plus axitinib in patients with advanced RCC and determined the optimal dose for further testing.Experimental Design: Patients received dalantercept 0.6, 0.9, or 1.2 mg/kg subcutaneously every 3 weeks plus axitinib 5 mg by mouth twice daily until disease progression or intolerance.Results: Twenty-nine patients were enrolled in the dose escalation (n = 15) and expansion (n = 14) cohorts. There were no dose-limiting toxicities or grade 4/5 treatment-related adverse events. In addition to common VEGFR tyrosine kinase inhibitor effects, such as fatigue and diarrhea, commonly seen treatment-related adverse events were peripheral edema, epistaxis, pericardial effusion, and telangiectasia. The objective response rate by RECIST v1.1 was 25% with responses seen at all dose levels. The overall median progression-free survival was 8.3 months.Conclusions: The combination of dalantercept plus axitinib is well tolerated and associated with clinical activity. On the basis of safety and efficacy results, the 0.9 mg/kg dose level was chosen for further study in a randomized phase II trial of dalantercept plus axitinib versus placebo plus axitinib. Clin Cancer Res; 23(14); 3557–65. ©2016 AACR.

Supplementary Table 1, Supplementary Figures 1 and 2

The paper by Singh et al. shows that silibinin inhibits prostate cancer growth in the TRAMP mouse model. Among its other properties, silibinin inhibits the progression of prostate cancer by decreasing angiogenesis and tumor invasion. Identification of the biological effects of herbal remedies such as silibinin is critical for their development as anticancer treatments.

Purpose: Enzalutamide is a second-generation androgen receptor (AR) inhibitor that has improved overall survival (OS) in metastatic castration-resistant prostate cancer (CRPC). However, nearly all patients develop resistance. We designed a phase II multicenter study of enzalutamide in metastatic CRPC incorporating tissue and blood biomarkers to dissect mechanisms driving resistance. Patients and Methods: Eligible patients with metastatic CRPC underwent a baseline metastasis biopsy and then initiated enzalutamide 160 mg daily. A repeat metastasis biopsy was obtained at radiographic progression from the same site when possible. Blood for circulating tumor cell (CTC) analysis was collected at baseline and progression. The primary objective was to analyze mechanisms of resistance in serial biopsies. Whole-exome sequencing was performed on tissue biopsies. CTC samples underwent RNA sequencing. Results: A total of 65 patients initiated treatment, of whom 22 (33.8%) had received prior abiraterone. Baseline biopsies were enriched for alterations in AR (mutations, amplifications) and tumor suppression genes (PTEN, RB1, and TP53), which were observed in 73.1% and 92.3% of baseline biopsies, respectively. Progression biopsies revealed increased AR amplifications (64.7% at progression vs. 53.9% at baseline) and BRCA2 alterations (64.7% at progression vs. 38.5% at baseline). Genomic analysis of baseline and progression CTC samples demonstrated increased AR splice variants, AR-regulated genes, and neuroendocrine markers at progression. Conclusions: Our results demonstrate that a large proportion of enzalutamide-treated patients have baseline and progression alterations in the AR pathway and tumor suppressor genes. We demonstrate an increased number of BRCA2 alterations post-enzalutamide, highlighting the importance of serial tumor sampling in CRPC.

Plasma cell-free DNA (cfDNA) variant analysis is commonly used in many cancer subtypes. Cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq) has shown high sensitivity for cancer detection. To date, studies have not compared the sensitivity of both methods in a single cancer subtype. cfDNA from 40 metastatic RCC (mRCC) patients was subjected to targeted panel variant analysis. For 34 of 40, cfMeDIP-seq was also performed. A separate cohort of 38 mRCC patients were used in cfMeDIP-seq analysis to train an RCC classifier. cfDNA variant analysis detected 21 candidate variants in 11 of 40 mRCC patients (28%), after exclusion of 2 germline variants and 6 variants reflecting clonal hematopoiesis. Among 23 patients with parallel tumor sequencing, cfDNA analysis alone identified variants in 9 patients (39%), while cfDNA analysis focused on tumor sequencing variant findings improved the sensitivity to 52%. In 34 mRCC patients undergoing cfMeDIP-seq, cfDNA variant analysis identified variants in 7 (21%), while cfMeDIP-seq detected all mRCC cases (100% sensitivity) with 88% specificity in 34 control subjects. In 5 patients with cfDNA variants and serial samples, variant frequency correlated with response to therapy. cfMeDIP-seq is significantly more sensitive for mRCC detection than cfDNA variant analysis. However, cfDNA variant analysis may be useful for monitoring response to therapy.

Despite widespread use of taxanes, mechanisms of action and resistance in vivo remain to be established, and there is no way of predicting who will respond to therapy. This study examined prostate cancer (PCa) xenografts and patient samples to identify in vivo mechanisms of taxane action and resistance. Docetaxel drug-target engagement was assessed by confocal anti-tubulin immunofluorescence to quantify microtubule bundling in interphase cells and aberrant mitoses. Tumor biopsies from metastatic PCa patients obtained 2 to 5 days after their first dose of docetaxel or cabazitaxel were processed to assess microtubule bundling, which correlated with clinical response. Microtubule bundling was evident in PCa xenografts 2 to 3 days after docetaxel treatment but was decreased or lost with acquired resistance. Biopsies after treatment with leuprolide plus docetaxel showed extensive microtubule bundling as did biopsies obtained 2 to 3 days after initiation of docetaxel or cabazitaxel in 2 patients with castration-resistant PCa with clinical responses. In contrast, microtubule bundling in biopsies 2 to 3 days after the first dose of docetaxel was markedly lower in 4 nonresponding patients. These findings indicate that taxanes target both mitotic and interphase cells in vivo and that resistance is through mechanisms that impair drug-target engagement. Moreover, the findings suggest that microtubule bundling after initial taxane treatment may be a predictive biomarker for clinical response.

There are no biomarkers predictive of resistance to docetaxel or cabazitaxel validated for patients with metastatic castration-resistant prostate cancer (mCRPC). We assessed the association between ABCB1 amplification and primary resistance to docetaxel or cabazitaxel for patients with mCRPC, using circulating cell-free DNA (cfDNA). Patients with ≥1 plasma sample drawn within 12 months before starting docetaxel (cohort A) or cabazitaxel (cohort B) for mCRPC were identified from the Dana–Farber Cancer Institute IRB approved database. Sparse whole genome sequencing was performed on the selected cfDNA samples and tumor fractions were estimated using the computational tool ichorCNA. We evaluated the association between ABCB1 amplification or other copy number alterations and primary resistance to docetaxel or cabazitaxel. Of the selected 176 patients, 45 samples in cohort A and 21 samples in cohort B had sufficient tumor content. No significant association was found between ABCB1 amplification and primary resistance to docetaxel (p = 0.58, odds ratio (OR) = 1.49) or cabazitaxel (p = 0.97, OR = 1.06). No significant association was found between exploratory biomarkers and primary resistance to docetaxel or cabazitaxel. In this study, ABCB1 amplification did not predict primary resistance to docetaxel or cabazitaxel for mCRPC. Future studies including ABCB1 amplification in a suite of putative biomarkers and a larger cohort may aid in drawing definitive conclusions.

Resistance to tyrosine kinase inhibitors of the vascular endothelial growth factor receptor inevitably develops in most patients with metastatic kidney cancer. Our recent findings demonstrate that addition of angiotensin-(1-7) peptide can be a potential therapy that delays such resistance.

Purpose: The use of VEGFR TKIs for the adjuvant treatment of renal cell carcinoma (RCC) remains controversial. We investigated the effects of adjuvant VEGFR TKIs on circulating cytokines in the ECOG-ACRIN 2805 (ASSURE) trial. Experimental Design: Patients with resected high-risk RCC were randomized to sunitinib, sorafenib, or placebo. Plasma from 413 patients was analyzed from post-nephrectomy baseline, 4 weeks, and 6 weeks after treatment initiation. Mixed effects and Cox proportional hazards models were used to test for changes in circulating cytokines and associations between disease-free survival (DFS) and cytokine levels. Results: VEGF and PlGF increased after 4 weeks on sunitinib or sorafenib (P < 0.0001 for both) and returned to baseline at 6 weeks on sunitinib (corresponding to the break in the sunitinib schedule) but not sorafenib (which was administered continuously). sFLT-1 decreased after 4 weeks on sunitinib and 6 weeks on sorafenib (P < 0.0001). sVEGFR-2 decreased after both 4 and 6 weeks of treatment on sunitinib or sorafenib (P < 0.0001). Patients receiving placebo had no significant changes in cytokine levels. CXCL10 was elevated at 4 and 6 weeks on sunitinib and sorafenib but not on placebo. Higher baseline CXCL10 was associated with worse DFS (HR 1.41 per log increase in CXCL10, Bonferroni-adjusted P = 0.003). This remained significant after adjustment for T-stage, Fuhrman grade, and ECOG performance status. Conclusions: Among patients treated with adjuvant VEGFR TKIs for RCC, drug–host interactions mediate changes in circulating cytokines. Elevated baseline CXCL10 was associated with worse DFS. Studies to understand functional consequences of these changes are under way.

Several therapeutic options are available for metastatic RCC, but responses are almost never complete, and resistance to therapy develops in the vast majority of patients. Consequently, novel treatments are needed to combat resistance to current therapies and to improve patient outcomes. We have applied integrated transcriptome and proteome analyses to identify cathepsin B (CTSB), a cysteine proteinase of the papain family, as one of the most highly upregulated gene products in established human RCC xenograft models of resistance to vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI). We used established RCC models to test the significance of CTSB in the progression of renal cancer. Our evaluation of CTSB showed that stable CTSB knockdown suppressed RCC growth in vitro and in vivo. Stable over-overexpression of wild-type CTSB (CTSBwt/hi), but not of an CTSB active site mutant (CTSBN298A), rescued cell growth in CTSB knockdown cells and abolished the efficacy of VEGFR TKI treatment. Genome-wide transcriptome profiling of CTSB knockdown cells demonstrated significant effects on multiple metabolic and stem cell-related pathways, with ALDHA1A (ALDH1) as one of the most significantly downregulated genes. Importantly, survival analysis across 16 major TCGA cancers revealed that CTSB overexpression is associated with low rates of three and five year patient survival rates (P = 2.5e–08, HR = 1.4). These data strongly support a contribution of CTSB activity to RCC cell growth and tumorigenicity. They further highlight the promise of CTSB inhibition in development of novel combination therapies designed to improve efficacy of current TKI treatments of metastatic RCC.

Angiotensin converting enzyme 2 (ACE2) is an enzyme that belongs to the renin-angiotensin system (RAS) and antagonizes the classical angiotensin (Ang) II/angiotensin II receptor type 1 (AT1) receptor pathway. Here, we report that higher ACE2 expression correlates with better overall survival in patients with clear cell renal cell carcinoma (ccRCC). Moreover, ACE2 has inhibitory effects on tumor proliferation in ccRCC in vitro and in preclinical animal models of ccRCC. We further show that Ang-(1-7), a heptapeptide generated by ACE2, is the likely mediator of this effect. Vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGFR-TKI) treatment of ccRCC xenografts decreased ACE2 expression, and combination treatment with VEGFR-TKI and Ang-(1-7) generated additive suppression of tumor growth and improved survival outcomes. Last, the addition of Ang-(1-7) to programmed death-ligand 1 (PD-L1) pathway inhibitor and VEGFR-TKI showed further growth suppression in an immunocompetent RCC model. Together, these results suggest that targeting the ACE2/Ang-(1-7) axis is a promising therapeutic strategy against ccRCC.

Purpose: No circulating biomarkers are currently available to identify patients at highest risk of recurrence after nephrectomy for renal cell carcinoma (RCC). Kidney injury molecule-1 (KIM-1) is overexpressed in RCC and its ectodomain circulates in plasma. We investigated whether plasma KIM-1 is a prognostic biomarker in patients with localized RCC after nephrectomy. Experimental Design: The ECOG-ACRIN E2805 (ASSURE) trial evaluated adjuvant sunitinib, sorafenib, or placebo in resected high-risk RCC. KIM-1 levels were measured from banked plasma at trial enrollment 4–12 weeks after nephrectomy. Lognormal accelerated failure time models were used to test for association between KIM-1 and disease-free survival (DFS) as well as overall survival (OS). Results: Plasma from 418 patients was analyzed. Higher post-nephrectomy KIM-1 was associated with worse DFS across all study arms after adjustment for Fuhrman grade, T stage, N stage, and tumor histology [survival time ratio 0.56 for 75th vs. 25th percentile of KIM-1; 95% confidence interval (CI), 0.42–0.73; P < 0.001]. The association between KIM-1 and DFS was stronger among patients with pathologic nodal involvement (Pinteraction = 0.0086). The addition of post-nephrectomy KIM-1 improved the concordance of clinical prognostic models [Stage, Size, Grade, and Necrosis (SSIGN) concordance 0.57 vs. 0.43, P = 0.05; UCLA International Staging System (UISS) concordance 0.60 vs. 0.40, P = 0.0005]. Higher post-nephrectomy KIM-1 was also associated with worse OS after multivariable adjustment (survival time ratio 0.71 for 75th vs. 25th percentile of KIM-1; 95% CI, 0.56–0.91; P < 0.001). Conclusions: Post-nephrectomy plasma KIM-1 is associated with DFS and OS in RCC, and may be a biomarker for microscopic residual disease.

Background Tumor perfusion may inform therapeutic response and resistance in metastatic renal cell carcinoma (RCC) treated with antiangiogenic therapy. Purpose To determine if arterial spin labeled (ASL) MRI perfusion changes are associated with tumor response and disease progression in metastatic RCC treated with vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs). Materials and Methods In this prospective study (ClinicalTrials.gov identifier: NCT00749320), metastatic RCC perfusion was measured with ASL MRI before and during sunitinib or pazopanib therapy between October 2008 and March 2014. Objective response rate (ORR) and progression-free survival (PFS) were calculated. Perfusion was compared between responders and nonresponders at baseline, at week 2, after cycle 2 (12 weeks), after cycle 4 (24 weeks), and at disease progression and compared with the ORR by using the Wilcoxon rank sum test and with PFS by using the log-rank test. Results Seventeen participants received sunitinib (mean age, 59 years ± 7.0 [standard deviation]; 11 men); 11 participants received pazopanib (mean age, 63 years ± 6.6; eight men). Responders had higher baseline tumor perfusion than nonresponders (mean, 404 mL/100 g/min ± 213 vs 199 mL/100 g/min ± 136; P = .02). Perfusion decreased from baseline to week 2 (-53 mL/100 g/min ± 31; P < .001), after cycle 2 (-65 mL/100 g/min ± 25; P < .001), and after cycle 4 (-79 mL/100 g/min ± 15; P = .008). Interval reduction in perfusion at those three time points was not associated with ORR (P = .63, .29, and .27, respectively) or PFS (P = .28, .27, and .32). Perfusion increased from cycle 4 to disease progression (51% ± 11; P < .001). Conclusion Arterial spin labeled perfusion MRI may assist in identifying responders to vascular endothelial growth factor receptor tyrosine kinase inhibitors and may help detect early evidence of disease progression in patients with metastatic renal cell carcinoma. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Goh and De Vita in this issue.

Purpose Androgen deprivation therapy (ADT) is often used as adjuvant treatment with radiation therapy (RT) for intermediate-risk prostate cancer. ADT is associated with multiple side effects, including weight gain, loss of libido, and hot flashes. In contrast, antiandrogen monotherapy has been generally better tolerated. This study aimed to assess the effectiveness of enzalutamide (an antiandrogen) monotherapy with RT for the treatment of intermediate-risk prostate cancer. Methods and Materials This trial was an open-label, phase 2 study of 6 months of enzalutamide monotherapy with external beam RT for intermediate-risk prostate cancer. Enzalutamide was initiated 2 months before external beam RT. The primary endpoint was prostate-specific antigen (PSA) response measured at the end of enzalutamide administration at the 6-month timepoint. Secondary endpoints included assessment of toxicity and changes in anthropomorphic body measurement, sexual function, and metabolism. The sample size was 64 patients. The hypothesis was that if ≥60% of the patients did not achieve a PSA nadir of ≤0.2 ng/mL, the study results would be deemed negative. Results The results met the prespecified endpoint for efficacy in that PSA values ≤0.2 ng/mL were observed in 49 of 64 patients (77%), and 60 of 64 patients (94%) had PSA values ≤0.5ng/mL. The most frequent adverse events were hypertension and gynecomastia. There were no changes in anthropomorphic body measurements and only modest erectile dysfunction. Conclusions Using PSA response as an endpoint, enzalutamide monotherapy may be as effective as ADT in combination with external beam RT for patients with intermediate-risk prostate cancer, and it is associated with fewer side effects. Randomized trials comparing enzalutamide with ADT are justified.

Blockade of the PD1 pathway is a broadly effective cancer therapy, but additional immune-inhibitory pathways contribute to tumor immune evasion. HERV–H LTR-associating 2 (HHLA2; also known as B7H5 and B7H7) is a member of the B7 family of immunoregulatory ligands that mediates costimulatory effects through its interaction with the CD28 family member transmembrane and immunoglobulin domain containing 2 (TMIGD2). However, HHLA2 has also been known to have inhibitory effects on T cells. Here, we report that we have identified killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) as an inhibitory receptor for HHLA2 in T cells and natural killer (NK) cells and have generated HHLA2 and KIR3DL3 antibodies that block the immune-inhibitory activity of HHLA2, preserving the costimulatory signal. It is known that HHLA2 is frequently expressed in several tumor types, including clear cell renal cell carcinoma (ccRCC). We found that HHLA2 expression was nonoverlapping with PDL1 expression in ccRCC, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PDL1. Blockade of both the PD1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion. See related Spotlight on p. 128

Introduction Genomic events leading to activation of mechanistic target of rapamycin (mTOR) are common in renal cell carcinoma (RCC). Everolimus is an allosteric mTOR inhibitor with efficacy in metastatic RCC. We characterized the genomic profile of RCC tumors from metastatic sites and assessed whether particular alterations correlate with clinical response to everolimus. Patients and Methods An open-label, single-arm phase 2 biomarker study of everolimus 10 mg daily was conducted in metastatic RCC patients. Needle biopsy or metastasectomy was performed on metastatic tumors before everolimus initiation. Next-generation sequencing was performed using a targeted hybrid capture panel detecting alterations within exons and key introns of ≥ 300 cancer-associated genes. Disease assessments were obtained every 8 weeks using standard radiographic modalities and evaluated by Response Evaluation Criteria in Solid Tumors criteria. Results Objective response was seen in 1 (4.2%) of 24 patients. Two patients (8.3%) had stable disease lasting > 6 months. Median (90% confidence interval) overall and progression-free survival were 20.1 (8.6, NA) and 3.8 (2.4, 5.4) months, respectively. Next-generation sequencing was successful on 18 pretreatment specimens and 3 on-treatment specimens. Alterations in the phosphatidylinositol 3-kinase–protein kinase B–mammalian target of rapamycin (PI3K-AKT-mTOR) pathway were identified in 8 (44%) of 18 pretreatment samples. An mTOR E2419D mutation was identified in the patient who experienced partial response. Alterations in VHL, PBRM1, SETD2, KDM5C, and ATM were common in the RCC metastases before initiation of everolimus. Conclusion Nearly half of heavily pretreated RCC metastases may harbor mutations in components of the PI3K-AKT-mTOR pathway. Commonly mutated genes in primary RCC were also altered at a high frequency in RCC metastases.

The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (VHL) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC). Keywords: immunocompetent mouse model, ccRCC, RENCA, kidney cancer, CRISPR/Cas9, non-integrated lentivirus, immunotherapy

In preclinical models, c-Met promotes survival of renal cancer cells through the regulation of programmed death-ligand 1 (PD-L1). However, this relationship in human clear cell renal cell carcinoma (ccRCC) is not well characterized. We evaluated c-Met expression in ccRCC patients using paired primary and metastatic samples and assessed the association with PD-L1 expression and other clinical features. Areas with predominant and highest Fuhrman nuclear grade (FNG) were selected. c-Met expression was evaluated by IHC using an anti-Met monoclonal antibody (MET4 Ab) and calculated by a combined score (CS, 0–300): intensity of c-Met staining (0–3) x % of positive cells (0–100). PD-L1 expression in tumor cells was previously assessed by IHC and PD-L1+ was defined as PD-L1 > 0% positive cells. Our cohort consisted of 45 pairs of primary and metastatic ccRCC samples. Overall, c-Met expression was higher in metastatic sites compared to primary sites (average c-Met CS: 55 vs. 28, p = 0.0003). Higher c-Met expression was associated with higher FNG (4 vs. 3) in primary tumors (average c-Met CS: 52 vs. 20, p = 0.04). c-Met expression was numerically greater in PD-L1+ vs. PD-L1- tumors. Higher c-Met expression in metastatic sites compared to primary tumors suggests that testing for biomarkers of response to c-Met inhibitors should be conducted in metastases. While higher c-Met expression in PD-L1+ tumors requires further investigation, it supports exploring these targets in combination clinical trials.

Tumors induce their heterogeneous vasculature by secreting vascular endothelial growth factor (VEGF)-A. Anti-VEGF/VEGF receptor (VEGFR) drugs treat cancer, but the underlying mechanisms remain unclear. An adenovirus expressing VEGF-A (Ad-VEGF-A164) replicates the tumor vasculature in mice without tumor cells. Mother vessels (MV) are the first angiogenic vessel type to form in tumors and after Ad-VEGF-A164. Multiday treatments with a VEGF trap reverted MV back to normal microvessels. We now show that, within hours, a single dose of several anti-VEGF drugs collapsed MV to form glomeruloid microvascular proliferations (GMP), accompanied by only modest endothelial cell death. GMP, common in many human cancers but of uncertain origin, served as an intermediary step in MV reversion to normal microvessels. The vasodisruptive drug combretastatin CA4 also targeted MV selectively but acted differently, extensively killing MV endothelium. Antivascular changes were quantified with a novel Evans blue dye assay that measured vascular volumes. As in tumors, Ad-VEGF-A164 strikingly increased endothelial nitric oxide synthase (eNOS) expression. The eNOS inhibitor N(G)-Nitro-l-arginine methyl ester mimicked anti-VEGF/VEGFR drugs, rapidly collapsing MV to GMP. Inhibition of eNOS reduces synthesis of its vasodilatory product, nitric oxide, leading to arterial contraction. Patients and mice receiving anti-VEGF/VEGFR drugs develop hypertension, reflecting systemic arterial contraction. Together, anti-VEGF/VEGFR drugs act in part by inhibiting eNOS, causing vasocontraction, MV collapse to GMP, and subsequent reversion of GMP to normal microvessels, all without extensive vascular killing.

In 2017, there is no adjuvant systemic therapy proven to increase overall survival in non-metastatic renal cell carcinoma (RCC). The anti-PD-1 antibody nivolumab improves overall survival in metastatic treatment refractory RCC and is generally tolerable. Mouse solid tumor models have revealed a benefit with a short course of neoadjuvant PD-1 blockade compared to adjuvant therapy. Two ongoing phase 2 studies of perioperative nivolumab in RCC patients have shown preliminary feasibility and safety with no surgical delays or complications. The recently opened PROSPER RCC trial (A Phase 3 RandOmized Study Comparing PERioperative Nivolumab vs. Observation in Patients with Localized Renal Cell Carcinoma Undergoing Nephrectomy; EA8143) will examine if the addition of perioperative nivolumab to radical or partial nephrectomy can improve clinical outcomes in patients with high risk localized and locally advanced RCC. With the goal of increasing cure and recurrence-free survival (RFS) rates in non-metastatic RCC, we are executing a three-pronged, multidisciplinary approach of presurgical priming with nivolumab followed by resection and adjuvant PD-1 blockade. We plan to enroll 766 patients with clinical stage ≥T2 or node positive M0 RCC of any histology in this global, randomized, unblinded, phase 3 National Clinical Trials Network study. The investigational arm will receive two doses of nivolumab 240 mg IV prior to surgery followed by adjuvant nivolumab for 9 months. The control arm will undergo the current standard of care: surgical resection followed by observation. Patients are stratified by clinical T stage, node positivity, and histology. The trial is powered to detect a 14.4% absolute benefit in the primary endpoint of RFS from the ASSURE historical control of 55.8% to 70.2% at 5 years (HR = 0.70). The study is also powered to detect a significant overall survival benefit (HR 0.67). Key safety, feasibility, and quality of life endpoints are incorporated. PROSPER RCC exemplifies team science with a host of planned correlative work to investigate the impact of the baseline immune milieu and changes after neoadjuvant priming on clinical outcomes.

Purpose: Renal cell carcinoma (RCC) has the potential for cure with surgery when diagnosed at an early stage. Kidney injury molecule-1 (KIM-1) has been shown to be elevated in the plasma of RCC patients. We aimed to test whether plasma KIM-1 could represent a means of detecting RCC prior to clinical diagnosis. Experimental Design: KIM-1 concentrations were measured in prediagnostic plasma from 190 RCC cases and 190 controls nested within a population-based prospective cohort study. Cases had entered the cohort up to 5 years before diagnosis, and controls were matched on cases for date of birth, date at blood donation, sex, and country. We applied conditional logistic regression and flexible parametric survival models to evaluate the association between plasma KIM-1 concentrations and RCC risk and survival. Results: The incidence rate ratio (IRR) of RCC for a doubling in KIM-1 concentration was 1.71 [95% confidence interval (CI), 1.44–2.03, P = 4.1 × 10−23], corresponding to an IRR of 63.3 (95% CI, 16.2–246.9) comparing the 80th to the 20th percentiles of the KIM-1 distribution in this sample. Compared with a risk model including known risk factors of RCC (age, sex, country, body mass index, and tobacco smoking status), a risk model additionally including KIM-1 substantially improved discrimination between cases and controls (area under the receiver-operating characteristic curve of 0.8 compared with 0.7). High plasma KIM-1 concentrations were also associated with poorer survival (P = 0.0053). Conclusions: Plasma KIM-1 concentrations could predict RCC incidence up to 5 years prior to diagnosis and were associated with poorer survival. Clin Cancer Res; 24(22); 5594–601. ©2018 AACR.

BACKGROUND In a prior open-label study, the combination of dalantercept, a novel antiangiogenic targeting activin receptor-like kinase 1 (ALK1), plus axitinib was deemed safe and tolerable with a promising efficacy signal in patients with advanced renal cell carcinoma (RCC). METHODS In the current phase 2, randomized, double-blind, placebo-controlled study, patients with clear cell RCC previously treated with 1 prior angiogenesis inhibitor were randomized 1:1 to receive axitinib plus dalantercept versus axitinib plus placebo. Randomization was stratified by the type of prior therapy. The primary endpoint was progression-free survival (PFS). Secondary endpoints were PFS in patients with ≥2 prior lines of anticancer therapy, overall survival, and the objective response rate. RESULTS Between June 10, 2014, and February 23, 2017, a total of 124 patients were randomly assigned to receive axitinib plus dalantercept (59 patients) or placebo (65 patients). The median PFS was not found to be significantly different between the treatment groups (median, 6.8 months vs 5.6 months; hazard ratio, 1.11 [95% CI, 0.71-1.73; P = .670]). Neither group reached the median overall survival (hazard ratio, 1.39 [95% CI, 0.70-2.77; P = .349]). The objective response rate was 19.0% (11 of 58 patients; 95% CI, 9.9%-31.4%) in the dalantercept plus axitinib group and 24.6% (15 of 61 patients; 95% CI, 14.5%-37.3%) in the placebo plus axitinib group. At least 1 treatment-emergent adverse event of ≥grade 3 was observed in 59% of patients (34 of 58 patients) in the dalantercept group and 64% of patients (39 of 61 patients) in the placebo group. One treatment-related death occurred in the placebo plus axitinib group. CONCLUSIONS Although well tolerated, the addition of dalantercept to axitinib did not appear to improve treatment-related outcomes in previously treated patients with advanced RCC.