Toward a CRISPR-based mouse model of Vhl-deficient clear cell kidney cancer: Initial experience and lessons learned.

CRISPR is revolutionizing the ability to do somatic gene editing in mice for the purpose of creating new cancer models. Inactivation of the VHL tumor suppressor gene is the signature initiating event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). Such tumors are usually driven by the excessive HIF2 activity that arises when the VHL gene product, pVHL, is defective. Given the pressing need for a robust immunocompetent mouse model of human ccRCC, we directly injected adenovirus-associated viruses (AAVs) encoding sgRNAs against VHL and other known/suspected ccRCC tumor suppressor genes into the kidneys of C57BL/6 mice under conditions where Cas9 was under the control of one of two different kidney-specific promoters (Cdh16 or Pax8) to induce kidney tumors. An AAV targeting Vhl, Pbrm1, Keap1, and Tsc1 reproducibly caused macroscopic ccRCCs that partially resembled human ccRCC tumors with respect to transcriptome and cell of origin and responded to a ccRCC standard-of-care agent, axitinib. Unfortunately, these tumors, like those produced by earlier genetically engineered mouse ccRCCs, are HIF2 independent.

A pathology foundation model for cancer diagnosis and prognosis prediction.

Histopathology image evaluation is indispensable for cancer diagnoses and subtype classification. Standard artificial intelligence methods for histopathology image analyses have focused on optimizing specialized models for each diagnostic task1,2. Although such methods have achieved some success, they often have limited generalizability to images generated by different digitization protocols or samples collected from different populations3. Here, to address this challenge, we devised the Clinical Histopathology Imaging Evaluation Foundation (CHIEF) model, a general-purpose weakly supervised machine learning framework to extract pathology imaging features for systematic cancer evaluation. CHIEF leverages two complementary pretraining methods to extract diverse pathology representations: unsupervised pretraining for tile-level feature identification and weakly supervised pretraining for whole-slide pattern recognition. We developed CHIEF using 60,530 whole-slide images spanning 19 anatomical sites. Through pretraining on 44 terabytes of high-resolution pathology imaging datasets, CHIEF extracted microscopic representations useful for cancer cell detection, tumour origin identification, molecular profile characterization and prognostic prediction. We successfully validated CHIEF using 19,491 whole-slide images from 32 independent slide sets collected from 24 hospitals and cohorts internationally. Overall, CHIEF outperformed the state-of-the-art deep learning methods by up to 36.1%, showing its ability to address domain shifts observed in samples from diverse populations and processed by different slide preparation methods. CHIEF provides a generalizable foundation for efficient digital pathology evaluation for patients with cancer.

Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity.

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

Generation and Characterization of Ex Vivo Expanded Tumor-infiltrating Lymphocytes From Renal Cell Carcinoma Tumors for Adoptive Cell Therapy.

Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance antitumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma.

Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity. Herein, we characterized the epigenomic landscape of sRCC by profiling 107 epigenomic libraries from tissue and plasma samples from 50 patients with RCC and healthy volunteers. By profiling histone modifications and DNA methylation, we identified highly recurrent epigenomic reprogramming enriched in sRCC. Furthermore, CRISPRa experiments implicated the transcription factor FOSL1 in activating sRCC-associated gene regulatory programs, and FOSL1 expression was associated with the response to ICIs in RCC in two randomized clinical trials. Finally, we established a blood-based diagnostic approach using detectable sRCC epigenomic signatures in patient plasma, providing a framework for discovering epigenomic correlates of tumor histology via liquid biopsy.

Perioperative nivolumab versus observation in patients with renal cell carcinoma undergoing nephrectomy (PROSPER ECOG-ACRIN EA8143): an open-label, randomised, phase 3 study.

BACKGROUND: The standard of care for patients with intermediate-to-high risk renal cell carcinoma is partial or radical nephrectomy followed by surveillance. We aimed to investigate use of nivolumab before nephrectomy followed by adjuvant nivolumab in patients with high-risk renal cell carcinoma to determine recurrence-free survival compared with surgery only. METHODS: In this open-label, randomised, phase 3 trial (PROSPER EA8143), patients were recruited from 183 community and academic sites across the USA and Canada. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-1, with previously untreated clinical stage T2 or greater or Tany N+ renal cell carcinoma of clear cell or non-clear cell histology planned for partial or radical nephrectomy. Selected patients with oligometastatic disease, who were disease free at other disease sites within 12 weeks of surgery, were eligible for inclusion. We randomly assigned (1:1) patients using permuted blocks (block size of 4) within stratum (clinical TNM stage) to either nivolumab plus surgery, or surgery only followed by surveillance. In the nivolumab group, nivolumab 480 mg was administered before surgery, followed by nine adjuvant doses. The primary endpoint was investigator-reviewed recurrence-free survival in patients with renal cell carcinoma assessed in all randomly assigned patients regardless of histology. Safety was assessed in all randomly assigned patients who started the assigned protocol treatment. This trial is registered with ClinicalTrials.gov, NCT03055013, and is closed to accrual. FINDINGS: Between Feb 2, 2017, and June 2, 2021, 819 patients were randomly assigned to nivolumab plus surgery (404 [49%]) or surgery only (415 [51%]). 366 (91%) of 404 patients assigned to nivolumab plus surgery and 387 (93%) of 415 patients assigned to surgery only group started treatment. Median age was 61 years (IQR 53-69), 248 (30%) of 819 patients were female, 571 (70%) were male, 672 (88%) were White, and 77 (10%) were Hispanic or Latino. The Data and Safety Monitoring Committee stopped the trial at a planned interim analysis (March 25, 2022) because of futility. Median follow-up was 30·4 months (IQR 21·5-42·4) in the nivolumab group and 30·1 months (21·9-41·8) in the surgery only group. 381 (94%) of 404 patients in the nivolumab plus surgery group and 399 (96%) of 415 in the surgery only group had renal cell carcinoma and were included in the recurrence-free survival analysis. As of data cutoff (May 24, 2023), recurrence-free survival was not significantly different between nivolumab (125 [33%] of 381 had recurrence-free survival events) versus surgery only (133 [33%] of 399; hazard ratio 0·94 [95% CI 0·74-1·21]; one-sided p=0·32). The most common treatment-related grade 3-4 adverse events were elevated lipase (17 [5%] of 366 patients in the nivolumab plus surgery group vs none in the surgery only group), anaemia (seven [2%] vs nine [2%]), increased alanine aminotransferase (ten [3%] vs one [<1%]), abdominal pain (four [1%] vs six [2%]), and increased serum amylase (nine [2%] vs none). 177 (48%) patients in the nivolumab plus surgery group and 93 (24%) in the surgery only group had grade 3-5 adverse events due to any cause, the most common of which were anaemia (23 [6%] vs 19 [5%]), hypertension (27 [7%] vs nine [2%]), and elevated lipase (18 [5%] vs six [2%]). 48 (12%) of 404 patients in the nivolumab group and 40 (10%) of 415 in the surgery only group died, of which eight (2%) and three (1%), respectively, were determined to be treatment-related. INTERPRETATION: Perioperative nivolumab before nephrectomy followed by adjuvant nivolumab did not improve recurrence-free survival versus surgery only followed by surveillance in patients with high-risk renal cell carcinoma. FUNDING: US National Institutes of Health National Cancer Institute and Bristol Myers Squibb.

Impact of renal cell carcinoma molecular subtypes on immunotherapy and targeted therapy outcomes.

Saliby et al. show that a machine learning approach can accurately classify clear cell renal cell carcinoma (RCC) into distinct molecular subtypes using transcriptomic data. When applied to tumors biospecimens from the JAVELIN Renal 101 (JR101) trial, a benefit is observed with immune checkpoint inhibitor (ICI)-based therapy across all molecular subtypes.

Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

PD-1 Expression on Intratumoral Regulatory T Cells Is Associated with Lack of Benefit from Anti-PD-1 Therapy in Metastatic Clear-Cell Renal Cell Carcinoma Patients.

PURPOSE: Programmed cell death protein 1 (PD-1) expression on CD8+TIM-3-LAG-3- tumor-infiltrating cells predicts positive response to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC). Because inhibition of PD-1 signaling in regulatory T cells (Treg) augments their immunosuppressive function, we hypothesized that PD-1 expression on tumor-infiltrating Tregs would predict resistance to PD-1 inhibitors. EXPERIMENTAL DESIGN: PD-1+ Tregs were phenotyped using multiparametric immunofluorescence in ccRCC tissues from the CheckMate-025 trial (nivolumab: n = 91; everolimus: n = 90). Expression of CD8, PD-1, TIM-3, and LAG-3 was previously determined (Ficial and colleagues, 2021). Clinical endpoints included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: In the nivolumab (but not everolimus) arm, high percentage of PD-1+ Tregs was associated with shorter PFS (3.19 vs. 5.78 months; P = 0.021), shorter OS (18.1 vs. 27.7 months; P = 0.013) and marginally lower ORR (12.5% vs. 31.3%; P = 0.059). An integrated biomarker (PD-1 Treg/CD8 ratio) was developed by calculating the ratio between percentage of PD-1+Tregs (marker of resistance) and percentage of CD8+PD-1+TIM-3-LAG-3- cells (marker of response). In the nivolumab (but not everolimus) arm, patients with high PD-1 Treg/CD8 ratio experienced shorter PFS (3.48 vs. 9.23 months; P < 0.001), shorter OS (18.14 vs. 38.21 months; P < 0.001), and lower ORR (15.69% vs. 40.00%; P = 0.009). Compared with the individual biomarkers, the PD-1 Treg/CD8 ratio showed improved ability to predict outcomes to nivolumab versus everolimus. CONCLUSIONS: PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC, suggesting that targeting Tregs may synergize with PD-1 inhibition. A model that integrates PD-1 expression on Tregs and CD8+TIM-3-LAG-3- cells has higher predictive value.

Regulation of HHLA2 expression in kidney cancer and myeloid cells.

BACKGROUND: The immune checkpoint HERV-H LTR-associating 2 (HHLA2) is expressed in kidney cancer and various other tumor types. Therapeutics targeting HHLA2 or its inhibitory receptor KIR3DL3 are being developed for solid tumors, including renal cell carcinoma (RCC). However, the regulation of HHLA2 expression remains poorly understood. A better understanding of HHLA2 regulation in tumor cells and the tumor microenvironment is crucial for the successful translation of these therapeutic agents into clinical applications. METHODS: Flow cytometry and quantitative real-time PCR were used to analyze HHLA2 expression in primary kidney tumors ex vivo and during in vitro culture. HHLA2 expression in A498 and 786-O ccRCC cell lines was examined in vitro and in subcutaneous tumor xenografts in NSG mice. Monocytes and dendritic cells were analyzed for HHLA2 expression. We tested a range of cytokines and culture conditions, including hypoxia, to induce HHLA2 expression. RESULTS: Analysis of HHLA2 expression revealed that HHLA2 is expressed on tumor cells in primary kidney tumors ex vivo; however, its expression gradually diminishes during a 4-week in vitro culture period. A498 and 786-O ccRCC tumor cell lines do not express HHLA2 in vitro, but HHLA2 expression was observed when grown as subcutaneous xenografts in NSG immunodeficient mice. Induction experiments using various cytokines and culture conditions failed to induce HHLA2 expression in A498 and 786-O tumor cell lines in vitro. Analysis of HHLA2 expression in monocytes and dendritic cells demonstrated that only IL-10 and BMP4, along with IL-1ß and IL-6 to a lesser extent, modestly enhanced HHLA2 protein and mRNA expression. CONCLUSIONS: HHLA2 expression is induced on kidney cancer cells in vivo by a tumor microenvironmental signal that is not present in vitro. HHLA2 expression is differentially regulated in kidney cancer epithelial cells and monocytes. Cytokines, particularly IL10, that induce HHLA2 expression in monocytes fail to upregulate HHLA2 expression in tumor cell lines in vitro. These findings underscore the importance of the interplay between tumor cell and tumor microenvironmental signals in the regulation of HHLA2. Further investigation is warranted to elucidate the mechanisms involved in HHLA2 regulation and its implications for therapeutic development.

Spatially aware deep learning reveals tumor heterogeneity patterns that encode distinct kidney cancer states.

Clear cell renal cell carcinoma (ccRCC) is molecularly heterogeneous, immune infiltrated, and selectively sensitive to immune checkpoint inhibition (ICI). However, the joint tumor-immune states that mediate ICI response remain elusive. We develop spatially aware deep-learning models of tumor and immune features to learn representations of ccRCC tumors using diagnostic whole-slide images (WSIs) in untreated and treated contexts (n = 1,102 patients). We identify patterns of grade heterogeneity in WSIs not achievable through human pathologist analysis, and these graph-based "microheterogeneity" structures associate with PBRM1 loss of function and with patient outcomes. Joint analysis of tumor phenotypes and immune infiltration identifies a subpopulation of highly infiltrated, microheterogeneous tumors responsive to ICI. In paired multiplex immunofluorescence images of ccRCC, microheterogeneity associates with greater PD1 activation in CD8+ lymphocytes and increased tumor-immune interactions. Our work reveals spatially interacting tumor-immune structures underlying ccRCC biology that may also inform selective response to ICI.

Immune dysfunction revealed by digital spatial profiling of immuno-oncology markers in progressive stages of renal cell carcinoma and in brain metastases.

BACKGROUND: The tumor microenvironment (TME) contributes to cancer progression and treatment response to therapy, including in renal cell carcinoma (RCC). Prior profiling studies, including single-cell transcriptomics, often involve limited sample sizes and lack spatial orientation. The TME of RCC brain metastases, a major cause of morbidity, also remains largely uncharacterized. METHODS: We performed digital spatial profiling on the NanoString GeoMx platform using 52 validated immuno-oncology markers on RCC tissue microarrays representing progressive stages of RCC, including brain metastases. We profiled 76 primary tumors, 27 adjacent histologically normal kidney samples, and 86 metastases, including 24 brain metastases. RESULTS: We observed lower immune checkpoint (TIM-3 and CTLA-4), cytolytic (GZMA and GZMB), and T cell activation (CD25) protein expression in metastases compared with primary tumors in two separate cohorts. We also identified changes in macrophages in metastases, with brain metastases-susceptible patients showing less M1-like, inflammatory macrophage markers (HLA-DR and CD127) in metastatic samples. A comparison of brain metastases to extracranial metastases revealed higher expression of the anti-apoptotic, BCL-2-family protein BCL-XL and lower expression of the innate immune activator STING in brain metastases. Lower TIM-3 and CD40 in the TME of brain metastases appear to be associated with longer survival, a finding that requires further validation. CONCLUSIONS: Compared with primary tumors, RCC metastases, including brain metastases, express lower levels of numerous markers of immune activation and current or investigational therapeutic targets. Our findings may have important implications for designing future biomarker and treatment studies and may aid in development of brain metastases-specific therapies.

KDM6A epigenetically regulates subtype plasticity in small cell lung cancer.

Small cell lung cancer (SCLC) exists broadly in four molecular subtypes: ASCL1, NEUROD1, POU2F3 and Inflammatory. Initially, SCLC subtypes were thought to be mutually exclusive, but recent evidence shows intra-tumoural subtype heterogeneity and plasticity between subtypes. Here, using a CRISPR-based autochthonous SCLC genetically engineered mouse model to study the consequences of KDM6A/UTX inactivation, we show that KDM6A inactivation induced plasticity from ASCL1 to NEUROD1 resulting in SCLC tumours that express both ASCL1 and NEUROD1. Mechanistically, KDM6A normally maintains an active chromatin state that favours the ASCL1 subtype with its loss decreasing H3K4me1 and increasing H3K27me3 at enhancers of neuroendocrine genes leading to a cell state that is primed for ASCL1-to-NEUROD1 subtype switching. This work identifies KDM6A as an epigenetic regulator that controls ASCL1 to NEUROD1 subtype plasticity and provides an autochthonous SCLC genetically engineered mouse model to model ASCL1 and NEUROD1 subtype heterogeneity and plasticity, which is found in 35-40% of human SCLCs.

Circulating and Intratumoral Immune Determinants of Response to Atezolizumab plus Bevacizumab in Patients with Variant Histology or Sarcomatoid Renal Cell Carcinoma.

Renal cell carcinoma (RCC) of variant histology comprises approximately 20% of kidney cancer diagnoses, yet the optimal therapy for these patients and the factors that impact immunotherapy response remain largely unknown. To better understand the determinants of immunotherapy response in this population, we characterized blood- and tissue-based immune markers for patients with variant histology RCC, or any RCC histology with sarcomatoid differentiation, enrolled in a phase II clinical trial of atezolizumab and bevacizumab. Baseline circulating (plasma) inflammatory cytokines were highly correlated with one another, forming an "inflammatory module" that was increased in International Metastatic RCC Database Consortium poor-risk patients and was associated with worse progression-free survival (PFS; P = 0.028). At baseline, an elevated circulating vascular endothelial growth factor A (VEGF-A) level was associated with a lack of response (P = 0.03) and worse PFS (P = 0.021). However, a larger increase in on-treatment levels of circulating VEGF-A was associated with clinical benefit (P = 0.01) and improved overall survival (P = 0.0058). Among peripheral immune cell populations, an on-treatment decrease in circulating PD-L1+ T cells was associated with improved outcomes, with a reduction in CD4+PD-L1+ [HR, 0.62; 95% confidence interval (CI), 0.49-0.91; P = 0.016] and CD8+PD-L1+ T cells (HR, 0.59; 95% CI, 0.39-0.87; P = 0.009) correlated with improved PFS. Within the tumor itself, a higher percentage of terminally exhausted (PD-1+ and either TIM-3+ or LAG-3+) CD8+ T cells was associated with worse PFS (P = 0.028). Overall, these findings support the value of tumor and blood-based immune assessments in determining therapeutic benefit for patients with RCC receiving atezolizumab plus bevacizumab and provide a foundation for future biomarker studies for patients with variant histology RCC receiving immunotherapy-based combinations.

Pathological Outcomes of Patients With Advanced Renal Cell Carcinoma Who Receive Nephrectomy Following Immunotherapy.

BACKGROUND: Even though cytoreductive nephrectomy (CN) was once the standard of care for patients with advanced renal cell carcinoma (RCC), its role in treatment has not been well analyzed or defined in the era of immunotherapy (IO). MATERIALS AND METHODS: This study analyzed pathological outcomes in patients with advanced or metastatic RCC who received IO prior to CN. This was a multi-institutional, retrospective study of patients with advanced or metastatic RCC. Patients were required to receive IO monotherapy or combination therapy prior to radical or partial CN. The primary endpoint assessed surgical pathologic outcomes, including American Joint Committee on Cancer (AJCC) staging and frequency of downstaging, at the time of surgery. Pathologic outcomes were correlated to clinical variables using a Wald-chi squared test from Cox regression in a multi-variable analysis. Secondary outcomes included objective response rate (ORR) defined by response evaluation criteria in solid tumors (RECIST) version 1.1 and progression-free survival (PFS), which were estimated using the Kaplan-Meier method with reported 95% CIs. RESULTS: Fifty-two patients from 9 sites were included. Most patients were male (65%), 81% had clear cell histology, 11% had sarcomatoid differentiation. Overall, 44% of patients experienced pathologic downstaging, and 13% had a complete pathologic response. The ORR immediately prior to nephrectomy was stable disease in 29% of patients, partial response in 63%, progressive disease in 4%, and 4% unknown. Median follow-up for the entire cohort was 25.3 months and median PFS was 3.5 years (95% CI, 2.1-4.9). CONCLUSIONS: IO-based interventions prior to CN in patients with advanced or metastatic RCC demonstrates efficacy, with a small fraction of patients showing a complete response. Additional prospective studies are warranted to investigate the role of CN in the modern IO-era.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.