Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer, but a comprehensive description of its genomic landscape is lacking. We report the whole genome sequencing of 778 ccRCC patients enrolled in the 100,000 Genomes Project, providing the most detailed somatic mutational landscape to date. We identify new driver genes, which as well as emphasising the major role of epigenetic regulation in ccRCC highlight additional biological pathways extending opportunities for drug repurposing. Genomic characterisation identified patients with divergent clinical outcome; higher number of structural copy number alterations associated with poorer prognosis, whereas VHL mutations were independently associated with a better prognosis. The twin observations that higher T-cell infiltration is associated with better outcome and that genetically predicted immune evasion is not common supports the rationale for immunotherapy. These findings should inform personalised surveillance and treatment strategies for ccRCC patients.

Background Checkpoint inhibitor-induced hepatitis (CPI-hepatitis) is an emerging problem with the widening use of CPIs in cancer immunotherapy. Here, we developed a mouse model to characterize the mechanism of CPI-hepatitis and to therapeutically target key pathways driving this pathology. Methods C57BL/6 wild-type (WT) mice were dosed with toll-like receptor (TLR)9 agonist (TLR9-L) for hepatic priming combined with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) plus anti-programmed cell death 1 (PD-1) (“CPI”) or phosphate buffered saline (PBS) control for up to 7 days. Flow cytometry, histology/immunofluorescence and messenger RNA sequencing were used to characterize liver myeloid/lymphoid subsets and inflammation. Hepatocyte damage was assessed by plasma alanine transaminase (ALT) and cytokeratin-18 (CK-18) measurements. In vivo investigations of CPI-hepatitis were carried out in Rag2−/− and Ccr2rfp/rfp transgenic mice, as well as following anti-CD4, anti-CD8 or cenicriviroc (CVC; CCR2/CCR5 antagonist) treatment. Results Co-administration of combination CPIs with TLR9-L induced liver pathology closely resembling human disease, with increased infiltration and clustering of granzyme B+perforin+CD8+ T cells and CCR2+ monocytes, 7 days post treatment. This was accompanied by apoptotic hepatocytes surrounding these clusters and elevated ALT and CK-18 plasma levels. Liver RNA sequencing identified key signaling pathways (JAK-STAT, NF-ΚB) and cytokine/chemokine networks (Ifnγ, Cxcl9, Ccl2/Ccr2) as drivers of CPI-hepatitis. Using this model, we show that CD8+ T cells mediate hepatocyte damage in experimental CPI-hepatitis. However, their liver recruitment, clustering, and cytotoxic activity is dependent on the presence of CCR2+ monocytes. The absence of hepatic monocyte recruitment in Ccr2rfp/rfp mice and CCR2 inhibition by CVC treatment in WT mice was able to prevent the development and reverse established experimental CPI-hepatitis. Conclusion This newly established mouse model provides a platform for in vivo mechanistic studies of CPI-hepatitis. Using this model, we demonstrate the central role of liver infiltrating CCR2+ monocyte interaction with tissue-destructive CD8+ T cells in the pathogenesis of CPI-hepatitis and highlight CCR2 inhibition as a novel therapeutic target.

Cancer is an evolutionary process, and an evolutionary framework can be used to decipher cancer initiation, progression and treatment resistance. In the context of VHL disease with repeat evolution of malignancies across different tissue contexts we can examine the factors that constrain tumour evolution. In the largest analysis of VHL-related tumours to date (1321 tumours across 132 patients), we observe the second hits on the background of germline VHL mutation vary and include loss of chromosome 3p, narrow deletion of VHL locus, VHL mutation or methylation. Pheochromocytomas and paragangliomas (PPGL) and solid renal tumours show a higher frequency of 3p loss, while pancreatic neuroendocrine tumors (PNETs) and renal cysts, exhibited a higher frequency of somatic VHL mutations, without 3p loss. Tumour size and progression were contingent on the nature of the second hit, suggesting potential evolutionary "dead-ends" where the tumor initiating event does not involve 3p loss. The type of germline VHL mutation (whether a complete deletion or missense) determined the distribution of cancers and their burden, but beyond this, multiple renal tumours in individual patients did not converge onto the same trajectory. This observation suggests that following malignant transformation evolutionary contingency and chance dominate tumour evolution. Metastases from renal tumours were driven by loss of chromosomes 9p and 14q, reminiscent of sporadic renal tumours, suggesting that acquisition of metastatic competence is associated with a high degree of bottlenecking and highlighting these events as important targets for metastasis prevention. Citation Format: Scott Shepherd, Alex Coulton, Cathy Vocke, Chris Ricketts, Marston Linehan, Samra Turajlic. The cell of origin is the dominant constraint in evolution of VHL related neoplasms [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr IA025.

To maximize the impact of precision medicine approaches, it is critical to accurately identify genetic variants in cancer-associated genes with functional consequences. Yet, our knowledge of rare variants conferring clinically relevant phenotypes and the mechanisms through which they act remains highly limited. A tumor suppressor gene exemplifying the challenge of variant interpretation is VHL. VHL encodes an E3 ubiquitin ligase that regulates the cellular response to hypoxia. Germline pathogenic variants in VHL predispose patients to tumors including clear cell renal cell carcinoma (ccRCC) and pheochromocytoma, and somatic VHL mutations are frequently observed in sporadic renal cancer. Here, we optimize and apply Saturation Genome Editing (SGE) to assay nearly all possible single nucleotide variants (SNVs) across VHL’s coding sequence. To delineate mechanisms, we quantify mRNA dosage effects over time and compare effects in isogenic cell lines. Function scores for 2,268 VHL SNVs identify a core set of pathogenic alleles driving ccRCC with perfect accuracy, inform differential risk across tumor types, and reveal novel mechanisms by which variants impact function. These results have immediate utility for classifying VHL variants encountered in both germline testing and tumor profiling and illustrate how precise functional measurements can resolve pleiotropic and dosage-dependent genotype-phenotype relationships across complete genes.

Despite patients with stage II melanoma being relatively high risk, contributing to 30% of melanoma-associated deaths, there remains a relatively low individual chance of melanoma recurrence. Therefore, strategies are required to better select those patients at highest risk of recurrence. We have developed assays using circulating tumour DNA (ctDNA) to detect minimal residual disease (MRD) or molecular recurrence of melanoma. Originally, the DETECTION trial was designed to perform ctDNA sampling in addition to clinical follow-up in patients with stage IIB/C melanoma. Those with ctDNA detected were randomized 1 : 1 in a double-blind fashion to continue routine follow-up with the investigators’ choice of treatment if they developed disease recurrence or were unblinded and treated with nivolumab. Since its opening, adjuvant therapy has been licensed, with trials showing approximately 6% absolute reduction in distant metastasis in stage IIB/C melanoma with 1 year of antiprogrammed cell death protein 1 therapy; however, grade 3 or 4 toxicity was observed in 16% of patients, with 25% experiencing lifelong endocrine disorders. Better enrichment of patients at high risk of recurrence, and avoiding treatment toxicity in those who do not require it, therefore remains highly relevant. As DETECTION had only started recruitment, there was opportunity to redesign it, which we present here. We have designed tumour-informed assays targeting BRAF (V600E/K/R), NRAS (G12D, Q61K/L/R) and the H-TERT promoter region (−124 and −146) mutations for ctDNA detection using droplet digital polymerase chain reaction. DETECTION has been redesigned as a phase III trial with the primary objectives of elucidating (i) whether MRD/molecular relapse following curative intent surgery can be identified earlier than clinical relapse and (ii) whether early treatment of molecular recurrence based on ctDNA detection is noninferior to adjuvant therapy in preventing distant metastasis. Patients (n = 1000) with stage IIB/C/III/IV resected BRAF/NRAS/TERT promoter mutant cutaneous melanoma, ECOG 0/1 and adequate organ function, with complete resection performed within 12 weeks and radiological/clinical disease-free status confirmed and no prior immune/targeted therapy will be included. Patients will be randomized 1 : 1 to either 1 year of adjuvant therapy (arm A) or longitudinal ctDNA monitoring (arm B) and treatment only if a local recurrence (surgery followed by 1 year of adjuvant therapy with ongoing ctDNA monitoring) or ctDNA detection (2 years of the investigators’ choice of therapy). The primary endpoint is distant metastasis-free survival. CtDNA is a useful tool to monitor for MRD/molecular relapse. The DETECTION trial will assess whether it can be used to safely monitor patients and systemically treat only those at highest risk of melanoma progression.

Introduction: SETD2, a histone H3-K36 trimethyltransferase, is necessary for regulation of proper intron splicing. SETD2 is frequently inactivated in kidney cancer. We hypothesized that SETD2 deficiency would cause aberrant translation of retained introns (ATaRI) that could serve as potential immunotherapeutic targets in SETD2-mutant states. Methods and Results: We detected increased presence of retained introns in SETD2-mutant vs WT tumors in two publicly available renal cell carcinoma RNA-seq data sets. We hypothesized that if intronic sequences translated into proteins, accumulation of misfolded proteins would activate the Unfolded Protein Response (UPR). The UPR pathway was strongly transcriptionally enriched as measured by GSEA, as were several immunotherapy-relevant pathways, suggesting that tumors are inflamed, possibly related to the antigenic nature of translated introns. To investigate this further, we generated Setd2-isogenic mouse renal cancer cells using CRISPR in the RENCA cell line. RNAseq followed by GSEA confirmed upregulation of UPR pathway transcriptional signature in the Setd2-null condition, supporting our observation in human tumors. Indicators of UPR activation such as cleavage of ATF6 and increase in ATF4 and Xbp1 levels in Setd2 deficient vs. unedited RENCA cells, as well as nuclear translocation of ATF6 in SETD2-mutant human kidney cancer samples validates these findings. We then measured the presence of ATaRI-derived peptides in Setd2-mutant RENCA cells using mass spectrometry, detecting 47 such peptides. Using publicly available proteomics data from one human data set, we preliminarily identified peptides translated from 151 introns that are retained in SETD2-mutant cases. Applying netMHCpan to both the human and murine data, we found that detectable ATaRI peptides were predicted to generate multiple 10-amino acid-long peptides which strongly bind to the 8 murine MHC or to the 20 most common human HLA receptors. We propose that these presentable peptides might activate an immune response to SETD2-mutant renal cell carcinoma. Conclusions: We identified intron retention which may cause activation of the UPR as a feature of SETD2-mutant kidney cancer. ATaRI peptides are detectable and should be presented to the adaptive immune system. These features may represent a new therapeutic vulnerability for exploitation as a rationale for personalized medicine. Citation Format: Marya T. Kozinova, Alexander Metz, Robert Uzzo, Janusz Franco-Barraza, Michael Slifker, Jessica Peskin, Angel Fernández Sanromán, Samra Turajlic, Edna Cukierman, Phillip Phillip Abbosh. SETD2 loss in renal cell carcinoma generates peptides from aberrantly translated retained introns. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6794.

In many areas of oncology, we lack sensitive tumor-burden monitoring to guide critical decision making. While circulating tumor DNA (ctDNA) promises to enable disease monitoring, this approach is limited by the sparsity of ctDNA in the plasma. To overcome this challenge, error-corrected deep targeted sequencing has been proposed. Nonetheless, this framework is limited by the low number of genomic equivalents (GEs, ~103/mL of plasma), imposing a ceiling on effective sequencing depth. We have previously shown that genome-wide mutational integration through plasma whole genome sequencing (WGS) can sever the dependency between available GEs and assay sensitivity (Zviran et al, 2020). In this approach, tumor-informed mutational profiles are applied to plasma WGS, allowing detection of tumor fractions as low as 10−5. However, the higher cost of WGS limits practical depth of coverage (20-30X) and may limit broad adoption. Lower costs may thus allow for enhanced ctDNA cancer monitoring via WGS. We therefore applied emerging lower-cost WGS (1USD/Gb, Almogy et al, 2022) to plasma from 7 patients with metastatic cancer at ~115x coverage depth. Read depth profiling and error rates were comparable between matched Ultima and standard platform datasets. Integration of deep learning architectures for signal to noise enrichment (Widman et al, biorxiv, 2022) with deeper WGS coverage enabled ctDNA detection at the parts per million range. We reasoned that lower sequencing cost can be harnessed for duplex error-corrected WGS. Proof-of-concept experiments in mouse PDX samples showed ~1,500x decrease in errors. Applied to the plasma of stage IV melanoma patients (n=5), we obtained error rates ~10−7. We used this approach to tackle the challenging context of cancer monitoring in early-stage melanoma without matched tumor sequencing. While in uncorrected WGS, de novo mutation calling yielded limited ability to detect melanoma specific mutations, duplex-corrected WGS allowed us to harness melanoma mutational signatures for disease monitoring without matched tumor profiling. Analytic validation of our assay showed sensitive and specific cancer detection when the concentration of ctDNA was at 10−4 concentrations. Applied to a cohort of stage III melanoma patients with negative ctDNA detection using previously described methods, we detected ctDNA in all cases (n=4), demonstrating enhanced sensitivity using duplex WGS. These data demonstrate the exciting potential of low cost WGS for ultra-sensitive ctDNA cancer monitoring. In the tumor-informed setting, deeper sequencing increased sensitivity for mutational profile detection. Moreover, the application of duplex error-correction at genome scale allowed for sensitive cancer monitoring without matched tumor profiles. We envision that the era of low-cost sequencing will empower ultra-sensitive cancer monitoring via WGS, with transformative impact on cancer care. Citation Format: Alexandre P. Cheng, Adam J. Widman, Anushri Arora, Itai Rusinek, William F. Hooper, Rebecca Murray, Daniel Halmos, Theophile Langanay, Giorgio Inghirami, Soren Germer, Melissa Marton, Adrienne Helland, Rob Furatero, Jaime McClintock, Lara Winterkorn, Zoe Steinsnyder, Yohyoh Wang, Srinivas Rajagopalan, Asrar I. Alimohamed, Murtaza S. Malbari, Ashish Saxena, Margaret K. Callahan, Dennie T. Frederick, Lavinia Spain, Ariel Jaimovich, Doron Lipson, Samra Turajlic, Michael C. Zody, Nasser K. Altorki, Jedd D. Wolchok, Michael A. Postow, Nicolas Robine, Genevieve Boland, Dan A. Landau. Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5709.