CRISPR-Cas9 screening identifies KRAS-induced COX-2 as a driver of immunotherapy resistance in lung cancer.

Oncogenic KRAS impairs anti-tumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of how oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive cyclooxygenase-2 (COX-2) in cancer cells. Oncogenic KRAS potently induced COX-2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX-2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX-2/PGE2 remodeled the tumor microenvironment by inducing pro-inflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX-2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX-2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer.

Untranslated regions (UTRs) are a potential novel source of neoantigens for personalised immunotherapy.

Background: Neoantigens, mutated tumour-specific antigens, are key targets of anti-tumour immunity during checkpoint inhibitor (CPI) treatment. Their identification is fundamental to designing neoantigen-directed therapy. Non-canonical neoantigens arising from the untranslated regions (UTR) of the genome are an overlooked source of immunogenic neoantigens. Here, we describe the landscape of UTR-derived neoantigens and release a computational tool, PrimeCUTR, to predict UTR neoantigens generated by start-gain and stop-loss mutations. Methods: We applied PrimeCUTR to a whole genome sequencing dataset of pre-treatment tumour samples from CPI-treated patients (n = 341). Cancer immunopeptidomic datasets were interrogated to identify MHC class I presentation of UTR neoantigens. Results: Start-gain neoantigens were predicted in 72.7% of patients, while stop-loss mutations were found in 19.3% of patients. While UTR neoantigens only accounted 2.6% of total predicted neoantigen burden, they contributed 12.4% of neoantigens with high dissimilarity to self-proteome. More start-gain neoantigens were found in CPI responders, but this relationship was not significant when correcting for tumour mutational burden. While most UTR neoantigens are private, we identified two recurrent start-gain mutations in melanoma. Using immunopeptidomic datasets, we identify two distinct MHC class I-presented UTR neoantigens: one from a recurrent start-gain mutation in melanoma, and one private to Jurkat cells. Conclusion: PrimeCUTR is a novel tool which complements existing neoantigen discovery approaches and has potential to increase the detection yield of neoantigens in personalised therapeutics, particularly for neoantigens with high dissimilarity to self. Further studies are warranted to confirm the expression and immunogenicity of UTR neoantigens.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Fcγ receptors and immunomodulatory antibodies in cancer.

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

Whole genome sequencing refines stratification and therapy of patients with clear cell renal cell carcinoma.

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.

Quantifying the impact of immunotherapy on RNA dynamics in cancer.

BACKGROUND: Checkpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer types. Resolving intratumor transcriptomic changes induced by CPI may improve our understanding of the mechanisms of sensitivity and resistance. METHODS: We assembled a cohort of longitudinal pre-therapy and on-therapy samples from 174 patients treated with CPI across six cancer types by leveraging transcriptomic sequencing data from five studies. RESULTS: Meta-analyses of published RNA markers revealed an on-therapy pattern of immune reinvigoration in patients with breast cancer, which was not discernible pre-therapy, providing biological insight into the impact of CPI on the breast cancer immune microenvironment. We identified 98 breast cancer-specific correlates of CPI response, including 13 genes which are known IO targets, such as toll-like receptors TLR1, TLR4, and TLR8, that could hold potential as combination targets for patients with breast cancer receiving CPI treatment. Furthermore, we demonstrate that a subset of response genes identified in breast cancer are already highly expressed pre-therapy in melanoma, and additionally we establish divergent RNA dynamics between breast cancer and melanoma following CPI treatment, which may suggest distinct immune microenvironments between the two cancer types. CONCLUSIONS: Overall, delineating longitudinal RNA dynamics following CPI therapy sheds light on the mechanisms underlying diverging response trajectories, and identifies putative targets for combination therapy.

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.

Pan-cancer T cell atlas links a cellular stress response state to immunotherapy resistance.

Tumor-infiltrating T cells offer a promising avenue for cancer treatment, yet their states remain to be fully characterized. Here we present a single-cell atlas of T cells from 308,048 transcriptomes across 16 cancer types, uncovering previously undescribed T cell states and heterogeneous subpopulations of follicular helper, regulatory and proliferative T cells. We identified a unique stress response state, TSTR, characterized by heat shock gene expression. TSTR cells are detectable in situ in the tumor microenvironment across various cancer types, mostly within lymphocyte aggregates or potential tertiary lymphoid structures in tumor beds or surrounding tumor edges. T cell states/compositions correlated with genomic, pathological and clinical features in 375 patients from 23 cohorts, including 171 patients who received immune checkpoint blockade therapy. We also found significantly upregulated heat shock gene expression in intratumoral CD4/CD8+ cells following immune checkpoint blockade treatment, particularly in nonresponsive tumors, suggesting a potential role of TSTR cells in immunotherapy resistance. Our well-annotated T cell reference maps, web portal and automatic alignment/annotation tool could provide valuable resources for T cell therapy optimization and biomarker discovery.

Translational Studies Using the MALT1 Inhibitor (S)-Mepazine to Induce Treg Fragility and Potentiate Immune Checkpoint Therapy in Cancer.

Introduction: Regulatory T cells (Tregs) play a critical role in the maintenance of immune homeostasis but also protect tumors from immune-mediated growth control or rejection and pose a significant barrier to effective immunotherapy. Inhibition of MALT1 paracaspase activity can selectively reprogram immune-suppressive Tregs in the tumor microenvironment to adopt a proinflammatory fragile state, which offers an opportunity to impede tumor growth and enhance the efficacy of immune checkpoint therapy (ICT). Methods: We performed preclinical studies with the orally available allosteric MALT1 inhibitor (S)-mepazine as a single-agent and in combination with anti-programmed cell death protein 1 (PD-1) ICT to investigate its pharmacokinetic properties and antitumor effects in several murine tumor models as well as patient-derived organotypic tumor spheroids (PDOTS). Results: (S)-mepazine demonstrated significant antitumor effects and was synergistic with anti-PD-1 therapy in vivo and ex vivo but did not affect circulating Treg frequencies in healthy rats at effective doses. Pharmacokinetic profiling revealed favorable drug accumulation in tumors to concentrations that effectively blocked MALT1 activity, potentially explaining preferential effects on tumor-infiltrating over systemic Tregs. Conclusions: The MALT1 inhibitor (S)-mepazine showed single-agent anticancer activity and presents a promising opportunity for combination with PD-1 pathway-targeted ICT. Activity in syngeneic tumor models and human PDOTS was likely mediated by induction of tumor-associated Treg fragility. This translational study supports ongoing clinical investigations (ClinicalTrials.gov Identifier: NCT04859777) of MPT-0118, (S)-mepazine succinate, in patients with advanced or metastatic treatment-refractory solid tumors.

The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.

Integrated transcriptome landscape of ALS identifies genome instability linked to TDP-43 pathology.

Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

Genomic-transcriptomic evolution in lung cancer and metastasis.

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

The Immunopeptidome from a Genomic Perspective: Establishing the Noncanonical Landscape of MHC Class I-Associated Peptides.

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development.

Clonal somatic copy number altered driver events inform drug sensitivity in high-grade serous ovarian cancer.

Chromosomal instability is a major challenge to patient stratification and targeted drug development for high-grade serous ovarian carcinoma (HGSOC). Here we show that somatic copy number alterations (SCNAs) in frequently amplified HGSOC cancer genes significantly correlate with gene expression and methylation status. We identify five prevalent clonal driver SCNAs (chromosomal amplifications encompassing MYC, PIK3CA, CCNE1, KRAS and TERT) from multi-regional HGSOC data and reason that their strong selection should prioritise them as key biomarkers for targeted therapies. We use primary HGSOC spheroid models to test interactions between in vitro targeted therapy and SCNAs. MYC chromosomal copy number is associated with in-vitro and clinical response to paclitaxel and in-vitro response to mTORC1/2 inhibition. Activation of the mTOR survival pathway in the context of MYC-amplified HGSOC is statistically associated with increased prevalence of SCNAs in genes from the PI3K pathway. Co-occurrence of amplifications in MYC and genes from the PI3K pathway is independently observed in squamous lung cancer and triple negative breast cancer. In this work, we show that identifying co-occurrence of clonal driver SCNA genes could be used to tailor therapeutics for precision medicine.

Immune Determinants of the Association between Tumor Mutational Burden and Immunotherapy Response across Cancer Types.

The FDA has recently approved a high tumor mutational burden (TMB-high) biomarker, defined by ≥10 mutations/Mb, for the treatment of solid tumors with pembrolizumab, an immune checkpoint inhibitor (ICI) that targets PD1. However, recent studies have shown that this TMB-high biomarker is only able to stratify ICI responders in a subset of cancer types, and the mechanisms underlying this observation have remained unknown. The tumor immune microenvironment (TME) may modulate the stratification power of TMB (termed TMB power), determining if it will be predictive of ICI response in a given cancer type. To systematically study this hypothesis, we inferred the levels of 31 immune-related factors characteristic of the TME of different cancer types in The Cancer Genome Atlas. Integration of this information with TMB and response data of 2,277 patients treated with anti-PD1 identified key immune factors that determine TMB power across 14 different cancer types. We find that high levels of M1 macrophages and low resting dendritic cells in the TME characterized cancer types with high TMB power. A model based on these two immune factors strongly predicted TMB power in a given cancer type during cross-validation and testing (Spearman Rho = 0.76 and 1, respectively). Using this model, we predicted the TMB power in nine additional cancer types, including rare cancers, for which TMB and ICI response data are not yet publicly available. Our analysis indicates that TMB-high may be highly predictive of ICI response in cervical squamous cell carcinoma, suggesting that such a study should be prioritized. SIGNIFICANCE: This study uncovers immune-related factors that may modulate the relationship between high tumor mutational burden and ICI response, which can help prioritize cancer types for clinical trials.

Frequency of pathogenic germline variants in cancer susceptibility genes in 1336 renal cell carcinoma cases.

Renal cell carcinoma (RCC) occurs in a number of cancer predisposition syndromes, but the genetic architecture of susceptibility to RCC is not well defined. We investigated the frequency of pathogenic and likely pathogenic (P/LP) germline variants in cancer susceptibility genes (CSGs) within a large series of unselected RCC participants. Whole-genome sequencing data on 1336 RCC participants and 5834 controls recruited to the UK 100 000 Genomes Project, a nationwide multicentre study, was analyzed to identify rare P/LP short variants (single nucleotide variants and insertions/deletions ranging from 1 to 50 base pairs) and structural variants in 121 CSGs. Among 1336 RCC participants [mean: 61.3 years (±12 SD), range: 13-88 years; 64% male], 85 participants [6.4%; 95% CI (5.1, 7.8)] had one or more P/LP germline variant in a wider range of CSGs than previously recognized. A further 64 intragenic variants in CSGs previously associated with RCC were classified as a variant of uncertain significance (VUS) (24 'hot VUSs') and were considered to be of potential clinical relevance as further evaluation might results in their reclassification. Most patients with P variants in well-established CSGs known to predispose to renal cell carcinoma (RCC-CSGs) were aged <50 years. Burden test analysis for filtered variants in CSGs demonstrated a significant excess of CHEK2 variants in European RCC participants compared with the healthy European controls (P = 0.0019). Approximately, 6% of the patients with RCC unselected for family history have a germline variant requiring additional follow-up analysis. To improve diagnostic yield, we suggest expanding the panel of RCC-CSGs tested to include CHEK2 and all SDHx subunits and raising the eligibility criteria for age-based testing.

Classifying cGAS-STING Activity Links Chromosomal Instability with Immunotherapy Response in Metastatic Bladder Cancer.

The cGAS-STING pathway serves a critical role in anticancer therapy. Particularly, response to immunotherapy is likely driven by both active cGAS-STING signaling that attracts immune cells, and by the presence of cancer neoantigens that presents as targets for cytotoxic T cells. Chromosomal instability (CIN) is a hallmark of cancer, but also leads to an accumulation of cytosolic DNA that in turn results in increased cGAS-STING signaling. To avoid triggering the cGAS-STING pathway, it is commonly disrupted by cancer cells, either through mutations in the pathway or through transcriptional silencing. Given its effect on the immune system, determining the cGAS-STING activation status prior to treatment initiation is likely of clinical relevance. Here, we used combined expression data from 2,307 tumors from five cancer types from The Cancer Genome Atlas to define a novel cGAS-STING activity score based on eight genes with a known role in the pathway. Using unsupervised clustering, four distinct categories of cGAS-STING activation were identified. In multivariate models, the cGAS-STING active tumors show improved prognosis. Importantly, in an independent bladder cancer immunotherapy-treated cohort, patients with low cGAS-STING expression showed limited response to treatment, while patients with high expression showed improved response and prognosis, particularly among patients with high CIN and more neoantigens. In a multivariate model, a significant interaction was observed between CIN, neoantigens, and cGAS-STING activation. Together, this suggests a potential role of cGAS-STING activity as a predictive biomarker for the application of immunotherapy. Significance: The cGAS-STING pathway is induced by CIN, triggers inflammation and is often deficient in cancer. We provide a tool to evaluate cGAS-STING activity and demonstrate clinical significance in immunotherapy response.