The high cost and attrition rate of drug development underscore the need for more effective strategies for therapeutic target discovery. Here, we present a network medicine-based machine learning framework that integrates single-cell transcriptomics, bulk multi-omic profiles, genome-wide CRISPR perturbation screens, and protein-protein interaction networks to systematically prioritise disease-specific targets. Applied to clear cell renal cell carcinoma, the framework successfully recovered established targets and predicted five therapeutic candidates, with subsequent in vitro validation demonstrating that among these, ENO2 inhibition had the strongest anti-tumour effect, followed by LRRK2, a repurposing candidate with phase III Parkinson’s disease inhibitors. The proposed approach advances target discovery by moving beyond single-feature, single-modality heuristics to a scalable, machine learning-driven strategy that is generalisable across diseases.

Background Clear cell renal cell carcinoma (ccRCC) is a highly heterogeneous cancer requiring a large number of biopsies to correctly characterize the tumor. Multiple biopsies are rarely feasible in the clinical setting, and therefore imaging methods offer the potential to evaluate the whole tumor non-invasively. For example, metabolic imaging offers the potential to probe the altered metabolism and metabolic heterogeneity that is characteristic of ccRCC. In this study we have explored and validated the use of hyperpolarized carbon-13 MRI (HP- 13 C-MRI) as a non-invasive clinical tool to probe metabolic heterogeneity in ccRCC patients and to more accurately identify which metabolic pathways are altered in vivo . Methods 58 tumor and healthy tissues biopsies were acquired postoperatively from 6 ccRCC patients imaged following injection of hyperpolarized [1- 13 C]pyruvate. MRI parameters were correlated with the metabolomic (146 metabolites) and transcriptomic (2523 metabolic genes) data obtained from these biopsies, split across 34 metabolic pathways. The results were used to generate metabologram projections as a visual representation of these metabolic differences. For each metabolic pathway, we generated a novel metabolic consensus scoring system for the identification of key altered metabolic pathways in ccRCC and their relationship to the imaging parameters. Results We show that the apparent exchange constant between pyruvate and lactate ( k PL ) and the lactate to pyruvate ratio (LP r ) on MRI can be used to measure differential metabolic pathways: they correlated positively with glycolysis and the pentose phosphate pathway, negatively with the TCA cycle, while also correlating with other pathways. Dichotomizing the imaged signal based on high and low k PL measurements was sufficient to discriminate metabolic distinct regions on biopsy and this could be a simple tool to assess metabolism clinically. Furthermore, metabolic heterogeneity increased in regions with a higher k PL and could be used to assess metabolic divergence. Conclusion This work validated the role of HP- 13 C-MRI to measure not only glycolysis, but also a range of other altered metabolic pathways in ccRCC. This could improve tumor stratification and provide novel methods to monitor treatment response. Metabolic imaging can also be used to guide biopsy acquisition based on metabolic alterations, and therefore could improve tumour characterization.

Fumarate hydratase-deficient renal cell carcinoma (FHd-RCC) is a rare and aggressive renal cancer subtype characterised by increased fumarate accumulation and upregulated lactate production. Renal tumours demonstrate significant intratumoral metabolic heterogeneity, which may contribute to treatment failure. Emerging non-invasive metabolic imaging techniques have clinical potential to more accurately phenotype tumour metabolism and its heterogeneity. In this case study we have used hyperpolarised 13C-pyruvate MRI (HP 13C-MRI) to assess 13C-lactate generation in a patient with an organ-confined FHd-RCC. Post-operative tissue samples were co-registered with imaging and underwent sequencing, IHC staining, and mass spectrometry imaging (MSI). HP 13C-MRI reveals two metabolically distinct tumour regions. The 13C-lactate-rich region shows a high lactate/pyruvate ratio and slightly lower fumarate on MSI compared to the other tumour region, as well as increased CD8 + T cell infiltration, and genetic dedifferentiation. Compared to the normal kidney, the vascularity in the tumour is decreased, while immune cell fraction is markedly higher. This study shows the potential of metabolic HP 13C-MRI to characterise FHd-RCC and how targeting of biopsies to regions of metabolic dysregulation could be used to obtain the tumour samples of greatest clinical significance, which in turn can inform on early and successful response to treatment. Horvat-Menih et al. use hyperpolarised 13C-pyruvate MRI (HP 13C-MRI) to assess 13C-lactate generation in a patient with a fumarate hydratase-deficient renal cell carcinoma (FHd-RCC), and correlate imaging findings with genetic and metabolic analysis on post-operative tissue samples. HP 13C-MRI reveals two metabolically distinct tumour regions. Kidney cancer resulting from a genetic mutation of a protein responsible for sugar metabolism, called “fumarate hydratase” is a rare and aggressive cancer. To understand this complex cancer better, our study aimed to assess the sugar metabolism directly in a patient bearing this tumour, using a specialised imaging technique. In addition, we performed biological analyses of the tumour. The results revealed two regions with different biology and aggressiveness within the same tumour. This shows the potential of the imaging technique to detect regions of varying aggressiveness within the same tumour, which could guide biopsies to obtain tumour samples and inform upon early treatment decisions.

Human isotopic tracer studies are key for in vivo studies of cancer metabolism. Yet, the effects of sampling conditions on the tissue metabolome remain understudied. Here, we perform a 13C-glucose study coupled with metabolomic, transcriptomic, and proteomic profiling in patients with clear cell renal cell carcinoma (ccRCC) to assess the impact of ischaemia on tissues sampled intraoperatively and post-surgical resection, where tissues are exposed to varying degrees of warm ischaemia. Although several metabolic features were preserved, including suppressed TCA cycle activity, ischaemia masked other metabolic phenotypes of ccRCC, such as suppressed gluconeogenesis. Notably, normal kidneys were more metabolically susceptible to ischaemia than the ccRCC tumours. Despite their overall stability, ischaemia caused subtle changes in the proteome and transcriptome. Using orthotopic ccRCC-derived xenografts, we evidenced that prolonged ischaemia disrupted the tissue metabolome stability. Overall, minimising tissue ischaemia is pivotal in accurately profiling cancer metabolism in patient studies.

Venous tumour thrombus (VTT), where the primary tumour invades the renal vein and inferior vena cava, affects 10-15% of renal cell carcinoma (RCC) patients. Curative surgery for VTT is high-risk, but neoadjuvant therapy may improve outcomes. The NAXIVA trial demonstrated a 35% VTT response rate after 8 weeks of neoadjuvant axitinib, a VEGFR-directed therapy. However, understanding non-response is critical for better treatment. Here we show that response to axitinib in this setting is characterised by a distinct and predictable set of features. We conduct a multiparametric investigation of samples collected during NAXIVA using digital pathology, flow cytometry, plasma cytokine profiling and RNA sequencing. Responders have higher baseline microvessel density and increased induction of VEGF-A and PlGF during treatment. A multi-modal machine learning model integrating features predict response with an AUC of 0.868, improving to 0.945 when using features from week 3. Key predictive features include plasma CCL17 and IL-12. These findings may guide future treatment strategies for VTT, improving the clinical management of this challenging scenario.

Clear cell renal cell carcinoma (ccRCC) is characterised by significant genetic heterogeneity, which has diagnostic and prognostic implications. Very limited evidence is available regarding DNA methylation heterogeneity. We therefore generate sequence level DNA methylation data on 136 multi-region tumour and normal kidney tissue from 18 ccRCC patients, along with matched whole exome sequencing (85 samples) and gene expression (47 samples) data on a subset of samples. We perform a comprehensive systematic analysis of heterogeneity between patients, within a patient and within a sample. We demonstrate that bulk methylation data may be deconvoluted into cell-type-specific latent methylation components (LMCs), and that LMC1, which is likely to represent T cells, is associated with prognostic parameters. Differential epipolymorphism was noted between ccRCC and normal tissue in the promoter region of genes which are known to be associated with kidney cancer. This was externally validated in an independent cohort of 71 ccRCC and normal kidney tissues. Differential epipolymorphism in the gene promoter was a predictor of gene expression, after adjusting for average methylation. This represents the first evaluation of epipolymorphism in ccRCC and suggests that gains and losses in methylation disorder may have a functional relevance, gleaning important information on tumourigenesis.

Venous tumor thrombus (VTT) is present in 10-15% patients with renal cell carcinoma (RCC), where the primary tumor invades the renal vein and inferior vena cava, increasing the morbidity and mortality of curative surgery. Neoadjuvant anti-angiogenic therapies have shown potential to reduce VTT length and improve nephrectomy success rates. The NAXIVA trial, a phase II study of neoadjuvant axitinib in 20 RCC patients with VTT, demonstrated that 35% of patients experienced significant downstaging of VTT. However, the biological mechanisms driving response or resistance in the remaining 65% are unclear. Machine learning has the potential to uncover mechanistic relationships between features in these patients and guide biomarker discovery for early response.In this study, we present a predictive machine learning framework based on multi-modal integration of digital pathology, flow cytometry and plasma cytokine profiling, using tissue and blood samples from the NAXIVA trial. A machine learning model incorporating recursive feature elimination and logistic regression with stochastic gradient descent was developed to predict response. To avoid overfitting, a leave-one-out nested cross validation approach was used for 20 iterations, leaving one patient out at a time and training the model on the remaining 19 patients. Two separate model ensembles were produced, using baseline features (N=62) and features from both baseline and two weeks post-treatment initiation (N=69) respectively. We used a statistical consensus approach to identify key predictive factors.The baseline model achieved an AUC of 0.868, identifying tissue microvessel density, plasma IL-12p70 and plasma CCL17 as key predictive factors. Responding patients had lower plasma IL-12p70, lower CCL17 and higher microvessel density than non-responding patients. Bulk RNA-seq data from pre-treatment tumor biopsies revealed elevated IL-12R expression in non-responders. Incorporating features post-treatment initiation improved model performance (AUC = 0.945), with fold changes in PlGF and sTie-2, alongside baseline IL-12p70 and CCL17, emerging as key predictors. Responders showed greater PlGF and sTie-2 induction following treatment onset.In conclusion, our machine learning framework accurately predicts response to neoadjuvant axitinib in RCC patients with VTT. The high microvessel density and growth factor induction in responders suggests they exhibit an “angiogenic” phenotype (clusters 1/2 from IMmotion151), while higher cytokine levels in non-responders suggest an “immunogenic” phenotype (cluster 4). Early changes in PlGF and sTie-2 may serve as actionable biomarkers for patient stratification. Further validation in independent datasets is essential to facilitate clinical integration and improve personalized treatment strategies for RCC patients with VTT. Rebecca Wray, Hania Paverd, Ines Machado, Johanna Barbieri, Farhana Easita, Abigail R. Edwards, Ferdia A. Gallagher, Iosif A. Mendichovszky, Thomas J. Mitchell, Maike De La Roche, Jacqueline D. Shields, Stephan Ursprung, Lauren Wallis, Anne Y. Warren, Sarah J. Welsh, Mireia Crispin-Ortuzar, Grant D. Stewart, James O. Jones. Multi-modal machine learning identifies predictive biomarkers of response to neoadjuvant axitinib in renal cell carcinoma with venous tumor thrombus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1103.

Background: Early and accurate grading of renal cell carcinoma (RCC) improves patient risk stratification and has implications for clinical management and mortality. However, current diagnostic approaches using imaging and renal mass biopsy have limited specificity and may lead to undergrading. Methods: This study explored the use of hyperpolarised [1-13C]pyruvate MRI (HP 13C-MRI) to identify the most aggressive areas within the tumour of patients with clear cell renal cell carcinoma (ccRCC) as a method to guide biopsy targeting and to reduce undergrading. Six patients with ccRCC underwent presurgical HP 13C-MRI and conventional contrast-enhanced MRI. From the imaging data, three k-means clusters were computed by combining the kPL as a marker of metabolic activity, and the 13C-pyruvate signal-to-noise ratio (SNRPyr) as a perfusion surrogate. The combined clusters were compared to those derived from individual parameters and to those derived from the percentage of enhancement on the nephrographic phase (%NG). The diagnostic performance of each cluster was assessed based on its ability to predict the highest histological tumour grade in postsurgical tissue samples. The postsurgical tissue samples underwent immunohistochemical staining for the pyruvate transporter (monocarboxylate transporter 1, MCT1), as well as RNA and whole-exome sequencing. Results: The clustering approach combining SNRPyr and kPL demonstrated the best performance for predicting the highest tumour grade: specificity 85%; sensitivity 64%; positive predictive value 82%; and negative predictive value 68%. Epithelial MCT1 was identified as the major determinant of the HP 13C-MRI signal. The perfusion/metabolism mismatch cluster showed an increased expression of metabolic genes and markers of aggressiveness. Conclusions: This study demonstrates the potential of using HP 13C-MRI-derived metabolic clusters to identify intratumoral variations in tumour grade with high specificity. This work supports the use of metabolic imaging to guide biopsies to the most aggressive tumour regions and could potentially reduce sampling error.

While systemic anti-cancer therapy (SACT) has become the principal management option for many patients with metastatic RCC, there remains debate on the benefits of delayed cytoreductive nephrectomy (CN), and on whether immediate CN and tumour thrombectomy is the correct strategy when there is venous tumour thrombus (VTT) extending into the inferior vena cava (IVC). Given the poor prognosis associated with untreated VTT, the risks of rapid progression and the sequalae from venous congestion or distal embolism, urgent extirpative surgery may be beneficial, dependent on patient fitness. Alternatively, given the high clinical response rates of modern immunotherapy (IO)-based systemic treatment, there has been interest in downstaging cases of locally advanced RCC (including those with VTT) with neoadjuvant therapy [1]. We present a case where a patient with metastatic clear cell (cc)RCC and with Mayo Level IV IVC tumour thrombus had an excellent response to treatment with lenvatinib and pembrolizumab, which allowed for single-cavity extirpative surgery. A 69-year-old female of Eastern European descent presented in October 2022 with lower abdominal pain, constipation, and lethargy. A CT scan (Fig. 1A) identified a 95-mm right lower pole renal mass, with VTT extending to the right atrium (Mayo Level IV). She also had an enhancing 18-mm right adrenal nodule and three lung nodules (largest 9 mm) in keeping with metastatic disease. She had a past history of hypertension and smoking (~50 pack-years) and reported restriction in physical activity due to fatigue/lethargy (Eastern Cooperative Oncology Group [ECOG] Performance Status 1). Initial biochemistry found of note: haemoglobin 84 g/L (reference range: 118–158 g/L), platelets 643 × 109/L (reference range: 160–370 × 109/L), neutrophils 8.23 × 109/L (reference range: 1.50–7.70 × 109/L), corrected calcium 2.65 mmoL/L (reference range: 2.20–2.60 mmoL/L). Overall, she had poor-risk disease (score 5) per the International Metastatic RCC Database Consortium (IMDC). Ultrasound-guided renal biopsy was predominantly necrotic with focal International Society of Urological Pathology (ISUP) Grade 4 ccRCC. There was no evidence of sarcomatoid or rhabdoid morphology. After multidisciplinary team (MDT) review, and careful discussion with the patient via translator, as per international guidelines she was treated with lenvatinib and pembrolizumab. Between 3 and 6 months after treatment initiation, she developed IO-induced diabetes mellitus (treated with insulin), hypoadrenalism (treated with maintenance hydrocortisone), and Grade 2 diarrhoea, which responded to steroid treatment and pembrolizumab omission. Repeat CT scan at 6 months showed reduction in the size of the renal mass to 55 mm, along with resolution of the lung nodules and reduction in the extent of the tumour thrombus to the level of the hepatic veins (Mayo Level III). Pembrolizumab was restarted, but between 10 and 14 months after treatment initiation, she developed Grade 3 colitis, Grade 2 hepatitis (peak alanine transaminase 200 U/L [reference range 10–49 U/L]) and had a transient ischaemic attack. A CT scan at 15 months (Fig. 1B) showed minimal further reduction in the size of the renal mass (51 mm), and further shrinkage of the caval tumour thrombus (Mayo Level II), with a new 11-mm aortocaval node and a stable 15-mm right adrenal nodule. Given the multiple toxicities on systemic treatment, CN was again discussed with the patient. MRI confirmed the tumour thrombus level extending to 1.2 cm below the right hepatic vein. Following MDT discussion, she proceeded to right open CN, caval thrombectomy, right adrenalectomy and lymphadenectomy 16 months after initiation of SACT. While the case required mobilisation of the liver, caval thrombectomy was successfully undertaken using infra-renal, left renal and infra-hepatic caval cross-clamping (Fig. 2). She made an uneventful recovery and was discharged home on postoperative Day 8. Pathology confirmed 95% of the right renal lesion showed fibrosis, haemorrhage, microcalcification and tumour necrosis (Fig. 3). Focally the mass showed ISUP Grade 3 ccRCC with no sarcomatoid or rhabdoid morphology. There were no viable tumour cells within the renal vein or IVC thrombus, and the excised caval wall showed no evidence of malignancy. One lymph node showed ccRCC. The adrenal lesion was described as showing focal scarring and haemosiderin laden macrophages suspicious for prior metastatic focus, with an additional adrenocortical adenoma. Final stage was ypT1a N1. The patient was well at 3 months after surgery, with CT scan showing no evidence of recurrent or metastatic disease. With the exception of select patients with IMDC favourable-risk and low-volume metastatic disease, SACT has become the principal initial treatment option for patients with metastatic RCC [2]. While there are a number of ongoing or upcoming studies investigating the benefits of deferred CN with IO-based therapy (ClinicalTrials.gov identifiers: NCT03494816, NCT03977571, NCT05753839, NCT05941169, NCT06279403), a subgroup of particular interest are patients with metastatic RCC who have concurrent VTT, particularly involving the IVC. There may be clear benefits from undertaking urgent extirpative surgery in this setting, as is considered optimal for patients with VTT as part of locally advanced disease. Alternatively, in patients with established metastatic disease, the delivery of modern systemic therapy with proven overall survival benefits could be significantly delayed by perioperative morbidity resulting from CN. There is no Level 1 evidence to support the use of neoadjuvant SACT in cases of locally advanced RCC with VTT, or to guide the timing of CN in cases with concurrent VTT, particularly where that involves the IVC. This case demonstrates that the combination of IO and tyrosine kinase inhibitor (TKI) therapy can effectively downstage patients with VTT and metastatic disease, which facilitated operative management. The patient's case was regularly discussed at MDT meetings; she was initially judged to have poor-risk, albeit relatively low-volume, metastatic disease, and not to be fit for bicaval surgery involving cardiopulmonary bypass. Surgery was specifically re-discussed with the patient at 6 months, and at various points thereafter; however, she opted to continue systemic therapy. While numerous studies have demonstrated SACT, followed by delayed CN if indicated, is the preferable treatment sequence for many patients with metastatic disease [2], most studies do not comment on how many of their cohort had VTT, or on the extent of that VTT. Neoadjuvant treatment of patients with Level III or IV VTT is particularly attractive as downstaging has the potential to allow less invasive surgical intervention, as described in the case above. Existing data in this space demonstrate what is achievable by TKI alone, with 73% patients across multiple studies having a measurable decrease in VTT length (mean reduction 1.4 cm), 30% having a reduction in VTT level, and possible impacts on survival with little evidence of toxicity [3]. In the NAXIVA trial (ClinicalTrials.gov identifier: NCT03494816), the median (range) change in VTT length at 9 weeks was 20 (34 to 68 mm), with 41% patients having less extensive surgery performed than was planned prior to axitinib [4]. Given that TKI-IO combined regimens have been shown to have a superior effect on primary tumour and metastases than TKI therapy alone [2], there is compelling rationale for future neoadjuvant trials using these agents. Of note, one case series reported longer operative time and higher blood loss following neoadjuvant treatment [5]. This marries with our experience where many patients who have received IO-combined therapy prior to CN have an intense inflammatory reaction or fibrotic tissue, particularly around hilar vessels or the IVC. The case described also highlights potential toxicity of TKI-IO therapy; patients with immune-related adverse events may be a group to consider CN at a lower threshold, particularly where this is correlated with response to treatment. We believe the question of whether and when to perform CN in cases with VTT remains an open one [1]. Does downstaging with SACT, particularly with IO-combined regimens, have a clinically-relevant impact on staging of the tumour (i.e., less invasive surgery becomes possible)? What sequence of SACT and CN has the greatest effect on survival? What is the optimal time to offer delayed CN? How can we best assess pathological response (PR) following neoadjuvant treatment? Indeed, do patients need nephrectomy if complete PR has been achieved? Conversely, does SACT result in harm caused by the delay to extirpative surgery? Additional challenges are posed by the histological reporting of deferred CN cases as there is no agreed framework or defined criteria for PR (replacement of the tumour with post-treatment fibrosis, necrosis, and inflammation can lead to discrepancy between macroscopic size and microscopic stage). None of these questions have as yet been adequately answered. While most patients will have objective responses to systemic therapy, some will have primary progression and there is currently no ability to predict who these patients might be [6]. Identifying these patients will require collaboration between interested centres, comparing those who undergo immediate nephrectomy to those who undergo delayed nephrectomy or SACT alone. In this space, trials of deferred CN after IO-IO or TKI-IO combinations should include subgroup analyses of patients with VTT. We present a case where SACT effectively down staged a patient's VTT and metastatic disease allowing single-cavity CN to be undertaken. Whether immediate surgery, or initial SACT with delayed CN, is the correct approach for cases of metastatic disease with VTT remains an open question, and there is an urgent need for trials using IO-combined therapy in this space. Grant D. Stewart has received educational grants from Pfizer, AstraZeneca and Intuitive Surgical; consultancy fees from Pfizer, MSD, EUSA Pharma and CMR Surgical; Travel expenses from MSD and Pfizer; Speaker fees from Pfizer; Clinical lead (urology) National Kidney Cancer Audit and Topic Advisor for the National Institute for Health and Care Excellence (NICE) kidney cancer guideline. Kate Fife has received advisory, consultancy or speaker fees from ESAI, Ipsen, Merck, Eusa, MSD, Regeneron and Sanofi, conference support from Ipsen, MSD and EUSA, and Institutional research funding from Merck, Exelixis. No other conflicts of interest to declare.

Purpose Conventional renal mass biopsy approaches are inaccurate, potentially leading to undergrading. This study explored using hyperpolarised [1-13C]pyruvate MRI (HP 13C-MRI) to identify the most aggressive areas within the tumour of patients with clear cell renal cell carcinoma (ccRCC). Experimental design Six patients with ccRCC underwent presurgical HP 13C-MRI and conventional contrast-enhanced MRI. Three k-means clusters were computed by combining the k PL as a marker of metabolic activity, and the 13C-pyruvate signal-to-noise ratio (SNRPyr) as a perfusion surrogate. Combined clusters were compared to those derived from individual parameters and to those derived from percentage enhancement on nephrographic phase (%NG). The diagnostic performance of each cluster was assessed based on its ability to predict the highest histological tumour grade in postsurgical tissue samples. Tissues were further subject to MCT1 staining, RNA and whole-exome sequencing. Results Forty-four samples were collected in total. The clustering approach combining SNRPyr and k PL demonstrated the best performance for predicting highest tumour grade: specificity 85%; sensitivity 64%; positive predictive value 82%; and negative predictive value 68%. Epithelial MCT1 was identified as the major determinant of the HP 13C-MRI signal. The perfusion/metabolism mismatch cluster showed increased expression of metabolic genes and markers of aggressiveness, which may be due to genetic divergence. Conclusions This study demonstrates the potential of using HP 13C-MRI-derived metabolic clusters to identify intratumoral variations in tumour grade with high specificity. This work supports the use of metabolic imaging to guide biopsies to the most aggressive tumour regions, which could potentially reduce sampling error. ### Competing Interest Statement G.D.S. has received educational grants from Pfizer, AstraZeneca and Intuitive Surgical; consultancy fees from Pfizer, MSD, EUSA Pharma and CMR Surgical; Travel expenses from MSD and Pfizer; Speaker fees from Pfizer; Clinical lead (urology) National Kidney Cancer Audit and Topic Advisor for the NICE kidney cancer guideline. S.J.W. is a founder and director of Pinto Medical Consultancy. F.A.G. and J.D.K. have research grants from GlaxoSmithKline and AstraZeneca, and research support from GE Healthcare. F.A.G. has consulted for AstraZeneca on behalf of the University of Cambridge. ### Clinical Trial NCT03526809 ### Funding Statement This study was funded by Cancer Research UK (C19212/A27150, C19212/A29082, C19212/A16628). This research was supported by the Mark Foundation for Cancer Research (RG95043), Cancer Research UK Cambridge Centre (C9685/A25177 and CTRQQR-2021\100012), the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014 and NIHR203312). The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care. The authors have additional funding from the National Cancer Imaging Translational Accelerator (NCITA; C42780/A27066), the Cambridge Experimental Cancer Medicine Centre, the Mark Foundation Institute for Integrated Cancer Medicine (MFICM), and the Canadian Institute For Advanced Research (CIFAR). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: East of England - Cambridge East Research Ethics Committee gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data that support the findings of this study are available on reasonable request from the corresponding author, on condition that this will not be used to deanonymize the patients. The data are not publicly available due to them containing information that could compromise research participant privacy and consent.

Embryogenesis is a vulnerable time. Mutations in developmental cells can result in the wide dissemination of cells predisposed to disease within mature organs. We characterised the evolutionary history of four synchronous renal tumours from a 14-year-old girl using whole genome sequencing alongside single cell and bulk transcriptomic sequencing. Phylogenetic reconstruction timed the origin of all tumours to a multipotent embryonic cell committed to the right kidney, around 4 weeks post-conception. Biochemical and structural analysis of their shared MTOR mutation, absent from normal tissues, demonstrates enhanced protein flexibility, enabling a FAT domain hinge to dramatically increase activity of mTORC1 and mTORC2. Developmental mutations, not usually detected in traditional genetic screening, have vital clinical importance in guiding prognosis, targeted treatment, and family screening decisions for paediatric tumours.

IntroductionLocalised renal masses are an increasing burden on healthcare due to the rising number of cases. However, conventional imaging cannot reliably distinguish between benign and malignant renal masses, and renal mass biopsies are unable to characterise the entirety of the tumour due to sampling error, which may lead to delayed treatment or overtreatment. There is an unmet clinical need to develop novel imaging techniques to characterise renal masses more accurately. Renal tumours demonstrate characteristic metabolic reprogramming, and novel MRI methods have the potential to detect these metabolic perturbations, which may therefore aid accurate characterisation. Here, we present our study protocol for the investigation of the differential biology of benign and malignant renal masses using advanced MRI techniques (IBM-Renal).Methods and analysisIBM-Renal is a multiarm, single-centre, non-randomised, feasibility study with the aim to provide preliminary evidence for the potential role of the novel MRI techniques to phenotype localised renal lesions. 30 patients with localised renal masses will be recruited to three imaging arms, with 10 patients in each: (1) hyperpolarised [1-13C]-pyruvate MRI, (2) deuterium metabolic imaging (DMI) and (3) sodium MRI. The diagnosis will be made on samples acquired at biopsy or at surgery. The primary objective is the technical development of the novel MRI techniques, with the ultimate aim to understand whether these can identify differences between benign and malignant tumours, while the secondary objectives aim to assess how complementary the techniques are, and if they provide additional information. The exploratory objective is to link imaging findings with clinical data and molecular analyses for the biological validation of the novel MRI techniques.Ethics and disseminationThis study was ethically approved (UK REC HRA: 22/EE/0136; current protocol version 2.1 dated 11 August 2022). The plans for dissemination include presentations at conferences, publications in scientific journals, a doctoral thesis and patient and public involvement.Trial registration numberNCT06016075.

Abstract Background Accurate oncological diagnoses are essential to provide personalized and optimum care for patients. In children, renal tumors account for approximately one in 20 malignancies. Diagnostic workup in pediatric renal tumors is current focused on epidemiology, radiology, and histology, with a very limited role for molecular analysis outside of suspected cancer predisposition. Methods Retrospective clinical record review of seven pediatric and young adult renal tumor patients presenting to a single principal treatment center in the East of England, UK. Data collection was focused on their clinical presentation, radiology, histopathology, and molecular investigations including whole genome sequencing (WGS), treatment and outcomes. We analyzed the impact of molecular analysis on the care of these patients. Results Four patients presented with histologically difficult to classify renal tumors. Subsequent nephrectomy provided no additional information over biopsy in the two cases where a biopsy was performed first. Two young patients with histological concerns over renal cell carcinoma (RCC) had somatic mutations reported in Wilms tumor (WT) and responded well to WT treatment. One patient had histologically mixed features of papillary RCC and epithelial WT. WGS revealed a copy number profile consistent with papillary RCC as well as somatic WT changes and responded well and durably to chemo/radiotherapy, not expected for RCC alone. A young adult with an atypical RCC was shown to harbor a ERC1::CCNY fusion likely defining a novel entity. A further three cases, who did not have classical features of WT/cancer predisposition, were found to harbor mosaic/germline predisposition variants; allowing for appropriate treatment as per syndromic WT protocols. Discussion Diagnostic uncertainty is a challenge for oncologists, their patients, and families. Here we provide evidence that agnostic molecular analysis, including whole genomic sequencing (WGS), can be helpful in delineating such cases. In two children the molecular analysis helped to define the diagnosis as WT despite histological concerns for RCC. A novel tumor with mixed WT/RCC phenotype and genotype was responsive to chemo/radiotherapy. Molecular analysis thus improves the accuracy of diagnosis and helps to define novel entities. WT is well recognized to occur in the context of cancer predisposition syndromes. At present, genetics referrals and investigations are limited to those with suggestive family history or clinical features. Routinely undertaking molecular analysis will increase the rate of detection of underlying predisposition. We propose rapid turn-around molecular analysis for those undergoing pre-operative chemotherapy (as practiced in Europe) to identify patients and to plan for nephron-sparing surgery to reduce the risk of long-term renal replacement therapy. The molecular multidisciplinary team is crucial for the interpretation of routinely performed agnostic molecular analysis in children and young people with renal tumors given the evolving complexity of such cases. Citation Format: Sarah M. Leiter, Aisosa Guobadia, Ben Fleming, Thankamma Ajithkumar, James Armitage, Ruth Armstrong, GA Amos Burke, Charlotte Burns, Tanzina Chowdhury, Nicholas Coleman, Helen Hatcher, Gail Horan, Lisa Howell, Anna-May Long, Sarah McDonald, Thomas J. Mitchell, James C. Nicholson, Thomas Roberts, Grant D. Stewart, John A. Tadross, Patrick Tarpey, Claire Trayers, Jamie Trotman, James Watkins, Anne Y. Warren, Godran Vujanic, C. Elizabeth Hook, Sam Behjati, Matthew J. Murray. Molecular analysis improves the diagnosis of young people with renal tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A011.

Abstract Background Clinical imaging tools to probe aggressiveness of renal masses are lacking, and T2-weighted imaging as an integral part of magnetic resonance imaging protocol only provides qualitative information. We developed high-resolution and accelerated T2 mapping methods based on echo merging and using k-t undersampling and reduced flip angles (TEMPURA) and tested their potential to quantify differences between renal tumour subtypes and grades. Methods Twenty-four patients with treatment-naïve renal tumours were imaged: seven renal oncocytomas (RO); one eosinophilic/oncocytic renal cell carcinoma; two chromophobe RCCs (chRCC); three papillary RCCs (pRCC); and twelve clear cell RCCs (ccRCC). Median, kurtosis, and skewness of T2 were quantified in tumours and in the normal-adjacent kidney cortex and were compared across renal tumour subtypes and between ccRCC grades. Results High-resolution TEMPURA depicted the tumour structure at improved resolution compared to conventional T2-weighted imaging. The lowest median T2 values were present in pRCC (high-resolution, 51 ms; accelerated, 45 ms), which was significantly lower than RO (high-resolution; accelerated, p = 0.012) and ccRCC (high-resolution, p = 0.019; accelerated, p = 0.008). ROs showed the lowest kurtosis (high-resolution, 3.4; accelerated, 4.0), suggestive of low intratumoural heterogeneity. Lower T2 values were observed in higher compared to lower grade ccRCCs (grades 2, 3 and 4 on high-resolution, 209 ms, 151 ms, and 106 ms; on accelerated, 172 ms, 160 ms, and 102 ms, respectively), with accelerated TEMPURA showing statistical significance in comparison (p = 0.037). Conclusions Both high-resolution and accelerated TEMPURA showed marked potential to quantify differences across renal tumour subtypes and between ccRCC grades. Trial registration ClinicalTrials.gov, NCT03741426 . Registered on 13 November 2018. Relevance statement The newly developed T2 mapping methods have improved resolution, shorter acquisition times, and promising quantifiable readouts to characterise incidental renal masses. Graphical Abstract

Background: Accurate non-invasive subtyping of localised kidney tumours is an unmet clinical question in uro-oncology. Differentiation of benign renal oncocytomas (RO) from malignant chromophobe renal cell carcinomas (chRCC) is not possible using conventional imaging. Despite the importance of renal function for sodium regulation, little is known about sodium handling in kidney tumours. Purpose: Here we used non-invasive sodium MRI (23Na-MRI) to quantify sodium concentration and relaxation dynamics across a range of different kidney tumour subtypes and have correlated these findings with imaging surrogates for perfusion, hypoxia, and cellularity. Materials and Methods: Between January and April 2023, patients with localised renal masses were prospectively recruited and underwent 23Na and proton (1H) MRI at 3T to acquire 3D maps of B1, total sodium concentration (TSC), proton and sodium relaxation rates (R2*), and diffusion weighted imaging (DWI). Statistical analysis included comparison and correlation of quantified imaging parameters across kidney tumour subtypes. Results: Ten patients were included in the final analysis (mean age ± S.D. = 64 ± 8 years; 7:3 male:female ratio) encompassing seven ROs, two chRCCs, two clear cell RCCs (ccRCC), and one papillary RCC (pRCC). The TSC was significantly higher in the ROs compared to the chRCCs: 162 ± 58 mM vs. 71 ± 2 mM (P < 0.05). The mean TSC in ccRCC was 135 ± 59 mM, and 81 mM in pRCC. The 23Na-derived and 1H-derived R2* values showed a weak correlation (Spearman r = 0.17; P = 0.50). There was a significant inverse correlation between TSC and 1H-R2* (Spearman r = -0.39, P < 0.05), but TSC was independent of the DWI-derived imaging parameters. Conclusion: 23Na-MRI detected markedly different sodium concentrations within benign ROs and malignant chRCCs. In addition, the sodium signal inversely correlated with 1H-R2* as a surrogate for hypoxia. Therefore we have shown the feasibility and potential of 23Na-MRI for future research in renal tumours.### Competing Interest StatementGDS has received educational grants from Pfizer, AstraZeneca and Intuitive Surgical; consultancy fees from Pfizer, MSD, EUSA Pharma and CMR Surgical; Travel expenses from MSD and Pfizer; Speaker fees from Pfizer; Clinical lead (urology) National Kidney Cancer Audit and Topic Advisor for the NICE kidney cancer guideline. S.J.W. is a founder and director of Pinto Medical Consultancy. F.A.G. has research grants from GlaxoSmithKline and AstraZeneca, research support from GE Healthcare, and has consulted for AstraZeneca on behalf of the University of Cambridge.### Clinical TrialNCT06016075### Funding StatementThis study was funded by Cancer Research UK (C19212/A27150, C19212/A29082, C19212/A16628, EDDPMA-May22\100068),by the Mark Foundation for Cancer Research (RG95043), Cancer Research UK Cambridge Centre (C9685/A25177 and CTRQQR-2021\100012), the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014 and NIHR203312). The authors have additional funding from the National Cancer Imaging Translational Accelerator (NCITA; C42780/A27066), the Cambridge Experimental Cancer Medicine Centre, the Cancer Research UK Cambridge Centre, the Mark Foundation Institute for Integrated Cancer Medicine (MFICM), and the Canadian Institute For Advanced Research (CIFAR).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:East of England; Cambridge East Research Ethics Committee (REC), Health Research Authority (HRA), receiving the REC reference: 22/EE/0136I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Purpose: To establish and optimize abdominal deuterium MRSI in conjunction with orally administered 2H-labelled molecules. Methods: A flexible transmit-receive surface coil was used to image naturally abundant deuterium signal in phantoms and healthy volunteers and after orally administered 2H2O in a patient with a benign renal tumor (oncocytoma). Results: Water and lipid peaks were fitted with high confidence from both unlocalized spectra and from voxels within the liver, kidney, and spleen on spectroscopic imaging. Artifacts were minimal despite the high 2H2O concentration in the stomach immediately after ingestion, which can be problematic with the use of a volume coil. Conclusion: We have shown the feasibility of abdominal deuterium MRSI at 3 T using a flexible surface coil. Water measurements were obtained in healthy volunteers and images were acquired in a patient with a renal tumor after drinking 2H2O. The limited depth penetration of the surface coil may have advantages in characterizing early uptake of orally administered agents in abdominal organs despite the high concentrations in the stomach which can pose challenges with other coil combinations. ### Competing Interest Statement Rolf Schulte is an employee of GE Healthcare ### Funding Statement This research was supported by Cancer Research UK (EDDPMA-May22/100068, C19212/A27150), the Cancer Research UK Cambridge Centre, the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014), and the Marmaduke-Sheild Fund. MM acknowledges additional support from the Cambridge Experimental Cancer Medicine Centre, PW from the Gates Cambridge Trust (#OPP1144), JB from the National Cancer Imaging Translational Accelerator (NCITA). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Yorkshire & The Humber - Leeds East Research Ethics Committee number: 23/YH/0127 (healthy volunteers) East of England - Cambridge East REC: 22/EE/0136 (patient with oncocytoma) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma 1 . Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate 2 . However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING)–TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Most epithelial cells with cancer driver mutations fail to form tumors. This has been particularly well established by genetic analysis of the renal epithelium, where cells with tumor-initiating mutations in the von Hippel-Lindau tumor suppressor (VHL) only rarely develop into clear cell renal carcinomas (ccRCC). The progression of VHLmutant cells to malignancy can take decades, offering a wide window of opportunity for preventative intervention. However, the cellular programs that facilitate the malignant transformation of VHL null ccRCC precursor clones remain elusive. Using a genetically engineered mouse model that allows conditional inactivation of Vhl and Polybromo 1 (Pbrm1), the second most commonly mutated ccRCC gene after VHL, in the renal epithelium of adult mice, we have analyzed the phenotypic evolution of the earliest stages of renal carcinoma formation over months at single cell resolution. Transcriptomic analysis by single cell RNA-sequencing (scRNA-seq) revealed that the acute activation of the hypoxia signaling response, a predicted consequence of genetic Vhl inactivation, is followed by distinct transcriptomic state transitions that precede the emergence of early carcinoma cells. These transitions are associated with constitutive activation of transcriptional programs that normally mediate acute and transient responses to renal injury, and are progressively activated at a low level in aging normal renal epithelium. Analysis of DNA accessibility by the single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) identified widespread opening of chromatin regions in association with progression towards malignancy, revealing a chromatin footprint of tissue repair pathway activation. Analysis of human ccRCC samples, including early-stage cases from VHL mutation carriers, validated our observations at the transcriptomic and protein level. Furthermore, genetic perturbation experiments in transplantable ccRCC models demonstrated the functional role of the renal injury pathway in tumor re-initiation. These results are compatible with a model whereby activation of the renal epithelial injury pathway facilitates the early progression of mutant ccRCC precursor clones towards malignancy. That the same repair pathway is increasingly activated over time in normal renal epithelium points at a link between ageing and ccRCC development. An understanding of the causes of ageing-associated renal damage could provide opportunities for early intervention in ccRCC, especially in high-risk individuals. Citation Format: Shoko Hirosue, Jianfeng Ge, Leticia Castillon, Paulo Rodrigues, Dóra Bihary, Alyson Speed, Ludovic Wesolowski, Veronica Caraffini, Anne Y. Warren, Grant D. Stewart, Sarah J. Welsh, Thomas J. Mitchell, Athena Matakidou, Fatemeh S. Seyednasrollah, Shamith A. Samarajiwa, Sakari Vanharanta. Injury-associated transcriptional state transitions in early renal carcinogenesis [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 PR002.