The thymus is essential for establishing T cell diversity early in life, but undergoes profound involution with age and has therefore traditionally been regarded as largely nonfunctional in adults1,2. Here we propose that preserving thymic functionality is integral to adult health and longevity. We developed a deep learning framework to quantify thymic health from routine radiographic images and evaluated its association with longevity and risk of major age-associated diseases in two large prospective cohorts of asymptomatic adults: the National Lung Screening Trial (n = 25,031) and the Framingham Heart Study (n = 2,581). In both cohorts, thymic health varied markedly across the population. In the National Lung Screening Trial, higher thymic health was consistently associated with lower all-cause mortality, reduced lung cancer incidence and lower cardiovascular mortality over 12 years of follow-up after adjustment for age, sex, smoking and comorbidities. In the independent Framingham Heart Study cohort, higher thymic health was significantly associated with reduced cardiovascular mortality, independent of age, sex and smoking. Thymic health was further linked to systemic inflammation and metabolic dysregulation, and associated with modifiable lifestyle factors including smoking, obesity and physical activity. Together, these findings reposition the thymus as a central regulator of immune-mediated ageing and disease susceptibility in adulthood, highlighting its potential as a target for preventive and regenerative strategies to promote healthy ageing and longevity.

Although immunotherapy has revolutionized cancer treatment, many patients still experience limited benefit, highlighting the urgent need for improved biomarkers1. Although immunotherapy is founded on unleashing T cells2, most existing biomarkers remain tumour-centric and mainly overlook host immune competence. The thymus is a key immune organ that is crucial for T cell maturation, and we hypothesized that thymic functionality is associated with immunotherapy outcomes3. Here we show that thymic health, a radiographic measure of thymic functionality, is strongly associated with immunotherapy outcomes across several cancer types. Using a deep-learning framework applied to routine computed tomography images, we quantified thymic health in a pan-cancer cohort of 3,476 patients receiving immune checkpoint inhibitors. In patients with non-small cell lung cancer, higher thymic health was associated with reduced risks of progression and all-cause mortality. These associations remained significant across clinically relevant levels of programmed death ligand 1 (PD-L1) and tumour mutation burden. In the prospective TRACERx lung cancer study, thymic health was positively associated with T cell receptor diversity and T cell receptor excision circles, and correlated with immune-system signalling pathways, supporting radiographic thymic health as a proxy for thymic activity and adaptive immune competence. Analysis across patients with melanoma, breast cancer or renal cancer demonstrated pan-cancer relevance. Together, these findings identify thymic health as a previously unrecognized, tumour-agnostic determinant of immunotherapy efficacy, with potential implications for patient stratification, treatment timing and the development of immune-rejuvenating strategies in precision immuno-oncology.

Abstract Background The thymus plays a crucial role in early life, serving as the primary source of T-cell development, a vital component of adaptive immunity. Despite its central role in immune biology, the thymus is not currently considered an organ-at-risk in Radiotherapy (RT). RT, a cornerstone of treatment for locally advanced non-small cell lung cancer (NSCLC), has effects extending beyond local tumor control. In particular, it can trigger immunogenic cell death, releasing tumor antigens that prime systemic antitumor immunity. These immune-mediated effects are particularly important when RT is combined with immunotherapy, where consolidation immunotherapy has been shown to improve survival after chemoradiation. Methods In this multicohort study, we assessed whether thymic irradiation was associated with distant metastases and all-cause mortality across 882 locally-advanced NSCLC patients treated with RT (RTOG-0617 n=460 | HARVARD-CRT n=422). For each patient, we measured the mean thymic radiation dose (MTD). Thymic health was determined based on thymic radiographic representation on CT scans, automatically inferred using a self-supervised deep learning model. Associations with clinical outcomes were assessed using multivariable Cox models, adjusting for key clinical factors. Sensitivity analyses evaluated the incremental contribution of MTD. To demonstrate the clinical feasibility of thymic sparing with modern RT techniques and AI-based volume delineation, we replanned the RT treatment plan in a representative case with high cumulative thymic dose exposure. Results In RTOG patients with preserved thymic health, higher thymic radiation dose was progressively associated with worse clinical outcomes. An MTD of 35Gy was identified as the lowest dose linked to a significant deterioration in outcomes. This threshold was validated in both cohorts, where exceeding the 35Gy MTD in patients with preserved thymic function was associated with increased risk of distant metastases (RTOG-0617: adjusted hazard-ratio [aHR]=1.32; p=0.002 | HARVARD-CRT: aHR=2.15; p=0.018) and worse overall survival (RTOG-0617: aHR=1.20; p=0.002 | HARVARD-CRT: aHR=2.02; p=0.014). No significant associations emerged in patients with low thymic health prior to radiation therapy. One-year follow-up imaging demonstrated dose-dependent declines in AI-quantified thymic health, supporting a mechanistic link between thymic irradiation and loss of immune competence. In a feasibility test case, thymic dose was successfully reduced below 35 Gy without compromising tumor coverage or cardiopulmonary constraints. Conclusions Thymic radiation exposure was independently associated with higher risks of metastasis and death in NSCLC patients with preserved thymic function. These findings suggest that the thymus should be recognized as an organ-at-risk in radiotherapy and that thymus-sparing strategies may be crucial to preserve immune health and improve patient outcomes. Citation Format: Vasco Prudente, Simon Bernatz, Suraj Pai, Katelyn M. Atkins, Keno Bressem, Christian V. Guthier, Leonard Nürnberg, Christopher E. Kehayias, Christopher Abbosh, Charles Swanton, Mariam Jamal-Hanjani, Nicolai Juul Birkbak, Raymond H. Mak, Hugo Aerts. Thymic radiation and oncologic outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2327.

Abstract Background: Immune checkpoint inhibitors (ICIs) depend on effective T-cell priming and renewal, yet current biomarkers focus primarily on tumor-intrinsic features (PD-L1, TMB). Because the thymus maintains naïve T-cell output and repertoire diversity, we hypothesized that an individual's thymic health represents a host-intrinsic determinant of ICI benefit. Methods: We developed a self-supervised deep learning model to quantify thymic function, here termed thymic health, from routine CT scans. Thymic health was independently evaluated in 3,476 patients treated with ICI for tumors of various entities. Associations with PFS and OS were assessed using multivariable Cox models adjusting for key epidemiological and clinical factors. Sensitivity analyses evaluated the incremental contribution of thymic health. Biological validation was performed in treatment-naïve NSCLC patients from TRACERx (n=464) using T-cell receptor (TCR) sequencing and plasma proteomics. Results: In patients with NSCLC (n=1,218), high thymic health was associated with significantly reduced risk of progression (HR 0.65; 95% CI, 0.54-0.77) and death (HR 0.56; 95% CI, 0.46-0.68); preserved after full multivariate adjustments for sex, age, PD-L1, TMB, ECOG, histology, treatment line and stratification by treatment type (P<0.001). Adding thymic health improved model fit beyond demographic, clinical, and tumor-intrinsic variables (likelihood ratio P<0.001). Importantly, including thymic health did not attenuate PD-L1 or TMB effects, indicating complementary, non-redundant prognostic information. Across PD-L1 and TMB strata, higher thymic health was consistently associated with improved PFS and OS (P<0.03). In TRACERx, thymic health was positively associated with T cell receptor excision circles, i.e., T cell output, and correlated with greater peripheral and intratumoral TCR diversity and with proteomic signatures of adaptive immune activation, biologically supporting its role as a radiographic proxy for immune competence. Pan-cancer analyses across 2,258 ICI-treated patients (melanoma, renal, breast, and others) showed consistent positive associations between thymic health and improved outcomes. Conclusions: Thymic health was consistently associated with improved immunotherapy outcomes across cancer types and added independent information beyond PD-L1, TMB, and standard clinical variables. These findings support thymic health as a possible non-invasive, host-intrinsic biomarker with potential to refine patient stratification and advance precision immuno-oncology. Clinical trial identification: ClinicalTrials.gov: TRACERx, NCT01888601 Citation Format: Simon Bernatz, Vasco Prudente, Suraj Pai, Asbjørn Kjær, Alessandro Di Federico, Andrew Rowan, Selvaraju Veeriah, Lars Dyrskjøt, Leonard Nürnberg, João Victor M. Alessi, Patrick Ott, Elad Sharon, Allan Hackshaw, Nicholas McGranahan, Christopher Abbosh, Raymond H. Mak, Danielle S. Bitterman, Mark M. Awad, Biagio Ricciuti, Charles Swanton, Mariam Jamal-Hanjani, Nicolai Juul Birkbak, Hugo JWL Aerts. Radiographic thymic health as host-intrinsic determinant of immunotherapy outcomes across cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1185.

Background: Sublobar resection is increasingly recognized as an effective treatment for early-stage NSCLC. However, no studies to date have investigated the potential role of preoperative ctDNA detection in guiding surgical decisions, such as opting for sublobar resection, in stage I NSCLC. Methods: Patients with solid-dominant (CTR>0.5), clinical stage I NSCLC were prospectively recruited between March 2014 and December 2020. Pre-surgical plasma samples were analyzed using a tumor-na & iuml;ve, methylationbased cell-free DNA assay. The impact of sublobar resection versus lobectomy on recurrence-free survival (RFS) was assessed according to pre-surgical ctDNA status. Associations between pre-surgical ctDNA detection and clinicopathologic factors were also investigated. Results: The analysis included 544 patients (178 women [33 %]; median age 66 [IQR, 60-71] years). Pre-surgical ctDNA was detected in 188 (35 %) patients. In patients without presurgical ctDNA, sublobar resection did not significantly increase the risk of relapse (adjusted HR, 1.01, p = 0.98). However, among ctDNA-positive patients, sublobar resection was associated with an increased risk of relapse (adjusted HR, 2.25; 95 % CI, 1.12-4.54; p = 0.024). Patients with presurgical ctDNA had higher rates of nodal upstaging (OR, 3.58; p < 0.001) and exhibited higher pathologic grade (p = 0.021), perineural invasion (p < 0.001), and lymphovascular invasion (p < 0.001). Conclusions: Pre-surgical tumor-na & iuml;ve ctDNA analysis holds promise in identifying patients with aggressive tumors that may not be sufficiently managed with sublobar resection. This approach can help personalize treatment strategies, potentially improving outcomes for patients with early-stage NSCLC.

Neoantigen vaccines are under investigation for various cancers, including epidermal growth factor receptor (EGFR)-driven lung cancers1,2. We tracked the phylogenetic history of an EGFR mutant lung cancer treated with erlotinib, osimertinib, radiotherapy and a personalized neopeptide vaccine (NPV) targeting ten somatic mutations, including EGFR exon 19 deletion (ex19del). The ex19del mutation was clonal, but is likely to have appeared after a whole-genome doubling (WGD) event. Following osimertinib and NPV treatment, loss of the ex19del mutation was identified in a progressing small-cell-transformed liver metastasis. Circulating tumour DNA analyses tracking 467 somatic variants revealed the presence of this EGFR wild-type clone before vaccination and its expansion during osimertinib/NPV therapy. Despite systemic T cell reactivity to the vaccine-targeted ex19del neoantigen, the NPV failed to halt disease progression. The liver metastasis lost vaccine-targeted neoantigens through chromosomal instability and exhibited a hostile microenvironment, characterized by limited immune infiltration, low CXCL9 and elevated M2 macrophage levels. Neoantigens arising post-WGD were more likely to be absent in the progressing liver metastasis than those occurring pre-WGD, suggesting that prioritizing pre-WGD neoantigens may improve vaccine design. Data from the TRACERx 421 cohort3 provide evidence that pre-WGD mutations better represent clonal variants, and owing to their presence at multiple copy numbers, are less likely to be lost in metastatic transition. These data highlight the power of phylogenetic disease tracking and functional T cell profiling to understand mechanisms of immune escape during combination therapies.

The implementation of lung cancer screening programs across the world has drawn considerable attention to improving early-stage lung cancer detection and prognostication. Several blood-based assays detecting circulating tumor DNA (ctDNA) recently emerged as noninvasive methods to detect malignancies. However, their limited sensitivity and predictive value remain a hurdle to their clinical use. We aimed to evaluate the association between clinicopathological parameters and presurgical ctDNA detection in clinical stage I non-small cell lung cancer patients to further understand ctDNA shedding biology. The cohort included 180 adenocarcinomas (LUAD) and 80 squamous cell carcinomas (LUSC) stage I patients who underwent lung cancer resection. Patients' clinical and pathological features were collected. A multicancer early-detection test (GRAIL LLC) was used to detect ctDNA using targeted methylation patterns. The association between the cell-free DNA tumor methylated fraction (TMeF) and the clinicopathological predictors was evaluated using univariate and multivariate modeling. LUSC was associated with a higher TMeF than LUAD. Pathological stage, tumor grade, and tumor volume were key determinants of ctDNA detection in both LUSC and LUAD. In LUAD, ctDNA detection also correlated with histologic pattern composition, necrosis, acute inflammation, and, to a lesser degree, spread through alveolar spaces and lymphovascular invasion. Based on our results, we propose classification methods for both LUAD (using histologic pattern composition) and LUSC (using tumor grade and pathological stage) to identify patients likely to have high ctDNA levels. These results confirm previous findings and suggest that previously unidentified factors, including histologic pattern composition and acute inflammation, influence ctDNA levels. These results will help in understanding the ctDNA shedding process and may allow identification of patients eligible for ctDNA detection-based follow-up.

Biomarkers accurately informing prognostic assessment and therapeutic strategy are critical for improving patient outcome in oncology. Here, we apply a whole-genome, tumor-informed circulating tumor DNA (ctDNA) detection approach to address this challenge, leveraging 1,800 variants across 2,994 plasma samples from 431 patients with non-small cell lung cancer (NSCLC) from the TRACERx study. We show that ultrasensitive ctDNA detection below 80 parts per million both pre- and postoperatively is highly prognostic, and combinatorial analysis of the pre- and postoperative ctDNA status identifies an intermediate risk group, improving disease stratification. ctDNA kinetics demonstrate clinical utility during adjuvant therapy, where patients that "clear" ctDNA during adjuvant therapy experience improved outcomes. Moreover, characterization of patterns in postoperative ctDNA kinetics reveals insights into the timing, risk, and anatomical pattern of relapses. By incorporating longitudinal ultrasensitive ctDNA detection, we propose a refined schema for guiding the stratification and treatment recommendations in early stage NSCLC.

T cells are crucial effector cells in the endogenous defense against cancer, yet the clinical impact of their quantity, diversity, and dynamics remains underexplored. Here, we investigate the clinical relevance of the T cell receptor (TCR) repertoire in patients with bladder cancer. In advanced-stage disease, low pre-treatment peripheral TCR diversity is associated with worse overall survival (p = 0.024), particularly when coupled with low circulating T cell fractions (p = 0.00049). These low-diversity repertoires are dominated by hyper-expanded clones that persist throughout treatment. Further longitudinal analysis reveals reductions in TCR diversity after treatment, indicating adverse effects on the immune system. In early-stage disease, immunotherapy increases TCR diversity in patients with good outcomes. Furthermore, single-cell sequencing identifies most hyper-expanded clones as cytotoxic T cells, while non-expanded clones are predominantly naive T cells. Overall, this highlights TCR diversity as a promising biomarker, offering opportunities for tailored oncological treatments to enhance clinical outcomes.

Abstract Current practice for evaluating prognosis of resectable lung adenocarcinoma (LUAD) patients relies on the Tumor, Node, Metastasis staging system. Despite low relapse risk, post-recurrence survival in a stage I population is poor with a median post relapse survival of around 25 months. There is a need for a biomarker that can enrich for stage I patients with relapse risk to identify a population likely to benefit from adjuvant therapy. In this study, we aim to identify a predictive biomarker of relapse in early-stage lung cancer following surgical resection using AI-based computational pathology. The retrospective patient cohort analyzed in this study consisted of a total of 166 patients that underwent surgical resection for a clinical stage I LUAD. Out of these, 54 patients experienced disease recurrence within 5 years, and 112 patients had a confirmed disease-free period of at least 5 years post-surgery. For each patient, at least one digitized slide (average = 2.46) stained with haematoxylin and eosin (H&E) was available for analysis. The project was accepted by the IUCPQ ethics committee (2022-3751, 22138). Manual annotations delineating the tumor core (TC) were drawn by pathologists. We then applied several proprietary image analysis models capable of distinguishing epithelium, stroma, and necrosis within the tumor tissue as well as detecting tumor infiltrating lymphocytes (TILs) and cell nuclei. In this project, we additionally introduce a novel approach rooted in mathematical graph theory to encoding topological aspects of the tissue morphology. A cell-graph constructed from the positions of cell nuclei was used to calculate cell-level graph-theoretic measures which were then aggregated to the slide-level by computing the median. In total, 23 data readouts were obtained of which 14 were based on the standard image analysis pipeline alone, and a further 9 originated from our newly developed graph-based approach. We identified promising biomarkers using the Wilcoxon rank-sum test and the area under the ROC curve (AUROC); robustness of this evaluation was investigating using resampling methods, using 100 bootstrap samples and 100 repeats of 3-fold cross-validation, respectively. This allowed us to evaluate the prognostic value of the biomarkers with respect to 5-year relapse or death. We were able to identify the best prognostic biomarker for relapse to be the median weighted clustering coefficient (CC) across all cells in the TC area, which is derived from the graph-based analyses. The median CC was able to achieve a whole-cohort AUROC of 0.695 (average cross-validated AUROC = 0.675, 95% interval 61.7%-70.4%) as well as a relapse vs relapse-free Wilcoxon test p-value of 4.65x10-5 (bootstrapped average = 0.0002). In addition, we were also able to demonstrate that the biomarker shows some relation to the architecture patterns. Citation Format: Florian J. Song, Alma Andoni, Manal Kordahi, Sara Batelli, Armin Meier, Markus Schick, Emilie Mahieu, Günter Schmidt, Claire E. Myers, Michael Abadier, Abjihit Dasgupta, Christopher Abbosh, Darren Hodgson, Michèle Orain, Fabien C. Lamaze, Yohan Bossé, Philippe Joubert. Identifying poor prognosis stage I lung adenocarcinoma patients through novel morphological biomarker based on computational pathology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6176.

Background: Circulating tumor DNA (ctDNA) positivity at diagnosis, which is associated with worse outcomes in multiple solid tumors including stage I-III non-small cell lung cancer (NSCLC), may have utility to guide (neo) adjuvant therapy. Methods: In this retrospective study, 260 patients with clinical stage I NSCLC (180 adenocarcinoma, 80 squamous cell carcinoma) were allocated (2:1) to high- and low-risk groups based on relapse versus disease-free status <= 5 years post-surgery. We evaluated the association of preoperative ctDNA detection by a plasma-only targeted methylation-based multi-cancer early detection (MCED) test with NSCLC relapse <= 5 years post-surgery in the overall population, followed by histology-specific subgroup analyses. Results: Across clinical stage I patients, preoperative ctDNA detection did not associate with relapse within 5 years post-surgery. Sub-analyses confined to lung adenocarcinoma suggested a histology-specific association between ctDNA detection and outcome. In this group, ctDNA positivity tended to associate with relapse within 2 years, suggesting prognostic implications of MCED test positivity may be histology- and time-dependent in stage I NSCLC. Preoperative ctDNA detection was associated with upstaging of clinical stage I to pathological stage II-III NSCLC. Conclusions: Our findings suggest preoperative ctDNA detection in patients with resectable clinical stage I NSCLC using MCED, a pan-cancer screening test developed for use in an asymptomatic population, has no detectable prognostic value for relapse <= 5 years post-surgery. MCED detection may be associated with early adenocarcinoma relapse and increased pathological upstaging rates in stage I NSCLC. However, given the exploratory nature of these findings, independent validation is required.

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes.

Abstract T cells represent essential effector cells in the intrinsic antitumor immune response. Cancer-specific T cells can recognize cancer neoantigens through their T cell receptors (TCRs). The expansion of these clones is believed to be an early response to malignancy. We hypothesized that the T cell landscape offers insights into immune competency. We aimed to characterize the TCR repertoire in patients with bladder cancer and explore correlations with disease outcomes. We analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer (MIBC) treated with chemotherapy followed by radical cystectomy. Peripheral and tumor TCR repertoires were produced using ultradeep amplicon-based sequencing of the TCR beta chain. T cell fractions were inferred from whole exome sequencing data of blood DNA using TcellExTRECT. Latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. The T cell subtype compositions in tumor and blood were investigated in four patients with bladder cancer using the Chromium Single Cell Immune profiling kit from 10X Genomics. Low pre-treatment peripheral TCR diversity was associated with worse overall survival (HR = 2.3, p = 0.024, n = 119) in patients with MIBC, particularly when combined with a low fraction of circulating T cells (HR= 4.7, p = 0.00049, n = 67). We validated these findings in two independent cohorts of patients with MIBC (p = 0.01, n = 107; p = 0.042, n = 79). The low-diversity TCR repertoires were characterized by large expanded T cell clones that persisted over time. Longitudinal analysis indicated a potential adverse impact of treatment, evidenced by a reduction in TCR diversity and circulating T cells over time in patients with initially high-diversity repertoires. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections and cytomegalovirus DNA detection was strongly associated with low TCR diversity (p = 5.3 × 10−5). We observed a notable disparity between tumor and peripheral blood TCR repertoires. Single-cell sequencing revealed that regulatory T cells were prevalent in the tumor, whereas a combination of cytotoxic and naïve T cells dominated the blood. More explicitly, expanded clones in the blood were commonly annotated as terminally differentiated effector memory T cells expressing high levels of cytotoxic and exhausted genes. Our results suggest that high peripheral TCR diversity and high T cell fraction are markers of general immune competence, reflecting the ability to eradicate cancer and other diseases. In contrast, the expanded persistent clones predominating the low peripheral TCR diversity repertoires are likely exhausted and less likely to exhibit tumor specificity or effective disease-fighting capabilities. Our findings underline the crucial role of the immune system in determining disease outcomes and highlight the potential for improving immune health as a promising approach for future treatment and prevention. Citation Format: Nanna Kristjánsdóttir, Asbjørn Kjær, Iver Nordentoft, Randi I Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Trine Strandgaard, Deema Radif, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen B Jensen, Nicolai J. Birkbak, Lars Dyrskjøt. T cell receptor repertoire and diversity are prognostic markers in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR003.

Abstract Bladder cancer is the sixth most common cancer overall, and treatment often involves removal of the bladder, which comes with a significant reduction in quality of life. Within this cancer type, an easily applicable prognostic biomarker may have clinical utility, potentially stratifying patients into bladder-sparing treatments. Expansion of T cell populations targeting cancer-specific neoantigens is an early response to malignancy. To investigate if the T cell receptor (TCR) repertoire could serve as a biomarker for cancer progression, we analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer, treated with neoadjuvant chemotherapy before undergoing radical cystectomy. Peripheral and tumor TCR repertoires were produced using amplicon-based sequencing of the TCR beta chain. Additionally, T cell fraction was determined from whole exome sequencing data using TcellExTRECT, and latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. To explore the T cell repertoire subtypes, single-cell sequencing was performed on four fresh blood samples. In pre-treatment peripheral blood, low-diversity TCR repertoires were associated with worse survival (HR=2.3, p=0.024). Particularly poor outcome was observed in a subset of patients with both low TCR diversity and low fraction of circulating T cells (HR=4.7, p=5e-4). Low-diversity repertoires were dominated by expanded T cell clones. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections (p=2e-13), and cytomegalovirus DNA detection was strongly associated with decreased TCR diversity (p=5e-5). Additionally, tumor TCR sequencing revealed that expanded clones were rarely found in the tumor. Single-cell sequencing of peripheral blood found that most expanded clones consisted of cytotoxic T cells, while non-expanded clones mostly consisted of naive T cells. This indicates that low TCR diversity may represent a contraction of the naive T cell compartment, resulting in reduced immune competence. Finally, analysis of patient-matched longitudinal samples revealed that chemotherapy and cystectomy reduced TCR diversity in patients with high pre-treatment diversity (p=0.001), indicating that treatment negatively affected the immune system in the most immune-competent patients. Our results suggest that both low TCR diversity and low T cell fraction represent decreased immune competency. This could be associated with an elevated risk of progression due to a reduced ability to control cancer growth and limit systemic cancer cell dissemination. These findings underline that immune health is an important factor during malignant disease, even when patients are not treated with immunotherapies. Therefore, improving immune health could be a potential future goal for cancer treatment and prevention. Citation Format: Asbjørn Kjær, Nanna Kristjánsdóttir, Iver Nordentoft, Randi Istrup Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen Bjerggaard Jensen, Nicolai J Birkbak, Lars Dyrskjøt. Peripheral T cell receptor repertoire diversity is associated with outcome in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5216.

Circulating tumour DNA (ctDNA) detection of molecular residual disease (MRD) in solid tumours correlates strongly with patient outcomes and is being adopted as a new clinical standard. ctDNA levels are known to correlate with tumor volume, and although the absolute levels vary across indication and histology, its analysis is driving the adoption of MRD. MRD assays must detect tumor when imaging cannot and, as such, require very high sensitivity to detect the low levels of ctDNA found after curative intent therapy. The minimum threshold is 0.01% Tumour Fraction but current methods like Archer and Signatera are limited by detection sensitivity resulting in some patients receiving a false negative call thereby missing out on earlier therapeutic intervention. Multiple vendors are increasing the number of somatic variants tracked in tumour-informed and personalized NGS assays, from tens to thousands of variants. Most recently, assays using other biological features of ctDNA, e.g methylation or fragmentome, have been developed at the LOD required for clinical utility. These uniformed, or tumour-naive and non-personalised assays may be more easily, and therefore more rapidly, adopted in the clinic. However, this rapid development in MRD assay technology results in significant challenges in benchmarking these new technologies for use in clinical trials. This is further complicated by the fact that previous reference materials have focused on somatic variants, and do not retain all of the epigenomic features assessed by newer technologies. In this Comments and Controversy paper, we detail what is known and what remains to be determined for optimal reference materials of MRD methods and provide opinions generated during three-years of MRD technology benchmarking in AstraZeneca Translational Medicine to help guide the community conversation.