Metabolic processes are crucial in immune regulation, yet the impact of metabolic heterogeneity on immunological functions remains unclear. Integrating metabolomics into immunology allows the exploration of the interactions of multilayered features in the biological system and the molecular regulatory mechanism of these features. To elucidate such insight in lung squamous cell carcinoma (LUSC), we analyzed 106 LUSC tumor tissues. We performed high-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) to obtain spatial metabolic profiles, and immunohistochemistry to detect tumor-infiltrating T lymphocytes (TILs). Unsupervised k-means clustering and Simpson's diversity index were employed to assess metabolic heterogeneity, identifying five distinct metabolic tumor subpopulations. Our findings revealed that TILs are specifically associated with metabolite distributions, not randomly distributed. Integrating a validation cohort, we found that heterogeneity-correlated metabolites interact with CD8+ TIL-associated genes, affecting survival. High metabolic heterogeneity was linked to worse survival and lower TIL levels. Pathway enrichment analyses highlighted distinct metabolic pathways in each subpopulation and their potential responses to chemotherapy. This study uncovers the significant impact of metabolic heterogeneity on immune functions in LUSC, providing a foundation for tailoring therapeutic strategies.

RET fusions are significant oncogenic drivers, resulting from chromosomal rearrangements of the RET proto-oncogene with various partner genes. Understanding their structural characteristics is crucial for elucidating oncogenic potential and developing targeted therapies.

This study analyzed 21,023 tumor samples and 3716 peripheral blood samples from a Chinese pan-cancer cohort using DNA or RNA-targeted next-generation sequencing (NGS). 19,668 tissues underwent DNA-based fusion detection and 1355 NSCLC tissues underwent RNA-based fusion detection using a 733-gene panel. ctDNA-based targeted NGS was also performed on blood samples.

RET fusions were detected in 1.027 % of tissue samples, predominantly lung and thyroid cancers. Compared to Western populations (87 % in intron 11), Chinese patients show a shift toward the exon 10-11 region, with 30.79 % in intron 10. RET rearrangements were classified into four categories (Simple Reciprocal Inversion, Co-Fusion, Single Fusion-Common, Single Fusion-Rare) with unique mutational profiles and tumor mutational burden scores. RNA-based NGS revealed some DNA-detected rearrangements might not undergo transcription, while Co-Fusion indicated potential simultaneous transcription of multiple RET fusions. An NSCLC patient with KIF5B-RET and ATRNL1-RET co-fusions achieved 15-month progression-free survival on RET-targeted therapy.

This study underscores the importance of structural insights for developing targeted therapies against RET fusion-driven cancers and highlights the need for further investigation into complex RET fusion mechanisms to better understand RET-driven oncogenesis.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition associated with increased mortality among patients with cancer. CHIP mutations with high variant-allele frequencies can be detected in tumors, a phenomenon we term tumor-infiltrating clonal hematopoiesis (TI-CH). The frequency of TI-CH and its effect on tumor evolution are unclear.

We characterized CHIP and TI-CH in 421 patients with early-stage non-small-cell lung cancer (NSCLC) from the TRACERx study and in 49,351 patients from the MSK-IMPACT pan-cancer cohort. We studied the association of TI-CH with survival and disease recurrence and evaluated the functional effect of TET2-mutant CHIP on the biologic features of lung tumors.

Among patients with NSCLC, 42% of those with CHIP had TI-CH. TI-CH independently predicted an increased risk of death or recurrence, with an adjusted hazard ratio of 1.80 (95% confidence interval [CI], 1.23 to 2.63) as compared with the absence of CHIP and an adjusted hazard ratio of 1.62 (95% CI, 1.02 to 2.56) as compared with CHIP in the absence of TI-CH. Among patients with solid tumors, 26% of those with CHIP had TI-CH. TI-CH conferred a risk of death from any cause that was 1.17 times (95% CI, 1.06 to 1.29) as high as the risk with CHIP in the absence of TI-CH. TET2 mutations were the strongest genetic predictor of TI-CH; such mutations enhanced monocyte migration to lung tumor cells, fueled a myeloid-rich tumor microenvironment in mice, and resulted in the promotion of tumor organoid growth.

TI-CH increased the risk of disease recurrence or death among patients with NSCLC and the risk of death from any cause among patients with solid tumors. TI-CH remodeled the tumor immune microenvironment and accelerated tumor organoid growth, findings that support a role for an aging-related hematologic clonal proliferation in cancer evolution. (Funded by the Royal Society and others.).

The evolution of metastasis in humans is considerably less well understood than the biology of early carcinogenesis. For over a century, clinicians and scientists have been debating whether metastatic potential is the intrinsic property of a cancer, pre-determined by the molecular characteristics of the tumour founder cell, or whether metastatic capacity evolves in a stepwise fashion as the tumour grows, akin to the multistage accumulation of oncogenic alterations that give rise to the first cancer cell. In this Perspective, I examine how genetic analyses of primary tumours and matched metastases can distinguish between these two competing metastasis evolution models, with particular emphasis on the utility of metastatic randomness - a quantitative measure that reflects whether metastases arise from a random selection of primary tumour subclones or whether they are enriched for descendants of privileged lineages that have acquired pro-metastatic traits. Probable metastasis evolution trajectories in tumours with high and low baseline metastatic capacity are discussed, along with the role of seeding rates and selection at different metastatic host sites. Finally, I argue that trailblazing insights into human metastasis biology are immediately possible if we make a concerted effort to apply existing experimental and theoretical tools to the right patient cohorts.

Lung cancer (LC) remains the leading cause of cancer-related mortality worldwide, with most cases diagnosed at advanced stages, resulting in poor survival rates. Early detection significantly improves outcomes, yet current screening methods, such as low-dose computed tomography (LDCT), are limited by high false-positive rates, radiation exposure, and restricted eligibility criteria. This review highlights the transformative potential of genomic and molecular technologies in advancing the early detection of LC. Key innovations include liquid biopsy tools, such as circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) analysis, which offer minimally invasive approaches to detect tumor-specific genetic and epigenetic alterations. Emerging biomarkers, including methylation signatures, cfDNA fragmentomics, and multi-omics profiles, demonstrate improved sensitivity and specificity in identifying early-stage tumors. Advanced platforms like next-generation sequencing (NGS) and machine-learning algorithms further enhance diagnostic accuracy. Integrated approaches that combine genomic data with LDCT imaging and artificial intelligence (AI) show promise in addressing current limitations by improving risk stratification and nodule characterization. The review also explores multi-cancer early detection assays and precision diagnostic strategies tailored for diverse at-risk populations. By leveraging these advancements, clinicians can achieve earlier diagnoses, reduce unnecessary procedures, and ultimately decrease LC mortality.

The limited genomic data on non-muscle-invasive bladder cancer (NMIBC) hampers our understanding of its carcinogenesis and development. Specifically, Aristolochic acid (AA), a potent human carcinogenic compound from aristolochia plants and commonly found in Chinese herbal medicine, has been extensively documented as being closely associated with the onset and progression of bladder cancer. However, the field of AA-induced NMIBC remains largely unexplored in terms of its genomic and molecular characteristics, as well as clinical therapeutic strategies.

To bridge this knowledge gap, we conducted a comprehensive study using a cohort of 81 NMIBC samples. We performed whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to obtain detailed genomic and transcriptomic data. We subjected these datasets to genomic analysis and subtype analysis to gain valuable insights into NMIBC.

By temporally dissecting mutations in NMIBC specimens, we identified a comprehensive mutational landscape of NMIBC and the associations of these mutations with recurrence-free survival. Additionally, we discerned four genomic subtypes of NMIBC: AA-like, FGFR3/HRAS, FGFR3 & chr9Del, and genome instability (GI). The AA-like subtype presented a high frequency of gene mutations along with a pronounced AA mutagenesis signature of SBS22 (Fisher test: P-value 3.5e-4, OR 25.25) even after temporal dissection. The FGFR3/HRAS subtype exhibited FGFR3 or HRAS mutations with few copy number alterations (CNAs). The FGFR3 & chr9Del subtype was characterized by the co-occurrence of chr9p and chr9q deletions as well as FGFR3 mutations, while the GI subtype showed a high frequency of CNAs. Notably, the AA-like and GI subtypes demonstrated better outcomes after immunotherapy, whereas the FGFR3/HRAS subtype showed poorer outcomes.

Our findings provide novel perspectives on the genomics of NMIBC, unveiling four prominent genomic subtypes, each showing different outcomes following immunotherapy.

No. 2019PHB268-01 (retrospectively registered on February 14, 2020).

The rising incidence of multiple lung cancers (MLCs), encompassing multiple primary lung cancers (MPLCs) and intrapulmonary metastasis (IPM), poses two significant clinical challenges. First, distinguishing between MPLC and IPM remains difficult due to insufficiently accurate criteria and ambiguous integration of genetic testing. Second, standardized therapeutic protocols are still lacking. To address these issues, the Lung Cancer Expert Committee of China Anti-Cancer Association (CACA) assembled a multidisciplinary expert panel spanning thoracic surgery, pulmonary medicine, oncology, radiology, and pathology. Following a comprehensive literature review ending on October 23, 2024, the panel engaged in iterative discussions and conducted two rounds of expert voting, culminating in 25 evidence-based recommendations across five key domains: epidemiology, pre-treatment evaluation, definitive diagnostics, surgical treatment, and non-surgical treatment. This consensus provides clinicians with practical guidance to enhance diagnostic precision and therapeutic decision-making in MLC management while highlighting unmet needs to inform future guideline development.

MSI is a crucial biomarker for selecting CRC patients for immunotherapy. Here, we analyze the first results from the observational prospective trial BLOOMSI (NCT06414304), which investigated the impact of MSI/dMMR testing methods and baseline tumor heterogeneity on treatment outcomes. Thirty MSI/dMMR+ CRC patients, who were candidates for immunotherapy, were enrolled. Depending on the local test used for MSI/dMMR, central PCR/IHC was performed. Baseline FFPE and liquid biopsy (LB) were analyzed with NGS. ORR (objective response rate) in the ITT population was 50% (95% CI, 31.3-68.7%). Concordance between local/central dMMR/MSI testing was 81%, and the concordance of IHC, PCR, NGS/FFPE, and NGS/LB was 68.4%. The ORR was similar for IHC+, PCR+, NGS/FFPE+, and NGS/LB+ patients (55.6%, 55.6%, 55%, and 57.9%, respectively). The ORR among patients with discordant IHC/PCR results was 0%, and the ORR among patients with NGS/LB-ORR was 25% (2/8 CR). Next, we performed quantitative MSI analysis, reflecting the clonality of MSI+ tumor cells. Multivariate analysis identified MSI clonality in FFPE (HR 0.63, 95% CI, 0.39-0.99, p = 0.0487) and LB (HR 3.05, 95% CI, 2.01-4.65, p < 0.00001) as independent predictors of progression. The ORR in patients with high clonality (≥7%, n = 4, NGS/LB) was 25%. We describe baseline methodological predictors of non-response to immunotherapy and propose a strategy for selecting potential non-responders. These findings warrant further investigation.

Tractable, patient-relevant models are needed to investigate cancer progression and heterogeneity. Here, we report an alternative in vitro model of lung squamous cell carcinoma (LUSC) using primary human bronchial epithelial cells (hBECs) from three healthy donors. The co-operation of ubiquitous alterations (TP53 and CDKN2A loss) and components of commonly deregulated pathways including squamous differentiation (SOX2), PI3K signalling (PTEN) and the oxidative stress response (KEAP1) is investigated by generating hBECs harbouring cumulative alterations. Our analyses confirms that SOX2-overexpression initiates early preinvasive LUSC stages, and co-operation with the oxidative stress response and PI3K pathways to drive more aggressive phenotypes, with expansion of cells expressing LUSC biomarkers and invasive properties. This cooperation is consistent with the classical LUSC subtype. Importantly, we connect pathway dysregulation with gene expression changes associated with cell-intrinsic processes and immunomodulation. Our approach constitutes a powerful system to model LUSC and unravel genotype-phenotype causations of clinical relevance.

Subclonal reconstruction algorithms use bulk DNA sequencing data to quantify parameters of tumor evolution, allowing an assessment of how cancers initiate, progress and respond to selective pressures. We launched the ICGC-TCGA (International Cancer Genome Consortium-The Cancer Genome Atlas) DREAM Somatic Mutation Calling Tumor Heterogeneity and Evolution Challenge to benchmark existing subclonal reconstruction algorithms. This 7-year community effort used cloud computing to benchmark 31 subclonal reconstruction algorithms on 51 simulated tumors. Algorithms were scored on seven independent tasks, leading to 12,061 total runs. Algorithm choice influenced performance substantially more than tumor features but purity-adjusted read depth, copy-number state and read mappability were associated with the performance of most algorithms on most tasks. No single algorithm was a top performer for all seven tasks and existing ensemble strategies were unable to outperform the best individual methods, highlighting a key research need. All containerized methods, evaluation code and datasets are available to support further assessment of the determinants of subclonal reconstruction accuracy and development of improved methods to understand tumor evolution.

Tumors arise from uncontrolled cell proliferation driven by mutations in genes that regulate stem cell renewal and differentiation. Intestinal tumors, however, retain some hierarchical organization, maintaining both cancer stem cells (CSCs) and cancer differentiated cells (CDCs). This heterogeneity, coupled with cellular plasticity enabling CDCs to revert to CSCs, contributes to therapy resistance and relapse. Using genetically encoded fluorescent reporters in human tumor organoids, combined with our machine-learning-based cell tracker, CellPhenTracker, we simultaneously traced cell-type specification, metabolic changes, and reconstructed cell lineage trajectories during tumor organoid development. Our findings reveal distinctive metabolic phenotypes in CSCs and CDCs. We find that lactate regulates tumor dynamics, suppressing CSC differentiation and inducing dedifferentiation into a proliferative CSC state. Mechanistically, lactate increases histone acetylation, epigenetically activating MYC. Given that lactate's regulation of MYC depends on the bromodomain-containing protein 4 (BRD4), targeting cancer metabolism and BRD4 inhibitors emerge as a promising strategy to prevent tumor relapse.

The study examines Opisthorchis viverrini (OV)-related cholangiocarcinoma (CCA), a serious malignancy common in Southeast Asia. Through multi-regional whole-exome sequencing of 52 tumor samples and 13 adjacent tissues from 13 patients, significant intratumoral heterogeneity (ITH) and inter-patient heterogeneity are shown. Chronic liver fluke infection induces a distinct mutational landscape, with 48-90% of mutations concentrated in each region of the tumor. The average mutation burden is 95 non-synonymous mutations per area, exceeding previous CCA investigations. Critical driver mutations in TP53, SMAD4, and other genes underscore their significance in pathogenesis. Mutational markers elucidate mechanisms including spontaneous deamination and impaired DNA repair. Unique mutation patterns distinguish OV-associated CCA from other variants. Chromosomal instability in patient K110 signifies aggressive tumor behavior and unfavorable prognosis. Targetable mutations such as ERBB2 underscore the possibility for personalized therapeutics. These findings underscore the necessity for personalized strategies for treatment that target both trunk and branch mutations in endemic areas.

Transmembrane protein 71 (TMEM71) is implicated in multiple cellular physiological functions and has been demonstrated to be crucial in the advancement of different cancerous growths. However, its specific function in low-grade glioma (LGG) remains unclear.

We examined the expression patterns and prognostic importance of TMEM71 in various types of cancer by using pan-cancer analysis. The analyses of the correlations between TMEM71 expression and clinicopathological characteristics, prognosis, biological functions, immune characteristics, and genomic variations in LGG were conducted based on its expression patterns. Finally, the expression level and biological function of TMEM71 in LGG were verified by executing in vitro studies.

Abnormal elevation of TMEM71 expression level was associated with poor prognosis of many tumors including LGG. Both multivariate and univariate Cox regression analyses indicated that the expression of TMEM71 served as a standalone prognostic biomarker for LGG. The levels of TMEM71 expression were associated with immune-related characteristics, infiltration of immune cells, immune checkpoint genes (ICPGs) expression, and tumor mutation burden (TMB) in patients with LGG. In laboratory studies, elevated levels of TMEM71 were found to be involved in activating the JAK2/STAT3 pathway, promoting CCAAT/Enhancer Binding Protein D (CEBPD) expression, and ultimately affecting the growth and motility of LGG cells.

TMEM71 is an independent prognostic indicator for LGG and is strongly associated with the growth of LGG cells, positioning it as a potential novel target for treatment.

Immunotherapy, particularly immune checkpoint inhibitor therapy, has demonstrated clinical benefits in solid tumours. Despite its satisfactory clinical efficacy, it still faces several issues, such as limited eligibility, low response rates and cytotoxicity. Cancer epigenetics implies that tumour cells exhibit unique phenotypes because of their unique characteristics, thus reprogramming of the epigenome holds promise for cancer therapy. Epigenetic regulation plays an important role in regulating gene expression during tumour development and maintenance. Epigenetic regulators induce cancer cell cycle arrest, apoptosis and differentiation of cancer cells, thereby exerting anti-tumour effects. Recent studies have revealed a significant correlation between epigenetic regulatory factors and immune checkpoint therapy. Epigenetics can modulate various aspects of the tumour immune microenvironment and immune response to enhance the sensitivity of immunotherapy, such as lowering the concentration required and mitigating cytotoxicity. This review primarily discusses DNA methyltransferase inhibitors, histone deacetylase inhibitors, enhancer of zeste homolog 2 inhibitors and lysine-specific demethylase 1 inhibitors, which are associated with transcriptional repression. This repression alters the expression of genes involved in the immune checkpoint, thereby enhancing the effectiveness of immunotherapy. We also discuss the potential and challenges of tumour immunotherapy and highlight its advantages, application challenges and clinical research on integrating epigenetic regulatory factors with tumour immunotherapy.

Resistance to immune checkpoint inhibitors (ICIs) represents a major challenge for the effective treatment of non-small cell lung cancer (NSCLC). Tumor heterogeneity has been identified as an important mechanism of treatment resistance in cancer and has been increasingly implicated in ICI resistance. The diversity and clonality of tumor neoantigens, which represent the target epitopes for tumor-specific immune cells, have been shown to impact the efficacy of immunotherapy. Advances in genomic techniques have further enhanced our understanding of clonal landscapes within NSCLC and their evolution in response to therapy. In this review, we examine the role of tumor heterogeneity during immune surveillance in NSCLC and highlight its spatial and temporal evolution as revealed by modern technologies. We explore additional sources of heterogeneity, including epigenetic and metabolic factors, that have come under greater scrutiny as potential mediators of the immune response. We finally discuss the implications of tumor heterogeneity on the efficacy of ICIs and highlight potential strategies for overcoming therapeutic resistance.

Adoptive cell therapy (ACT) with TCR-engineered T-cells represents a promising alternative to TIL- or CAR-T therapies for patients with advanced solid cancers. Currently, selection of therapeutic TCRs critically depends on knowing the target antigens, a condition excluding most patients from treatment. Direct antigen-agnostic identification of tumor-specific T-cell clonotypes and TCR-T manufacturing using their TCRs can advance ACT for patients with aggressive solid cancers. We present a method to identify tumor-specific clonotypes from surgical specimens by comparing TCRβ-chain repertoires of TILs and adjacent tissue-resident lymphocytes. In six out of seven NSCLC-patients analyzed, our selection of tumor-specific clonotypes based on TIL-abundance and high tumor-to-nontumor frequency ratios was confirmed by gene expression signatures determined by scRNA-Seq. In three patients, we demonstrated that predicted tumor-specific clonotypes reacted against autologous tumors. For one of these patients, we engineered TCR-T cells with four candidate tumor-specific TCRs that showed reactivity against the patient's tumor and HLA-matched NSCLC cell lines. The TCR-T cells were then used to screen for candidate neoantigens and aberrantly expressed antigens. Three TCRs recognized recurrent driver-mutation KRAS Q61H-peptide ILDTAGHEEY presented by HLA-A*01:01. The TCRs were also dominant in a tumor relapse, one was found in cell free DNA. The finding of homologous TCRs in independent KRAS Q61H-positive cancers suggests a therapeutic opportunity for HLA-matched patients with KRAS Q61H-expressing tumors.

Cancer, characterized by the uncontrolled proliferation of cells, is one of the leading causes of death globally, with approximately one in five people developing the disease in their lifetime. While many driver genes were identified decades ago, and most cancers can be classified based on morphology and progression, there is still a significant gap in knowledge about genetic aberrations and nuclear DNA damage. The study of two critical groups of genes-tumor suppressors, which inhibit proliferation and promote apoptosis, and oncogenes, which regulate proliferation and survival-can help to understand the genomic causes behind tumorigenesis, leading to more personalized approaches to diagnosis and treatment. Aberration of tumor suppressors, which undergo two-hit and loss-of-function mutations, and oncogenes, activated forms of proto-oncogenes that experience one-hit and gain-of-function mutations, are responsible for the dysregulation of key signaling pathways that regulate cell division, such as p53, Rb, Ras/Raf/ERK/MAPK, PI3K/AKT, and Wnt/β-catenin. Modern breakthroughs in genomics research, like next-generation sequencing, have provided efficient strategies for mapping unique genomic changes that contribute to tumor heterogeneity. Novel therapeutic approaches have enabled personalized medicine, helping address genetic variability in tumor suppressors and oncogenes. This comprehensive review examines the molecular mechanisms behind tumor-suppressor genes and oncogenes, the key signaling pathways they regulate, epigenetic modifications, tumor heterogeneity, and the drug resistance mechanisms that drive carcinogenesis. Moreover, the review explores the clinical application of sequencing techniques, multiomics, diagnostic procedures, pharmacogenomics, and personalized treatment and prevention options, discussing future directions for emerging technologies.

Understanding lung cancer evolution can identify tools for intercepting its growth. In a landscape analysis of 1024 lung adenocarcinomas (LUAD) with deep whole-genome sequencing integrated with multiomic data, we identified 542 LUAD that displayed diverse clonal architecture. In this group, we observed an interplay between mobile elements, endogenous and exogenous mutational processes, distinct driver genes, and epidemiological features. Our results revealed divergent evolutionary trajectories based on tobacco smoking exposure, ancestry, and sex. LUAD from smokers showed an abundance of tobacco-related C:G>A:T driver mutations in KRAS plus short subclonal diversification. LUAD in never smokers showed early occurrence of copy number alterations and EGFR mutations associated with SBS5 and SBS40a mutational signatures. Tumors harboring EGFR mutations exhibited long latency, particularly in females of European-ancestry (EU_N). In EU_N, EGFR mutations preceded the occurrence of other driver genes, including TP53 and RBM10. Tumors from Asian never smokers showed a short clonal evolution and presented with heterogeneous repetitive patterns for the inferred mutational order. Importantly, we found that the mutational signature ID2 is a marker of a previously unrecognized mechanism for LUAD evolution. Tumors with ID2 showed short latency and high L1 retrotransposon activity linked to L1 promoter demethylation. These tumors exhibited an aggressive phenotype, characterized by increased genomic instability, elevated hypoxia scores, low burden of neoantigens, propensity to develop metastasis, and poor overall survival. Reactivated L1 retrotransposition-induced mutagenesis can contribute to the origin of the mutational signature ID2, including through the regulation of the transcriptional factor ZNF695, a member of the KZFP family. The complex nature of LUAD evolution creates both challenges and opportunities for screening and treatment plans.

Radiotherapy is an integral component in the treatment of many types of cancer, with approximately half of patients with cancer receiving radiotherapy. Systemic therapy applies pressure that can select for resistant tumor subpopulations, underscoring the importance of understanding how radiation impacts tumor evolution to improve treatment outcomes. We integrated temporal genomic profiling of 120 spatially distinct tumor regions from 20 patients with undifferentiated pleomorphic sarcomas (UPS), longitudinal circulating tumor DNA analysis, and evolutionary biology computational pipelines to study UPS evolution during tumorigenesis and in response to radiotherapy. Most unirradiated UPSs displayed initial linear evolution, followed by subsequent branching evolution with distinct mutational processes during early and late development. Metrics of genetic divergence between regions provided evidence of strong selection pressures during UPS development that further increased during radiotherapy. Subclone abundance changed after radiotherapy with subclone contraction tied to alterations in calcium signaling, and inhibiting calcium transporters radiosensitized sarcoma cells. Finally, circulating tumor DNA analysis accurately measured subclone abundance and enabled noninvasive monitoring of subclonal changes. These results demonstrate that radiation exerts selective pressures on UPSs and suggest that targeting radioresistant subclonal populations could improve outcomes after radiotherapy. Significance: Radiotherapy mediates tumor evolution by leading to the expansion of resistant subclonal cancer cell populations, indicating that developing approaches to target resistant subclones will be crucial to improve radiotherapy response.

Intratumour hypoxia is a feature of all heterogenous solid tumours. Increased levels or subregions of tumour hypoxia are associated with an adverse clinical prognosis, particularly when this co-occurs with genomic instability. Experimental evidence points to the acquisition of DNA and chromosomal alterations in proliferating hypoxic cells secondary to inhibition of DNA repair pathways such as homologous recombination, base excision repair and mismatch repair. Cell adaptation and selection in repair-deficient cells give rise to a model whereby novel single-nucleotide mutations, structural variants and copy number alterations coexist with altered mitotic control to drive chromosomal instability and aneuploidy. Whole-genome sequencing studies support the concept that hypoxia is a critical microenvironmental cofactor alongside the driver mutations in MYC, BCL2, TP53 and PTEN in determining clonal and subclonal evolution in multiple tumour types. We propose that the hypoxic tumour microenvironment selects for unstable tumour clones which survive, propagate and metastasize under reduced immune surveillance. These aggressive features of hypoxic tumour cells underpin resistance to local and systemic therapies and unfavourable outcomes for patients with cancer. Possible ways to counter the effects of hypoxia to block tumour evolution and improve treatment outcomes are described.

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.

In recent years, the successful use of tumor-infiltrating lymphocyte (TIL) therapy to treat melanoma not only culminated in a landmark Food and Drug Administration approval, but has also fueled the emergence of a new, rapidly growing field in TIL cellular immunotherapy surrounding novel enhancements in TIL design, refined manufacturing strategies to enrich for more potent TIL populations, as well as numerous clinical trials now investigating TIL therapy in additional solid tumor types beyond melanoma. This review provides a summary of the latest advances in TIL therapy and what lies ahead for the field. The first section explores several solid cancers that demonstrate the greatest potential for future indications of TIL therapy. The second section provides insight into the promising innovations for designing the next generation of TIL with greater specificity, persistence, safety, and function.

Metastatic disease is the leading cause of cancer-related death, despite recent advances in therapeutic interventions. Prior modeling approaches have accounted for the adaptive immune system's role in combating tumors, which has led to the development of stochastic models that explain cancer immunoediting and tumor-immune co-evolution. However, cancer immune-mediated dormancy, wherein the adaptive immune system maintains a micrometastatic population by keeping its growth in check, remains poorly understood. Immune-mediated dormancy can significantly delay the emergence (and therefore detection) of metastasis. An improved quantitative understanding of this process will thereby improve our ability to identify and treat cancer during the micrometastatic period. Here, we introduce a generalized stochastic model that incorporates the dynamic effects of immunomodulation within the tumor microenvironment on T cell-mediated cancer killing. This broad class of nonlinear birth-death model can account for a variety of cytotoxic T cell immunosuppressive effects, including regulatory T cells, cancer-associated fibroblasts, and myeloid-derived suppressor cells. We develop analytic expressions for the likelihood and mean time of immune escape. We also develop a method for identifying a corresponding diffusion approximation applicable to estimating population dynamics across a wide range of nonlinear birth-death processes. Lastly, we apply our model to estimate the nature and extent of immunomodulation that best explains the timing of disease recurrence in bladder and breast cancer patients. Our findings quantify the effects that stochastic tumor-immune interaction dynamics can play in the timing and likelihood of disease progression. Our analytical approximations provide a method of studying population escape in other ecological contexts involving nonlinear transition rates.

EGFR status assessment is mandatory for adjuvant decision-making of resected stage IB-IIIA non-squamous non-small cell lung cancer (NS-NSCLC). It is questionable whether single-gene RT-PCR versus next-generation sequencing (NGS) should be used for this evaluation. Moreover, co-occurring mutations have an impact on tumor behavior and may influence future therapeutic decision-making. We aimed to describe the clinico-pathological and molecular features, as well as the prognostic factors of resected EGFR-mutant NS-NSCLC evaluated with reflex NGS and RT-PCR, so as to compare the results of the two methods. We retrospectively included and collected data from patients with resected EGFR-mutant NS-NSCLC diagnosed in our institution between 2005 and 2024. Additional cases from another center were included. Tumors were analyzed using targeted NGS and RT-PCR. A total of 153 patients were selected. The median follow-up after surgery was 22 months. The positive percent agreement of RT-PCR compared to NGS for the detection of an EGFR mutation was 88%. Common single EGFR mutations (L858R/del19) were observed in 117/153 (77%) cases; 22/153 (14%) and 14/153 (9%) cases had uncommon single and compound EGFR mutations, respectively. 63/153 (41%) patients had a co-occurring mutation, including a TP53 mutation in 43/63 (68%) co-mutated patients. EGFR/TP53-mutant tumors were associated with positive PD-L1 expression compared to EGFR-mutant/TP53-wild-type tumors (62% vs 39%; p = 0.006). Shorter median event-free survival (EFS) in patients with an EGFR exon 18 mutation and those with TP53 exon 7 co-mutation was recorded. The EGFR status should be systematically evaluated using targeted NGS reflex testing for resected NS-NSCLC since future therapeutic decision-making could soon consider integrating the presence of co-occurring mutations.

Recognition and elimination of pathogens and cancer cells depend on the adaptive immune system. Thus, accurate quantification of immune subsets is vital for precision medicine. We present immune lymphocyte estimation from nucleotide sequencing (ImmuneLENS), which estimates T cell and B cell fractions, class switching and clonotype diversity from whole-genome sequencing data at depths as low as 5× coverage. By applying ImmuneLENS to the 100,000 Genomes Project, we identify genes enriched with somatic mutations in T cell-rich tumors, significant sex-based differences in circulating T cell fraction and demonstrated that the circulating T cell fraction in patients with cancer is significantly lower than in healthy individuals. Low circulating B cell fraction was linked to increased cancer incidence. Finally, circulating T cell abundance was more prognostic of 5-year cancer survival than infiltrating T cells.