Nat Commun. 2025 Sep 1;16(1):8161. doi: 10.1038/s41467-025-63538-4.

ABSTRACT

Immune checkpoint blockade (ICB) has improved outcomes for patients with head and neck squamous cell carcinoma (HNSCC), but predictive biomarkers remain limited. Here, we use a time-resolved, multi-omic approach in a murine HNSCC model to characterize peripheral immune responses to ICB. Single-cell transcriptomics and T/B cell receptor analyses reveal early on-treatment expansion of effector memory T and B cell repertoires in responders, preceding tumor regression. These dynamic immune features inform a composite transcriptional signature that accurately predicts ICB response in independent human HNSCC cohorts. LiBIO outperforms existing biomarkers and generalizes to melanoma, non-small cell lung cancer, and breast cancer without retraining. These findings suggest that early treatment-induced changes in circulating immune repertoires reflect the host's capacity to mount an effective antitumor response. This work provides a framework for leveraging transient peripheral immune dynamics to develop non-invasive, high-fidelity biomarkers for response to immunotherapy across cancer types.

PMID:40890155 | PMC:PMC12402333 | DOI:10.1038/s41467-025-63538-4

Cell. 2025 Aug 22:S0092-8674(25)00922-5. doi: 10.1016/j.cell.2025.08.008. Online ahead of print.

ABSTRACT

Performing total RNA profiling on formalin-fixed, paraffin-embedded (FFPE) samples, the predominant sample conservation method in clinical practice, remains challenging for current spatial transcriptomics techniques. Here, we introduce Stereo-seq V2, which employs random primers to capture and sequence RNAs in situ on FFPE sections and provides single-cell resolution. The random-priming-based strategy offers unbiased transcript capturing and uniform gene body coverage, which increase the sensitivity to marker genes, the efficiency of non-polyadenylation (poly(A)) RNA profiling, and immune repertoire coverage. We demonstrated the robust performance of Stereo-seq V2 on clinical FFPE samples using triple-negative breast cancer (TNBC) sections and identified tumor-specific alternative splicing events. In a Mycobacterium tuberculosis (Mtb)-infected mouse model, we monitored gene expression dynamics of host and pathogen transcriptomes simultaneously by utilizing Stereo-seq V2. We also assembled immune repertoires and identified Mtb-specific BCR clones, which could also be observed in human tuberculous lung samples. These results highlight Stereo-seq V2's potential in biomedical research and personalized medicine.

PMID:40882628 | DOI:10.1016/j.cell.2025.08.008

Cancers (Basel). 2025 Aug 15;17(16):2668. doi: 10.3390/cancers17162668.

ABSTRACT

Background/Objectives: Esophageal and esophagogastric junction adenocarcinoma (EADC-EGJA), which mainly develops from Barrett's esophagus (BE), low-grade dysplasia (LGD), and high-grade dysplasia (HGD), has a poor prognosis and several unmet clinical needs, among which is the detection of minimal residual disease (MRD) after endoscopic/surgical resection. Long interspersed nuclear element-1 (LINE-1), a surrogate marker of global methylation, is considered an emerging biomarker for MRD monitoring. The aim of this study was to determine, by LINE-1 methylation analysis, at which carcinogenesis step global methylation is affected and whether this biomarker could be followed in longitudinal to monitor the disease behavior post-surgery. Methods: Cell-free DNA of 90 patients with non-dysplastic Barrett's esophagus (NDBE), HGD/early EADC-EGJA, or locally advanced/advanced EADC-EGJA were analyzed for LINE-1 methylation, by Methylation-Sensitive Restriction Enzyme droplet digital PCR (MSRE-ddPCR). Twenty-six patients were longitudinally studied by repetitive blood sampling. Results: Global hypomethylation increased during carcinogenesis, with significant difference between locally advanced/advanced EADC-EGJA and NDBE patients (p = 0.028). Longitudinal cases confirmed the rareness of hypomethylation in NDBE cases. The majority of HGD/early EADC-EGJA and locally advanced/advanced EADC-EGJA patients showed methylation changes after resection according to clinical status. Conclusions: This study suggests that global hypomethylation occurs just prior to cancer invasiveness and that it is a promising biomarker to monitor MRD.

PMID:40867295 | PMC:PMC12384112 | DOI:10.3390/cancers17162668

Br J Cancer. 2025 Aug 23. doi: 10.1038/s41416-025-03139-6. Online ahead of print.

ABSTRACT

Non-small cell lung cancer (NSCLC) represents a heterogeneous group of malignancies characterised by diverse histological and molecular features. Some NSCLCs, particularly adenocarcinomas, harbour genomic alterations in receptor tyrosine kinases or downstream RAS/RAF signalling pathways, which are targets of effective therapies. NSCLCs lacking actionable genomic alterations often benefit from immune checkpoint inhibitors, though only a minority of patients achieve long-term survival. These tumours often carry alterations in tumour suppressor genes like TP53, KEAP1, STK11, or NF1, for which pharmacological strategies are still under investigation. This review explores emerging therapeutic opportunities unveiled by multi-omics studies in NSCLCs without actionable genomic alterations. Proteogenomic approaches-integrating genomic, transcriptomic and proteomic data-enable a comprehensive understanding of NSCLC molecular landscapes and signalling network dysregulation, helping to identify distinct tumour subtypes and potential therapeutic targets. These tumours exhibit alterations in cell cycle regulation, DNA repair, immune signalling, epigenetic modulation and metabolic and redox pathways. Although therapies targeting tumour suppressor genes like p53 remain highly anticipated, extending our understanding of the broader molecular landscape in these tumours may reveal novel vulnerabilities and inform the development of novel drugs or combination strategies. This could further advance precision oncology for NSCLC.

PMID:40849356 | DOI:10.1038/s41416-025-03139-6

Cell Rep. 2025 Aug 21;44(9):116191. doi: 10.1016/j.celrep.2025.116191. Online ahead of print.

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease, and new therapeutic strategies are urgently needed. Here, we conduct an integrative, genome-scale examination of genetic dependencies and cell surface targets using CRISPR-Cas screening and multi-omic data, including single-nucleus and spatial transcriptomic data from patient tumors. We systematically identify clinically tractable and biomarker-linked PDAC dependencies, including CDS2 as a synthetic lethal target in cancer cells expressing signatures of epithelial-to-mesenchymal transition. We examine biomarkers and co-dependencies of the KRAS oncogene, defining gene expression signatures of sensitivity and resistance associated with response to pharmacological inhibition of KRAS. mRNA and protein profiling reveal cell surface protein-encoding genes with robust expression in patient tumors and minimal expression in non-malignant tissues. Furthermore, we define intratumoral and interpatient heterogeneity of target gene expression and identify orthogonal targets that suggest combinatorial strategies. Collectively, this work identifies multiple targets that may inform therapeutic strategies for patients with PDAC.

PMID:40848256 | DOI:10.1016/j.celrep.2025.116191

EClinicalMedicine. 2025 Aug 12;87:103406. doi: 10.1016/j.eclinm.2025.103406. eCollection 2025 Sep.

ABSTRACT

BACKGROUND: Recurrence risk after curative surgery for colorectal liver metastases (CRLM) remains high, underlining the need to identify prognostic markers enabling more individualised treatment approaches.

METHODS: In the MIRACLE, a prospective, observational biomarker study, a total of 188 patients with isolated, resectable CRLM without (neo)adjuvant chemotherapy were included between October 2015 and December 2021. Blood samples were collected before surgery (baseline) and three weeks after surgery. The primary objective was to assess the potential association between postoperative circulating tumour DNA (ctDNA) detection and recurrence of disease for patients with resectable CRLM within one year after resection. The secondary objective was the association between recurrence of disease within one year and detection of circulating tumour cells (CTCs). Baseline ctDNA was measured by next generation sequencing using a targeted panel (Oncomine Colon cell-free DNA assay) and postoperatively by digital PCR on genetic variants found preoperatively with the Oncomine panel. CTCs were enumerated using the FDA-approved CellSearch system.

FINDINGS: ctDNA was detected in 117/187 patients (63%) at baseline, and 28/104 evaluable patients (27%) still had detectable ctDNA postoperatively. CTC enumeration resulted in positivity for 37/183 patients (20%) at baseline and 14/158 patients (9%) postoperatively. No association was found between 1-year recurrence-free survival (RFS) and the presence of CTCs or ctDNA at baseline. In contrast, patients with postoperative undetectable ctDNA had a significantly improved 1-year RFS compared to patients with postoperative ctDNA (54% [95% CI 44%-67%] vs. 25% [95% CI 13%-47%], log-rank p = 0.0011). Similarly, patients with postoperative detectable CTCs had a significantly shorter 1-year RFS compared to patients without postoperative CTCs (15% [95% CI 4%-55%] vs. 53% [95% CI 45%-62%], log-rank p 0.0004). Also in multivariable analysis, detectable ctDNA and CTCs after surgery remained independently associated with a shorter 1-year RFS (HR 2.35; 95% CI 1.34-4.11; p = 0.0028 and HR 2.98; 95% CI 1.56-5.71; p = 0.0010, respectively).

INTERPRETATION: This is the first study conducted in patients with resectable CRLM without (neo)adjuvant chemotherapy, which demonstrates the impact of postoperative detectable circulating tumour load on 1-year RFS. Postoperative ctDNA and CTC detection both represent strong, independent predictors for a shorter RFS after local treatment, as opposed to preoperative detection.

FUNDING: This work was supported by KWF Kankerbestrijding (Dutch Cancer Society, EMCR 2014-6340).

PMID:40838198 | PMC:PMC12361997 | DOI:10.1016/j.eclinm.2025.103406

Ann Rheum Dis. 2025 Aug 20:S0003-4967(25)04249-9. doi: 10.1016/j.ard.2025.07.016. Online ahead of print.

ABSTRACT

OBJECTIVES: Catastrophic antiphospholipid syndrome (CAPS) is a complement-driven thrombotic disorder, characterised by widespread thrombosis and multiorgan failure. We identified rare germline variants including complement receptor 1 (CR1) in 50% of patients with CAPS. Here, we define CR1 dysregulation mechanisms (genetic/epigenetic) underlying complement-mediated thrombosis in CAPS and support C5 inhibition as a potential therapy.

METHODS: We quantified CR1 expression by flow cytometry across haematopoietic cell types. CRISPR/Cas9 genome editing of TF-1 (erythroleukaemia) cells was performed to generate CR1 'knock-out' and 'knock-in' lines with patient-specific CR1 variants. Multiomics analysis was performed to investigate the role of methylation in patients with reduced CR1 expression. Functional impact of low CR1 was assessed by complement-mediated cell killing using modified Ham assay, cell-bound complement degradation products through flow cytometry, and circulatory immune complexes in serum samples through ELISA.

RESULTS: CR1 expression in erythrocytes was markedly reduced on CAPS erythrocytes (n = 9, 21.80%) compared to healthy controls (HCs; n = 35, 84.04%), with promoter hypermethylation emerging as a plausible epigenetic mechanism for CR1 downregulation. Novel germline variant (CR1-V2125L; rs202148801) mitigated CR1 expression and increased complement-mediated cell death of knock-in cell lines. Erythrocytes from the patient with the CR1-V2125L variant had low CR1 expression. Levels of circulating immune complexes, which are bound and cleared by CR1 on erythrocytes, were higher in acute CAPS (n = 3, 25.55 µg Eq/mL) than HCs (n = 3, 7.445 µg Eq/mL). Five patients were treated with C5 inhibition which mitigated thrombosis.

CONCLUSIONS: Genetic or epigenetic-mediated CR1 deficiency is a potential hallmark of CAPS and predicts response to C5 inhibition.

PMID:40841298 | DOI:10.1016/j.ard.2025.07.016

OMICS. 2025 Aug 19. doi: 10.1177/15578100251366980. Online ahead of print.

ABSTRACT

Ferroptosis, an iron-dependent form of oxidative cell death, plays a critical role in cancer progression and immune regulation. However, the functional connections of ferroptosis with specific immune cell types remain poorly defined, limiting the future possibilities to harness ferroptosis for cancer biology, diagnosis, and treatment. To address this knowledge gap, we conducted an integrated transcriptomic analysis to investigate ferroptosis-related immune dynamics in gastric cancer (GC). We utilized GC datasets from The Cancer Genome Atlas-stomach adenocarcinoma (n = 412) and the GSE66229 (n = 300) that were clustered into three GC immune subtypes based on single-sample Gene Set Enrichment Analysis scores of 29 immune gene sets. Bulk RNA-seq analysis revealed that the immune-inflamed subtype (HIS) of tumor samples in both GC datasets exhibited the highest ferroptosis enrichment and showed a positive correlation with activated mast cells and neutrophils. Given the regulatory role of mast cells in the tumor microenvironment (TME), particularly in recruiting neutrophils, we further examined their link to ferroptosis. In a fibroblast-mast cell coculture RNA-seq data (GSE223179), fibroblasts exhibited increased ferroptosis enrichment, supporting a mast cell-mediated influence. Single-cell RNA-seq data confirmed stronger interactions between mast cells and fibroblasts in GC compared to normal tissues. Specifically, they revealed a positive correlation between mast cell activity and ferroptosis enrichment in tumor-associated fibroblasts. In conclusion, these findings suggest that mast cells may promote ferroptosis in the TME through paracrine signaling, possibly via annexin and cyclophilin A. By uncovering this novel pathophysiological axis, our study reveals a previously unrecognized role of mast cells in regulating ferroptosis within the TME. The findings call for translational and experimental medical research and have potential implications for innovation toward GC diagnostics and therapeutics.

PMID:40831393 | DOI:10.1177/15578100251366980

Sci Prog. 2025 Jul-Sep;108(3):368504251362350. doi: 10.1177/00368504251362350. Epub 2025 Aug 19.

ABSTRACT

Breast cancer recurrence remains a major cause of mortality, with up to 30% of early-stage patients relapsing as incurable metastatic disease. Conventional surveillance with imaging and serum markers (CA15-3, CEA) lacks the sensitivity and specificity to detect minimal residual disease. This narrative review examines non-invasive biomarkers such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and exosomes and the technologies enhancing their performance. Droplet digital PCR and next-generation sequencing detect ctDNA at allele frequencies below 0.1%, identifying molecular relapse a median of 10-12 months before radiologic progression. Microfluidic and affinity-based platforms isolate CTCs with over 75% sensitivity in metastatic settings. Nanoengineered sensors and standardized workflows improve exosome isolation, revealing miRNA and protein signatures predictive of recurrence. Proteomic and metabolomic profiling identify dysregulated metabolic pathways and protein networks, offering functional insights that complement molecular assays. Integrative multi-omics approaches merge genomic, transcriptomic, proteomic and metabolomic data; machine-learning frameworks detect subtle patterns and correlations, enabling dynamic, personalized surveillance. By detecting molecular and functional biomarkers early, clinicians can tailor therapy, monitor treatment response and intervene promptly. Challenges include low analyte abundance, assay variability, high costs and lack of standardized protocols, limiting clinical adoption. Prospective validation in large cohorts is critical. We highlight ongoing clinical trials such as ctDNA-guided adjuvant therapy and CTC-driven stratification studies that aim to establish clinical utility. Non-invasive biomarker platforms could shift breast cancer follow-up from reactive detection to proactive intervention, ultimately improving survival and quality of life through personalized, real-time monitoring.

PMID:40831186 | PMC:PMC12368403 | DOI:10.1177/00368504251362350

J Clin Microbiol. 2025 Aug 19:e0058525. doi: 10.1128/jcm.00585-25. Online ahead of print.

ABSTRACT

Human papillomavirus (HPV) is comprised of >200 genotypes and has an ~8 kb, circular, double-stranded DNA genome. Transmission of HPV occurs through skin-to-skin contact and infection of squamous epithelial cells of cutaneous and mucosal surfaces. HPV genotypes are categorized as low- or high-risk (hrHPV) based on oncogenic potential. There are approximately 14 types of hrHPV that can cause several types of cancer, including HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC). Detection of HPV(+)OPSCC is traditionally accomplished using p16 immunohistochemistry (IHC) and HPV-specific testing, either DNA or RNA in situ hybridization (ISH) staining or DNA-based PCR of suspected tumor biopsy tissue. More recently, platelet-poor plasma (PPP) samples from patients with HPV(+)OPSCC have proven useful for detection and quantitation of fragments of HPV circulating tumor DNA (ctDNA). ctDNA has been shown to be useful in determining treatment response and monitoring for disease recurrence. In this study, a novel droplet digital PCR assay (ddPCR) was developed and validated for the detection and quantitation of ctDNA from 5 hrHPV genotypes in PPP. Analytical sensitivity ranged from 7.71 to 19.45 fragments of HPV ctDNA per milliliter of PPP across five hrHPV genotypes. In patients with confirmed primary or recurrent HPV(+)OPSCC or HPV(-)OPSCC, testing of corresponding PPP samples (n = 32) by ddPCR demonstrated 90.63% (29/32) overall agreement with p16/HPV-ISH biopsy results. Compared with reference ddPCR assays performed at outside laboratories, our ddPCR assay yielded 90% (9/10) overall agreement. This assay may provide clinicians with a tool for monitoring HPV ctDNA prior to, during, and after treatment of an HPV-associated cancer.

IMPORTANCE: At least 14 genotypes of human papillomavirus (HPV) have been identified to have high oncogenic potential. While molecular diagnostic testing for HPV is widely available for liquid cytologic cervical samples, testing is limited for other sample types, including liquid biopsy samples, such as platelet-poor plasma (PPP). With the rising incidence of HPV-associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC), laboratory testing is an essential part of patient diagnosis, management, and surveillance. Here, we summarize the development and analytical performance validation of a multiplexed, droplet digital PCR (ddPCR) assay for the detection and quantitation of HPV circulating tumor DNA (ctDNA) in PPP. This assay may provide clinicians with a tool to address minimal residual disease for patients with an HPV-associated cancer.

PMID:40827899 | DOI:10.1128/jcm.00585-25

Expert Rev Anticancer Ther. 2025 Aug 18. doi: 10.1080/14737140.2025.2549538. Online ahead of print.

ABSTRACT

INTRODUCTION: In the era of precision medicine, molecular biomarker testing is increasingly becoming standard of care for Non-Small Cell Lung Cancer (NSCLC) patients. Tissue and liquid biopsy-based Next-Generation Sequencing (NGS) is now highly recommended.

AREAS COVERED: Different NGS platforms emerged as a cost-effective strategy to perform a massive and parallel sequencing performing higher technical sensitivity than old generation technologies in detecting low abundant alterations in challenging diagnostic samples. NGS systems can detect single nucleotide variants (SNV), small insertions and deletions (indels), copy number alterations (CNAs) and structural variants (SVs) or gene fusions across selected druggable genes optimizing clinical administration of NSCLC patients. The diagnostic implementation of the most adequate NGS panel depending on several factors that could impact on the clinical utility of testing assay.

EXPERT OPINION: Promising advanced technologies are emerging as potentially integrative tools in personalized medicine. In this context, multi-omic evaluation including genomic, transcriptomic, fragmentomic and epigenomic signatures are under investigation to significantly modify clinical algorithm of NSCLC patients. On this basis, sequencing strategies may play a pivotal role in the implementation of a new predictive model for cancer diagnosis and prognosis.

PMID:40823981 | DOI:10.1080/14737140.2025.2549538