A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

CD4+ T cell activation distinguishes response to anti-PD-L1+anti-CTLA4 therapy from anti-PD-L1 monotherapy.

Cancer patients often receive a combination of antibodies targeting programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA4). We conducted a window-of-opportunity study in head and neck squamous cell carcinoma (HNSCC) to examine the contribution of anti-CTLA4 to anti-PD-L1 therapy. Single-cell profiling of on- versus pre-treatment biopsies identified T cell expansion as an early response marker. In tumors, anti-PD-L1 triggered the expansion of mostly CD8+ T cells, whereas combination therapy expanded both CD4+ and CD8+ T cells. Such CD4+ T cells exhibited an activated T helper 1 (Th1) phenotype. CD4+ and CD8+ T cells co-localized with and were surrounded by dendritic cells expressing T cell homing factors or antibody-producing plasma cells. T cell receptor tracing suggests that anti-CTLA4, but not anti-PD-L1, triggers the trafficking of CD4+ naive/central-memory T cells from tumor-draining lymph nodes (tdLNs), via blood, to the tumor wherein T cells acquire a Th1 phenotype. Thus, CD4+ T cell activation and recruitment from tdLNs are hallmarks of early response to anti-PD-L1 plus anti-CTLA4 in HNSCC.

Distinct immunometabolic signatures in circulating immune cells define disease outcome in acute-on-chronic liver failure.

BACKGROUND AND AIMS: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and nonrecovery in ACLF. APPROACH AND RESULTS: Here, we mapped the single-cell transcriptome of circulating immune cells from patients with ACLF and acute decompensated (AD) cirrhosis and healthy individuals. We further interrogate how these findings, as well as immunometabolic and functional profiles, associate with ACLF-recovery (ACLF-R) or nonrecovery (ACLF-NR). Our analysis unveiled 2 distinct states of classical monocytes (cMons). Hereto, ACLF-R cMons were characterized by transcripts associated with immune and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1 . Additional metabolomic and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program, and blunted activation among lymphoid populations in patients with ACLF-R. Conversely, ACLF-NR cMon featured elevated expression of inflammatory and stress response genes such as VIM , LGALS2 , and TREM1 , along with blunted metabolic activity and increased functionality. CONCLUSIONS: This study identifies distinct immunometabolic cellular states that contribute to disease outcomes in patients with ACLF. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or nonrecovery phenotypes, which may be harnessed as potential therapeutic targets in the future.

High dose vitamin D supplementation does not improve outcome in a cutaneous melanoma population: results of a randomized double-blind, placebo-controlled study (ViDMe trial).

BACKGROUND: Observational studies in cutaneous melanoma have indicated an inverse relationship between levels of 25-hydroxy vitamin D and Breslow thickness, as well as a protective effect of high 25- hydroxy vitamin D levels on clinical outcome. OBJECTIVES: To evaluate whether high dose vitamin D supplementation in curatively resected cutaneous melanoma reduces melanoma relapse. METHODS: In a prospective, randomized, double-blind, placebo-controlled trial, 436 patients with resected cutaneous melanoma stage IA to III (8th American Joint Committee on Cancer staging) were randomized. Among them, 218 received a placebo while 218 received monthly 100,000 IU cholecalciferol for a minimum of 6 months and a maximum of 42 months (treatment arm). Following randomization, patients were followed for a median of 52 months, with a maximum follow-up of 116 months. The primary endpoint was relapse-free survival. Secondary endpoints were melanoma-related mortality, overall survival, and the evolution of 25-hydroxy vitamin D serum levels over time. RESULTS: In our population (mean age 55 years, 54% female) Vitamin D supplementation increased 25- hydroxy vitamin D serum levels after 6 months of supplementation in the treatment arm by a median 17 ng/ml (95%CI: 9; 26) compared to 0 ng/ml (95%CI: -6; 8) in the placebo arm (P < 0.001; Wilcoxon test) and remained at a steady state during the whole treatment period. The estimated event rate for relapse-free survival at 72 months after inclusion was 26.51% in the vitamin D supplemented arm (95% CI: 19.37; 35.64) versus 20.70% (95%CI: 14.26; 29.52) in the placebo arm, [hazard ratio 1.27 (95%CI 0.79; 2.03), P = 0.32]. After adjusting for confounding factors (including baseline stage, body mass index, age, gender, and baseline season), the hazard ratio was 1.20 (95% CI 0.74; 1.94, P = 0.46). Deaths from progression of cutaneous melanoma and non-melanoma related deaths were similar in both vitamin D supplemented and placebo group (n = 10 and 11 and n = 3 and 2, respectively). No major adverse events were observed during the study. CONCLUSION: In cutaneous melanoma patients, monthly high dose vitamin D supplementation was safe, resulted in a sustained increase in 25-hydroxy vitamin D levels during the treatment period, but did not improve relapse-free survival, melanoma-related death or overall survival.

Single-cell RNA sequencing of cystic fibrosis liver disease explants reveals endothelial complement activation.

BACKGROUND & AIMS: Cystic fibrosis (CF) is considered a multisystemic disorder in which CF-associated liver disease (CFLD) is the third most common cause of mortality. Currently, no effective treatment is available for CFLD because its pathophysiology is still unclear. Interestingly, CFLD exhibits identical vascular characteristics as non-cirrhotic portal hypertension, recently classified as porto-sinusoidal vascular disorders (PSVD). METHODS: Since endothelial cells (ECs) are an important component in PSVD, we performed single-cell RNA sequencing (scRNA-seq) on four explant livers from CFLD patients to identify differential endothelial characteristics which could contribute to the disease. We comprehensively characterized the endothelial compartment and compared it with publicly available scRNA-seq datasets from cirrhotic and healthy livers. Key gene signatures were validated ex vivo on patient tissues. RESULTS: We found that ECs from CF liver explants are more closely related to healthy than cirrhotic patients. In CF patients we also discovered a distinct population of liver sinusoidal ECs-coined CF LSECs-upregulating genes involved in the complement cascade and coagulation. Finally, our immunostainings further validated the predominant periportal location of CF LSECs. CONCLUSIONS: Our work showed novel aspects of human liver ECs at the single-cell level thereby supporting endothelial involvement in CFLD, and reinforcing the hypothesis that ECs could be a driver of PSVD. Therefore, considering the vascular compartment in CF and CFLD may help developing new therapeutic approaches for these diseases.

A phase II, multicenter, open-label study of abemaciclib and letrozole in patients with estrogen receptor-positive rare ovarian cancer: ALEPRO trial.

BACKGROUND: Low-grade serous and endometrioid ovarian cancers and adult-type granulosa cell tumors are rare ovarian malignancies that show high estrogen receptor positivity. Recurrences of these subtypes of ovarian cancer are often treated with conventional chemotherapy, although response rates are disappointing. PRIMARY OBJECTIVE: To determine the overall response rate of the combination therapy of abemaciclib and letrozole in patients with estrogen receptor-positive rare ovarian cancers. STUDY HYPOTHESIS: The combination therapy of abemaciclib and letrozole will provide a clinically meaningful therapeutic benefit, with an overall response rate of >25%. TRIAL DESIGN: This is a phase II, international, multicenter, open-label, single-arm study to evaluate the efficacy and safety of abemaciclib and letrozole in patients with advanced, recurrent, and/or metastatic estrogen receptor-positive, rare ovarian cancer. The study will follow a tandem two-stage design. MAJOR INCLUSION/EXCLUSION CRITERIA: Patients must have histologically confirmed low-grade serous/endometrioid ovarian cancer or adult-type granulosa cell tumor with estrogen receptor positivity on immunohistochemistry. Patients need to have recurrent and measurable disease according to Radiologic Evaluation Criteria in Solid Tumors (RECIST) version 1.1. A maximum of two prior lines of endocrine therapy are allowed, and patients cannot have previously received a cyclin-dependent kinase inhibitor. Patients with platinum-refractory disease are not allowed in any stage of the study. PRIMARY ENDPOINT: Investigator-assessed confirmed overall response rate, defined as the proportion of patients with a complete or partial response according to RECIST v1.1. SAMPLE SIZE: 40 to 100 patients will be included, depending on the results of the interim analysis. Patients will be included in Belgium, France and the Netherlands. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: Patient recruitment will be completed by the end of 2025 and reporting of the final study results will be done by the end of 2027. TRIAL REGISTRATION NUMBER: NCT05872204.

Immune checkpoint biology in health & disease: Immune checkpoint biology and autoimmunity in cancer patients.

Immune checkpoints (ICs) play a central role in maintaining immune homoeostasis. The discovery that tumours use this physiological mechanism to avoid elimination by the immune system, opened up avenues for therapeutic targeting of ICs as a novel way of treating cancer. However, this therapy a new array of autoimmune side effects, termed immune-related adverse events (irAEs). In this narrative review, we first recapitulate the physiological function of ICs that are approved targets for cancer immunotherapy (CTLA-4, PD-(L)1 and LAG-3), as the groundwork to critically discuss current knowledge on irAEs. Specifically, we summarize clinical aspects and examine a molecular classification and predisposing factors of irAEs. Finally, we discuss irAE treatment, particularly emphasizing how molecular knowledge is changing the current treatment paradigm.

Applications of single-cell multi-omics in liver cancer.

Primary liver cancer, more specifically hepatocellular carcinoma (HCC), remains a significant global health problem associated with increasing incidence and mortality. Clinical, biological, and molecular heterogeneity are well-known hallmarks of cancer and HCC is considered one of the most heterogeneous tumour types, displaying substantial inter-patient, intertumoural and intratumoural variability. This heterogeneity plays a pivotal role in hepatocarcinogenesis, metastasis, relapse and drug response or resistance. Unimodal single-cell sequencing techniques have already revolutionised our understanding of the different layers of molecular hierarchy in the tumour microenvironment of HCC. By highlighting the cellular heterogeneity and the intricate interactions among cancer, immune and stromal cells before and during treatment, these techniques have contributed to a deeper comprehension of tumour clonality, hematogenous spreading and the mechanisms of action of immune checkpoint inhibitors. However, major questions remain to be elucidated, with the identification of biomarkers predicting response or resistance to immunotherapy-based regimens representing an important unmet clinical need. Although the application of single-cell multi-omics in liver cancer research has been limited thus far, a revolution of individualised care for patients with HCC will only be possible by integrating various unimodal methods into multi-omics methodologies at the single-cell resolution. In this review, we will highlight the different established single-cell sequencing techniques and explore their biological and clinical impact on liver cancer research, while casting a glance at the future role of multi-omics in this dynamic and rapidly evolving field.

Comparison of the single-cell and single-nucleus hepatic myeloid landscape within decompensated cirrhosis patients.

Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.

Cabozantinib Induces Isolated Hyperbilirubinemia in Renal Cell Carcinoma Patients carrying the UGT1A1*28 Polymorphism.

BACKGROUND: Genetic variants of UGT1A1, involved in glucuronidation and clearance of bilirubin, are associated with reduced bilirubin metabolization and drug-induced isolated hyperbilirubinemia. We studied the impact of the UGT1A1*28 polymorphism on drug-induced isolated hyperbilirubinemia in metastatic renal cell carcinoma patients treated with pazopanib, cabozantinib, and axitinib. METHODS: We genotyped the UGT1A1*28 TA6/TA6-TA6/TA7-TA7/TA7 polymorphism and correlated with median baseline, on-treatment and peak bilirubin levels during therapy, incidence of grade-1- or -2 (G1/2)-hyperbilirubinemia and time-to-G1-hyperbilirubinemia. RESULTS: Of the 66 patients treated with pazopanib, 29 received axitinib and 28 cabozantinib upon progression. Median baseline bilirubin was higher in TA7/TA7-carriers versus TA6/TA6+TA6/TA7-carriers at start of pazopanib (P < .0001), cabozantinib (P < .0001), and axitinib (P = .007). During pazopanib therapy, median bilirubin increased 1.4-fold in TA7/TA7+TA6/TA7-carriers but not in TA6/TA6-carriers. On cabozantinib, bilirubin increased 1.5-fold in TA7/TA7-carriers but not in TA6/TA6+TA6/TA7-carriers. Axitinib did not increase bilirubin in any genotype. Peak bilirubin in TA7/TA7- versus TA6/TA6+TA6/TA7-carriers was higher on pazopanib (P < .0001) or cabozantinib (P < .0001). With pazopanib, G1-hyperbilirubinemia occurred in 57% of TA7/TA7- and 12% of TA6/TA6+TA6/TA7-carriers (P = .0009) and G2-hyperbilirubinemia in 36% and 6% of the patients, respectively (P = .004). On cabozantinib, G1-hyperbilirubinemia occurred in 100% of TA7/TA7- and 5% of TA6/TA6+TA6/TA7-carriers (P < .0001) and G2-hyperbilirubinemia in 33% and 0% of the patients, respectively (P = .04). On axitinib, no correlation between the genotypes and G1/2-hyperbilirubinemia was observed. CONCLUSION: We validate the previously described impact of the UGT1A1*28 polymorphism on isolated bilirubin increase on pazopanib. We report for the first time that cabozantinib also interferes with UGT1A1 and causes isolated bilirubin increase.

A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

Single-cell Atlas of Penile Cancer Reveals TP53 Mutations as a Driver of an Aggressive Phenotype, Irrespective of Human Papillomavirus Status, and Provides Clues for Treatment Personalization.

BACKGROUND AND OBJECTIVE: TP53 loss-of-function (TP53LOF) mutations might be a driver of poor prognosis and chemoresistance in both human papillomavirus (HPV)-independent (HPV-) and HPV-associated (HPV+) penile squamous cell carcinoma (PSCC). Here, we aim to describe transcriptomic differences in the PSCC microenvironment stratified by TP53LOF and HPV status. METHODS: We used single-cell RNA sequencing (scRNA-seq) and T-cell receptor sequencing to obtain a comprehensive atlas of the cellular architecture of PSCC. TP53LOF and HPV status were determined by targeted next-generation sequencing and sequencing HPV-DNA reads. Six HPV+ TP53 wild type (WT), six HPV- TP53WT, and four TP53LOF PSCC samples and six controls were included. Immunohistochemistry and hematoxylin-eosin confirmed the morphological context of the observed signatures. Prognostic differences between patient groups were validated in 541 PSCC patients using Kaplan-Meier survival estimates. KEY FINDINGS AND LIMITATIONS: Patients with aberrant p53 staining fare much worse than patients with either HPV- or HPV+ tumors and WT p53 expression. Using scRNA-seq, we revealed 65 cell subtypes within 83 682 cells. TP53LOF tumors exhibit a partial epithelial-to-mesenchymal transition, immune-excluded, angiogenic, and morphologically invasive environment, underlying their aggressive phenotype. HPV- TP53WT tumors show stemness and immune exhaustion. HPV+ TP53WT tumors mirror normal epithelial maturation with upregulation of antibody-drug-conjugate targets and activation of innate immunity. Inherent to the scRNA-seq analysis, low sample size is a limitation and validation of signatures in large PSCC cohorts is needed. CONCLUSIONS AND CLINICAL IMPLICATIONS: This first scRNA-seq atlas offers unprecedented in-depth insights into PSCC biology underlying prognostic differences based on TP53 and HPV status. Our findings provide clues for testing novel biomarker-driven therapies in PSCC. PATIENT SUMMARY: Here, we analyzed tissues of penile cancer at the level of individual cells, which helps us understand why patients who harbor a deactivating mutation in the TP53 gene do much worse than patients lacking such a mutation. Such an analysis may help us tailor future therapies based on TP53 gene mutations and human papillomavirus status of these tumors.

Transcriptomic characterization of the histopathological growth patterns in breast cancer liver metastases.

Metastatic breast cancer (mBC) remains incurable and liver metastases (LM) are observed in approximately 50% of all patients with mBC. In some cases, surgical resection of breast cancer liver metastases (BCLM) is associated with prolonged survival. However, there are currently no validated marker to identify these patients. The interactions between the metastatic cancer cells and the liver microenvironment result in two main histopathological growth patterns (HGP): replacement (r-HGP), characterized by a direct contact between the cancer cells and the hepatocytes, and desmoplastic (d-HGP), in which a fibrous rim surrounds the tumor cells. In patients who underwent resection of BCLM, the r-HGP is associated with a worse postoperative prognosis than the d-HGP. Here, we aim at unraveling the biological differences between these HGP within ten patients presenting both HGP within the same metastasis. The transcriptomic analyses reveal overexpression of genes involved in cell cycle, DNA repair, vessel co-option and cell motility in r-HGP while angiogenesis, wound healing, and several immune processes were found overexpressed in d-HGP LM. Understanding the biology of the LM could open avenues to refine treatment of BC patients with LM.

Respiratory complex I regulates dendritic cell maturation in explant model of human tumor immune microenvironment.

BACKGROUND: Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. METHODS: Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity. RESULTS: We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity. CONCLUSIONS: Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

PI3K/mTOR inhibition induces tumour microenvironment remodelling and sensitises pS6high uterine leiomyosarcoma to PD-1 blockade.

BACKGROUND: Uterine leiomyosarcomas (uLMS) are aggressive tumours with poor prognosis and limited treatment options. Although immune checkpoint blockade (ICB) has proven effective in some 'challenging-to-treat' cancers, clinical trials showed that uLMS do not respond to ICB. Emerging evidence suggests that aberrant PI3K/mTOR signalling can drive resistance to ICB. We therefore explored the relevance of the PI3K/mTOR pathway for ICB treatment in uLMS and explored pharmacological inhibition of this pathway to sensitise these tumours to ICB. METHODS: We performed an integrated multiomics analysis based on TCGA data to explore the correlation between PI3K/mTOR dysregulation and immune infiltration in 101 LMS. We assessed response to PI3K/mTOR inhibitors in immunodeficient and humanized uLMS patient-derived xenografts (PDXs) by evaluating tumour microenvironment modulation using multiplex immunofluorescence. We explored response to single-agent and a combination of PI3K/mTOR inhibitors with PD-1 blockade in humanized uLMS PDXs. We mapped intratumoural dynamics using single-cell RNA/TCR sequencing of serially collected biopsies. RESULTS: PI3K/mTOR over-activation (pS6high) associated with lymphocyte depletion and wound healing immune landscapes in (u)LMS, suggesting it contributes to immune evasion. In contrast, PI3K/mTOR inhibition induced profound tumour microenvironment remodelling in an ICB-resistant humanized uLMS PDX model, fostering adaptive anti-tumour immune responses. Indeed, PI3K/mTOR inhibition induced macrophage repolarisation towards an anti-tumourigenic phenotype and increased antigen presentation on dendritic and tumour cells, but also promoted infiltration of PD-1+ T cells displaying an exhausted phenotype. When combined with anti-PD-1, PI3K/mTOR inhibition led to partial or complete tumour responses, whereas no response to single-agent anti-PD-1 was observed. Combination therapy reinvigorated exhausted T cells and induced clonal hyper-expansion of a cytotoxic CD8+ T-cell population supported by a CD4+ Th1 niche. CONCLUSIONS: Our findings indicate that aberrant PI3K/mTOR pathway activation contributes to immune escape in uLMS and provides a rationale for combining PI3K/mTOR inhibition with ICB for the treatment of this patient population.

Tumoral and stromal hMENA isoforms impact tertiary lymphoid structure localization in lung cancer and predict immune checkpoint blockade response in patients with cancer.

BACKGROUND: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. METHODS: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. FINDINGS: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTßR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTßR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTßR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. INTERPRETATION: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. FUNDING: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, "Ricerca Corrente" granted by the Italian Ministry of Health.

Analysis of cell free DNA to predict outcome to bevacizumab therapy in colorectal cancer patients.

To predict outcome to combination bevacizumab (BVZ) therapy, we employed cell-free DNA (cfDNA) to determine chromosomal instability (CIN), nucleosome footprints (NF) and methylation profiles in metastatic colorectal cancer (mCRC) patients. Low-coverage whole-genome sequencing (LC-WGS) was performed on matched tumor and plasma samples, collected from 74 mCRC patients from the AC-ANGIOPREDICT Phase II trial (NCT01822444), and analysed for CIN and NFs. A validation cohort of plasma samples from the University Medical Center Mannheim (UMM) was similarly profiled. 61 AC-ANGIOPREDICT plasma samples collected before and following BVZ treatment were selected for targeted methylation sequencing. Using cfDNA CIN profiles, AC-ANGIOPREDICT samples were subtyped with 92.3% accuracy into low and high CIN clusters, with good concordance observed between matched plasma and tumor. Improved survival was observed in CIN-high patients. Plasma-based CIN clustering was validated in the UMM cohort. Methylation profiling identified differences in CIN-low vs. CIN high (AUC = 0.87). Moreover, significant methylation score decreases following BVZ was associated with improved outcome (p = 0.013). Analysis of CIN, NFs and methylation profiles from cfDNA in plasma samples facilitates stratification into CIN clusters which inform patient response to treatment.

Lower respiratory tract single-cell RNA sequencing and neutrophil extracellular trap profiling of COVID-19-associated pulmonary aspergillosis: a single centre, retrospective, observational study.

BACKGROUND: COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus affecting patients who are critically ill with COVID-19. The pathophysiology and the role of neutrophil extracellular traps (NETs) in this infection are largely unknown. We aimed to characterise the immune profile, with a focus on neutrophils and NET concentrations, of critically ill patients with COVID-19, with or without CAPA. METHODS: We conducted a single-centre, retrospective, observational study in two patient cohorts, both recruited at University Hospitals Leuven, Belgium. We included adults aged 18 years or older who were admitted to the intensive care unit because of COVID-19 between March 31, 2020, and May 18, 2021, and who were included in the previous Contagious trial (NCT04327570). We investigated the immune cellular landscape of CAPA versus COVID-19 only by performing single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid. Bronchoalveolar lavage immune cell fractions were compared between patients with CAPA and patients with COVID-19 only. Additionally, we determined lower respiratory tract NET concentrations using biochemical assays in patients aged 18 years and older who were admitted to the intensive care unit because of severe COVID-19 between March 15, 2020, and Dec 31, 2021, for whom bronchoalveolar lavage was available in the hospital biobank. Bronchoalveolar lavage NET concentrations were compared between patients with CAPA and patients with COVID-19 only and integrated with existing data on immune mediators in bronchoalveolar lavage and 90-day mortality. FINDINGS: We performed scRNA-seq of bronchoalveolar lavage on 43 samples from 39 patients, of whom 36 patients (30 male and six female; 14 with CAPA) were included in downstream analyses. We performed bronchoalveolar lavage NET analyses in 59 patients (46 male and 13 female), of whom 26 had CAPA. By scRNA-seq, patients with CAPA had significantly lower neutrophil fractions than patients with COVID-19 only (16% vs 33%; p=0·0020). The remaining neutrophils in patients with CAPA preferentially followed a hybrid maturation trajectory characterised by expression of genes linked to antigen presentation, with enhanced transcription of antifungal effector pathways. Patients with CAPA also showed depletion of mucosal-associated invariant T cells, reduced T helper 1 and T helper 17 differentiation, and transcriptional defects in specific aspects of antifungal immunity in macrophages and monocytes. We observed increased formation of NETs in patients with CAPA compared with patients with COVID-19 only (DNA complexed with citrullinated histone H3 median 15 898 ng/mL [IQR 4588-86 419] vs 7062 ng/mL [775-14 088]; p=0·042), thereby explaining decreased neutrophil fractions by scRNA-seq. Low bronchoalveolar lavage NET concentrations were associated with increased 90-day mortality in patients with CAPA. INTERPRETATION: Qualitative and quantitative disturbances in monocyte, macrophage, B-cell, and T-cell populations could predispose patients with severe COVID-19 to develop CAPA. Hybrid neutrophils form a specialised response to CAPA, and an adequate neutrophil response to CAPA is a major determinant for survival in these patients. Therefore, measuring bronchoalveolar lavage NETs could have diagnostic and prognostic value in patients with CAPA. Clinicians should be wary of aspergillosis when using immunomodulatory therapy that might inhibit NETosis to treat patients with severe COVID-19. FUNDING: Research Foundation Flanders, KU Leuven, UZ Leuven, VIB, the Fundação para a Ciência e a Tecnologia, the European Regional Development Fund, la Caixa Foundation, the Flemish Government, and Horizon 2020.

Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome.

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Genome-wide association analyses of ovarian cancer patients undergoing primary debulking surgery identify candidate genes for residual disease.

Survival from ovarian cancer depends on the resection status after primary surgery. We performed genome-wide association analyses for resection status of 7705 ovarian cancer patients, including 4954 with high-grade serous carcinoma (HGSOC), to identify variants associated with residual disease. The most significant association with resection status was observed for rs72845444, upstream of MGMT, in HGSOC (p = 3.9 × 10-8). In gene-based analyses, PPP2R5C was the most strongly associated gene in HGSOC after stage adjustment. In an independent set of 378 ovarian tumours from the AGO-OVAR 11 study, variants near MGMT and PPP2R5C correlated with methylation and transcript levels, and PPP2R5C mRNA levels predicted progression-free survival in patients with residual disease. MGMT encodes a DNA repair enzyme, and PPP2R5C encodes the B56γ subunit of the PP2A tumour suppressor. Our results link heritable variation at these two loci with resection status in HGSOC.