Silvia Guil-Luna

Pregnancy-associated breast cancer (PABC) is an aggressive malignancy affecting young women during gestation, lactation, or the post-weaning period. Despite its clinical significance and poor prognosis, how these physiological stages shape tumor biology remains poorly understood. Here, we integrate reproductive context with molecular and immune profiling to define shared and stage-specific features of PABC and uncover potential therapeutic vulnerabilities. We conducted BC360 NanoString gene expression analysis and CIBERSORTx immune deconvolution in 106 breast cancer cases, including 57 PABC patients stratified by diagnosis stage (gestation, breastfeeding, or post-weaning) and 49 non-PABC controls. Our study identified a shared aggressive signature across all PABC cases, marked by increased proliferation and impaired DNA repair. Notably, breastfeeding patients (PABC-BF) emerged as a distinct subset, displaying an immune-inflamed tumor profile characterized by elevated inflammatory chemokines, enriched CD8⁺ T-cell and regulatory T-cell infiltration, and prominent TIGIT inhibitory checkpoint upregulation. These findings reveal lactation as a physiological framework that shapes tumor immunity, defining a distinct tumor-immune landscape which warrants further investigation for potential therapeutic approaches.

Pregnancy-associated breast cancer (PABC), diagnosed during or shortly after pregnancy, is a challenging entity with an aggressive biology and poor prognosis. This study analyzed the clinicopathological characteristics and gene expression profile of 33 PABC and 26 non-PABC patients using the nCounter BC360 Panel (NanoString). Notably, PABC showed a higher prevalence of basal-like tumors than non-PABC (48.48% vs 15.38%, p = 0.012) and displayed 73 differentially expressed genes (e.g., DEPDC1, CCNA2, PSAT1, CDKN3, and FAM83D), enriched in DNA repair and cell proliferation pathways. Through the PPI network, we also identified a cluster of cell-cycle regulation genes like MYC, FOXM1, or PTEN. Interestingly, differences emerged when comparing patients diagnosed during gestation (PABC-GS) and the postpartum period (PABC-PP), with PABC-PP showing increased expression of immune-related genes, including PD-1, and greater immune cell infiltration (Tregs, macrophages, neutrophils, B-cells). These findings suggest an enhanced proliferative capacity and impaired DNA repair in PABC, and underscore the role of immune infiltration in postpartum cases; providing insights into its aggressive nature and potential targets.

Background/Objectives: Gut microbiota interacts with nutrients, which may be relevant to assigning a microbial signature to colorectal cancer (CRC). We aim to evaluate the potential of gut microbiota combined with dietary habits in the early detection of pathological findings related to CRC in the course of a screening program. Methodology: The colonoscopy performed on 152 subjects positive for fecal occult blood test showed that 6 subjects had adenocarcinoma, 123 had polyps, and 23 subjects had no pathological findings. Gut microbiota was analyzed by 16S metagenomic. Caret package was used to build the classification models in R. Results: Random forest (RF) classifier models were used to test the potential of gut microbiota alone or combined with dietary habits as a biomarker to discern between individuals with CRC-related lesions (polyps or adenocarcinoma) versus individuals without pathological findings. RF classifier models yielded an area under the curve of 0.790 using gut microbiota data, 0.710 using dietary habits data, and 0.804 in the combined model including gut microbiota and dietary habits data. The abundance of Suterella, Oscillospirales, Proteobacteria, and Burkholderiales was highly discriminant between groups, together with the consumption of fruit and vegetables and the consumption of carbonated and/or sweetened beverages. Conclusions: Our results suggest that the interaction between gut microbiota and dietary habits is relevant when a microbial signature is used as a marker in CRC. Moreover, gut microbiota signature and information about the dietary habits of the individuals seem to be important for improving screening programs for the early detection of CRC.

Background: Pancreatic cancer is the most lethal cancer with a dismal prognosis mainly due to diagnosis at advanced stage and ineffective treatments. CA19-9 levels and computed tomography (CT) imaging are the main standard criteria for evaluating disease progression and treatment response. In this study we explored liquid biopsy-based epigenetic biomarkers for prognosis and monitoring disease in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). Methods: Plasma samples were collected from 44 mPDAC patients at the time of diagnosis, and in 15 of them, additional samples were obtained during follow-up of the disease. After cell-free DNA (cfDNA), isolation circulating levels of methylated NPTX2, SPARC, BMP3, SFRP1 and TFPI2 genes were measured using digital droplet PCR (ddPCR). BEAMing technique was performed for quantitation of RAS mutations in cfDNA, and CA19-9 was measured using standard techniques. Results: NPTX2 was the most highly and frequently methylated gene in cfDNA samples from mPDAC patients. Higher circulating NPTX2 methylation levels at diagnosis were associated with poor prognosis and efficiently stratified patients for prediction of overall survival (6.06% cut-off, p = 0.0067). Dynamics of circulating NPTX2 methylation levels correlated with disease progression and response to therapy and predicted better than CA19-9 the evolution of disease in mPDAC patients. Remarkably, in many cases the disease progression detected by CT scan was anticipated by an increase in circulating NPTX2 methylation levels. Conclusions: Our study supports circulating NPTX2 methylation levels as a promising liquid biopsy-based clinical tool for non-invasive prognosis, monitoring disease evolution and response to treatment in mPDAC patients.

S‐nitrosoglutathione reductase (GSNOR) is a denitrosylase enzyme that has been suggested to play a tumor suppressor role, although the mechanisms responsible are still largely unclear. In this study, we show that GSNOR deficiency in tumors is associated with poor prognostic histopathological features and poor survival in patients with colorectal cancer (CRC). GSNOR‐low tumors were characterized by an immunosuppressive microenvironment with exclusion of cytotoxic CD8⁺ T cells. Notably, GSNOR‐low tumors exhibited an immune evasive proteomic signature along with an altered energy metabolism characterized by impaired oxidative phosphorylation (OXPHOS) and energetic dependence on glycolytic activity. CRISPR‐Cas9‐mediated generation of GSNOR gene knockout (KO) CRC cells confirmed in vitro and in vivo that GSNOR‐deficiency conferred higher tumorigenic and tumor‐initiating capacities. Moreover, GSNOR‐KO cells possessed enhanced immune evasive properties and resistance to immunotherapy, as revealed following xenografting them into humanized mouse models. Importantly, GSNOR‐KO cells were characterized by a metabolic shift from OXPHOS to glycolysis to produce energy, as indicated by increased lactate secretion, higher sensitivity to 2‐deoxyglucose (2DG), and a fragmented mitochondrial network. Real‐time metabolic analysis revealed that GSNOR‐KO cells operated close to their maximal glycolytic rate, as a compensation for lower OXPHOS levels, explaining their higher sensitivity to 2DG. Remarkably, this higher susceptibility to glycolysis inhibition with 2DG was validated in patient‐derived xenografts and organoids from clinical GSNOR‐low tumors. In conclusion, our data support the idea that metabolic reprogramming induced by GSNOR deficiency is an important mechanism for tumor progression and immune evasion in CRC and that the metabolic vulnerabilities associated with the deficiency of this denitrosylase can be exploited therapeutically. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Introduction: Liquid biopsy based on the analysis of circulating cell-free DNA (cfDNA), as well as on detection of point mutations by digital droplet PCR (ddPCR), has revolutionized the research in oncology. In recent years, this technique has been pioneering in veterinary medicine since it is a minimally invasive approach with very promising results for characterization of tumors.Methods: The aim of this study was, firstly, to analyze the concentration and the fragmentation pattern of cfDNA of dogs with mammary tumors (n= 36) and healthy dogs (n= 5) and its correlation with clinicopathological data. Secondly, analysis ofTP53gene expression and the point mutation in the codon 245 were performed in cfDNA and in tumor tissues to assess their potential as plasma biomarkers.Results and discussion: Our results highlighted that those dogs with worse clinicopathological characteristics (simple or undifferentiated carcinomas, higher histological grade and presence of peritumoral inflammation) shown higher cfDNA concentration and higher concentrations of short-fragments (TP53gene could be detected neither in plasma nor tumor tissue, an increasedTP53expression was detected in animals with tumors bearing malignant characteristics. Finally, a high concordance withTP53gene expression in plasma and tumor tissue and cfDNA concentration was also found. The results derived from this work confirm the valuable potential of cfDNA and its fragments, as well as the analysis ofTP53expression in plasma as useful liquid biomarkers for clinical application in veterinary oncology.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a highly inflammatory microenvironment and liquid biopsy has emerged as a promising tool for the noninvasive analysis of this tumor. In this study, plasma was obtained from 58 metastatic PDAC patients, and neutrophil–lymphocyte ratio (NLR), platelet–lymphocyte ratio (PLR), circulating cell-free DNA (cfDNA) concentration, and circulating RAS mutation were determined. We found that NLR was significantly associated with both overall survival (OS) and progression-free survival. Remarkably, NLR was an independent risk factor for poor OS. Moreover, NLR and PLR positively correlated, and combination of both inflammatory markers significantly improved the prognostic stratification of metastatic PDAC patients. NLR also showed a positive correlation with cfDNA levels and RAS mutant allelic fraction (MAF). Besides, we found that neutrophil activation contributed to cfDNA content in the plasma of metastatic PDAC patients. Finally, a multi-parameter prognosis model was designed by combining NLR, PLR, cfDNA levels, RAS mutation, RAS MAF, and CA19-9, which performs as a promising tool to predict the prognosis of metastatic PDAC patients. In conclusion, our study supports the idea that the use of systemic inflammatory markers along with circulating tumor-specific markers may constitute a valuable tool for the clinical management of metastatic PDAC patients.