Anesthesia Research Centers

BACKGROUND & AIMS: Within the Liver Imaging Reporting and Data System (LI-RADS), LI-RADS Malignant (LR-M) lesions remain diagnostically challenging: imaging indicates malignancy but often fails to distinguish hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma (iCCA). Established serum-based tools such as GALAD are optimized for HCC but are not designed to resolve entity ambiguity within LR-M. In this pilot proof-of-concept study, we investigated whether small extracellular vesicle (EV) integrated with routine serological markers could resolve LR-M lesion situation.

METHODS: A rare LR-M cohort (HCC = 25, iCCA = 25) was evaluated using LASSO logistic regression and principal component analysis (PCA) to prioritize informative biomarkers. Hybrid models combined CD9+CD133/2+ and CD81+CD133/2+ EVs with alkaline phosphatase, CRP, CA19-9, and optional AFP. Models were internally validated using an 80:20 train-test split and assessed with bootstrap and Monte-Carlo perturbation (±5-20%).

RESULTS: Individual markers demonstrated limited discrimination (AUROC ≤ 0.82). Hybrid logistic regression models showed moderate internal discrimination (AUROC 0.86 with AFP; 0.91 without AFP). Translation into additive scoring systems using ROC-Youden-derived cut-offs yielded high internal AUROC estimates (e.g., 0.95-0.96), though these remain internally validated. A simplified five-point PRISM score retained comparable discriminatory performance (AUROC ∼0.91). In exploratory survival analysis among iCCA patients (n = 25), those with above-median CD9+CD133/2+ EV levels (n = 13) had shorter overall survival (median 91 vs. 389 days; HR 2.80, 95% CI 1.16-6.74; p = 0.005).

CONCLUSIONS: Integrated EV and serological profiling may enable minimally invasive differentiation between HCC and iCCA within LR-M lesions. By transforming ML-guided model discovery into a clinically interpretable paper-and-pencil additive score, we illustrate a translational pathway from computational discovery to practical application.

IMPACT AND IMPLICATIONS: This study provides a scientific rationale for integrating small extracellular vesicle (EV) phenotyping with conventional serum biomarkers to improve minimally invasive differentiation between hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) in LR-M lesions. By translating a machine learning (ML)_derived hybrid model into a simple additive scoring system, we demonstrate how ML can yield clinically interpretable tools that bridge computational discovery and bedside application. The findings are particularly relevant for hepatologists, oncologists, and radiologists managing patients in whom imaging remains indeterminate and biopsy carries procedural risk. The five-point model can be applied using routine laboratory values, providing a paper-and-pencil diagnostic aid that requires no specialized software or hardware. While external multicenter validation is necessary given the cohort size, this approach illustrates how transparent, interpretable machine learning can support precision diagnostics in primary liver cancer.

Lower urinary tract symptoms (LUTS) are prevalent among the aging population, and current medications offer limited efficacy. Improved therapies require a better understanding of the mechanisms underlying LUTS. Abnormal extracellular ATP levels and altered purinergic contractility have been reported in patients with LUTS, suggesting dysregulation of the purinergic pathways. CD39/ENTPD1 is a major regulator of purinergic metabolism, and patients with ENTPD1 loss-of-function mutations exhibit bladder hypomotility and incontinence, suggesting critical roles for ENTPD1 in bladder function. We have tested this hypothesis in an overexpression mouse model with human ENTPD1 knock-in (hCD39TG), in a hemizygous Entpd1 mouse model (Entpd1+/-) mimicking LUTS patients with impaired ATP hydrolysis, and in a homozygous Entpd1-deleted mouse model (Entpd1-/-) mimicking the complete ENTPD1 loss-of-function observed in patients. We have demonstrated that ENTPD1 dysregulation leads to profound abnormalities in bladder voiding phenotypes and urodynamics, along with impaired BSM contractility and altered purinergic receptor signaling. Furthermore, we have found that modulation of downstream P2Y12/adenosine A2b receptor signaling partially or fully restores normal bladder function. Finally, we have identified expression of ENTPD1 and associated purinergic proteins in human BSM cells, underscoring the critical role of ENTPD1 in human bladder function and highlighting its translational potential for the treatment of LUTS.

Infection with Mycobacterium tuberculosis can cause diverse lesions, such as necrotic pneumonia, which can contribute to tuberculosis progression and transmission between individuals. Despite advances in understanding the role of ATP-gated P2RX7 ion channels in the development of severe forms of the disease, the regulation of this important signaling pathway remains unclear. Herein, we show that the ectonucleotidase CD39 plays an essential regulatory role in tuberculosis progression by preventing lung tissue damage, bacterial dissemination, and excessive inflammatory responses. Mechanistically, through its enzymatic activity on the cellular surface, CD39 protects infected macrophages from undergoing necrotic death mediated by P2RX7 activation. Cell-intrinsic CD39 expression also hinders the establishment of other myeloid cells, such as neutrophils, in the infected lung. We proposed that, by protecting infected macrophages from P2RX7-mediated cell death and bacterial dissemination in the lung tissue, CD39 prevents the development of necrotic lesions. Altogether, these findings uncover a significant role for CD39 as an essential component of the molecular regulation underlying the development of severe tuberculosis.

Francesco Di Virgilio, one of the giants of purinergic signalling, died suddenly on September 22, 2024, which is an immense loss to so many friends and colleagues and especially to his beloved wife, Dorianna. Here, 1 year on, we pay tribute to him and his immense contribution to our field. Francesco graduated in medicine from the University of Padova, Italy, in 1979, with what became a lifelong interest in inflammation. He then held post-doctoral positions at University College London, Padova University, and Columbia University, New York, where he became acquainted with P2X7 receptors. He then returned to Padova as an Associate Professor of Molecular Pathology before moving to the University of Ferrara in 1992, where he set up a world-leading lab that studied the roles and underlying cellular mechanisms of the action of P2X7 receptors in inflammatory pathologies. Francesco published over 370 peer-reviewed articles, which have been cited > 35,000 times, giving him an H-index > 100. In addition, he filed several patents related to purinergic signalling. He also collaborated extensively, both within the University of Ferrara and worldwide, including in universities in the UK, Spain, Germany, the USA, and Brazil. Francesco was a man of great passion and intellect, who possessed scientific vision, intuition, and integrity, and he became the go-to world expert on P2X7 receptors and inflammation. We have lost a giant in the field and a dear friend, but he leaves behind an exceptional body of work, an outstanding legacy, and many friends who will miss him.

ENTPD5 and ENTPD6 are members of the CD39-ectonucleoside triphosphate diphosphohydrolase (CD39-ENTPD) family, which play an important role in modulating the purinergic signaling pathway. Most of the knowledge in this area has been obtained by studying CD39/ENTPD1, the prototype member of this family, and evaluating the translational potential by either treating inflammation directly with recombinant proteins or by using antagonists to elicit immune responses in cancer. ENTPD5 and ENTPD6, "orphan-type" ectonucleotidases, are understudied to date, although both are expressed at high levels in various tissues, where they appear involved in regulating signal transduction, cellular energy, and metabolism. ENTPD5 is abnormally overexpressed in several types of malignancies, including prostate, liver, lung, and ovarian cancers. ENTPD5 appears to promote protein glycosylation and folding in part by regulating UDP and UMP levels, thereby enhancing the survival and proliferation of somatic or cancer cells. As such, ENTPD5 has been considered a potential proto-oncogene and a therapeutic target in cancer treatment. In contrast, despite comparable functionality, the related ENTPD6 shows relatively stable expression across tissues in both normal and pathological conditions, with specific roles in cancer yet unclear. This review provides a comprehensive overview of these two understudied ectoenzymes, detailing their shared molecular structures and control of purinergic signal transduction. In addition, we explore different patterns of tissue and organelle expression of these ecto-enzymes and propose relevance to the modulation of cellular metabolism, as would be important in cancer. We review the sometimes conflicting evidence from experimental animal models and propose potential future clinical applications. This review offers insights into the roles of this distinct duo of ENTPD family members to support future basic and translational research in this field.