Aging is the greatest risk factor for nearly all major chronic diseases, including cardiovascular diseases, cancer, Alzheimer’s and other neurodegenerative diseases of aging. Age-related impairment of immune function (immunosenescence) is one important cause of age-related morbidity and mortality, which may extend beyond its role in infectious disease. One aspect of immunosenescence that has received less attention is age-related natural killer (NK) cell dysfunction, characterized by reduced cytokine secretion and decreased target cell cytotoxicity, accompanied by and despite an increase in NK cell numbers with age. Moreover, recent studies have revealed that NK cells are the central actors in the immunosurveillance of senescent cells, whose age-related accumulation is itself a probable contributor to the chronic sterile low-grade inflammation developed with aging (“inflammaging”). NK cell dysfunction is therefore implicated in the increasing burden of infection, malignancy, inflammatory disorders, and senescent cells with age. This review will focus on recent advances and open questions in understanding the interplay between systemic inflammation, senescence burden, and NK cell dysfunction in the context of aging. Understanding the factors driving and enforcing NK cell aging may potentially lead to therapies countering age-related diseases and underlying drivers of the biological aging process itself.

The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care.

Background: The accurate identification of high-grade dysplasia (HGD) or invasive cancer (advanced neoplasia, AN) in pancreatic cystic lesions (PCLs) is needed to identify PCLs that warrant surgical intervention. Cytologic evaluation of cyst fluid is widely-used but has poor sensitivity. We previously found that activity of the lysosomal serine protease tripeptidyl peptidase 1 (TPP1) is associated with mucinous PCLs that harbor AN (AUC 0.72). We aimed to identify additional functional biomarkers that would improve performance of our classifier for dysplastic grade in mucinous PCLs. Methods: We used a combination of global protease activity profiling and shotgun proteomics to identify differentiating markers between PCLs with low-grade dysplasia (LGD, n=3) and HGD (n=3). Candidate biomarkers were validated in an independent cohort of 28 clinically-annotated mucinous PCLs (HGD n=12, LGD n=16), by measuring both protein concentration (using ELISA) and enzymatic activity (using internally-quenched fluorescent substrates). We used a nested cross-validation approach to iteratively split our cohort into training and validation sets to predict HGD. Model accuracy was evaluated using area under the curve (AUC), sensitivity, specificity, and accuracy. Results: We identified 8 proteins that were highly abundant in mucinous PCLs with HGD. Among these were the inflammatory enzymes myeloperoxidase (MPO) and neutrophil elastase (ELANE), suggesting that differential activity levels could be exploited that would minimize fluid requirements and cost. Among 28 mucinous PCLs, a composite score that included both activity and mass of MPO performed the best for HGD in mucinous cysts (AUC 0.955, sensitivity 98%, specificity 93%). ELANE activity (AUC 0.76, sensitivity 69%, specificity 59%) and TPP1 activity (AUC 0.765, sensitivity 71%, specificity 74%) had modest performance. Conclusions: Our functional biomarkers accurately classified HGD among mucinous cysts. Functional biomarkers that require low sample volumes (<10μl) of cyst fluid have the potential to substantially improve clinical decision-making for patients with PCLs. Citation Format: Francesco Caiazza, Andre Lourenco, Patricia Conroy, Thomas Hoffmann, Sam L. Ivry, Tyler York, Gina Zhu, Audrey Mustoe, Anthony J. O'Donoghue, Charles S. Craik, Kimberly Kirkwood. Using functional biomarkers to accurately predict advanced neoplasia in pancreatic cystic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2226.

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