OBJECTIVE: The American Gastroenterological Association (AGA) recently reported evidence-based guidelines for the management of asymptomatic neoplastic pancreatic cysts. These guidelines advocate a higher threshold for surgical resection than prior guidelines and imaging surveillance for a considerable number of patients with pancreatic cysts. The aims of this study were to assess the accuracy of the AGA guidelines in detecting advanced neoplasia and present an alternative approach to pancreatic cysts.
METHODS: The study population consisted of 225 patients who underwent EUS-guided FNA for pancreatic cysts between January 2014 and May 2015. For each patient, clinical findings, EUS features, cytopathology results, carcinoembryonic antigen analysis, and molecular testing of pancreatic cyst fluid were reviewed. Molecular testing included the assessment of hotspot mutations and deletions for KRAS, GNAS, VHL, TP53, PIK3CA, and PTEN.
RESULTS: Diagnostic pathology results were available for 41 patients (18%), with 13 (6%) harboring advanced neoplasia. Among these cases, the AGA guidelines identified advanced neoplasia with 62% sensitivity, 79% specificity, 57% positive predictive value, and 82% negative predictive value. Moreover, the AGA guidelines missed 45% of intraductal papillary mucinous neoplasms with adenocarcinoma or high-grade dysplasia. For cases without confirmatory pathology, 27 of 184 patients (15%) with serous cystadenomas (SCAs) based on EUS findings and/or VHL alterations would continue magnetic resonance imaging (MRI) surveillance. In comparison, a novel algorithmic pathway using molecular testing of pancreatic cyst fluid detected advanced neoplasias with 100% sensitivity, 90% specificity, 79% positive predictive value, and 100% negative predictive value.
CONCLUSIONS: The AGA guidelines were inaccurate in detecting pancreatic cysts with advanced neoplasia. Furthermore, because the AGA guidelines manage all neoplastic cysts similarly, patients with SCAs will continue to undergo unnecessary MRI surveillance. The results of an alternative approach with integrative molecular testing are encouraging but require further validation.

Stimulation of the beta(2)-adrenergic receptor (beta(2)AR) in human embryonic kidney (HEK) 293 cells causes a transient activation of Extracellular Signal-Regulated Kinase (ERK) 1/2. One of the mechanisms proposed for this activation is a PKA-mediated phosphorylation of the beta(2)AR that switches receptor coupling from G(s) to G(i) and triggers internalization of the receptor. To examine these phenomena, we characterized agonist activation of ERK1/2 in HEK293 cells by the endogenous beta(2)AR and in HEK293 cells stably overexpressing either the wild-type beta(2)AR or a substitution mutant beta(2)AR (PKA(-)) that lacks the cyclic AMP-dependent protein kinase (PKA) consensus phosphorylation sites (S261A, S262A and S345A, S346A). As the baseline, we established that epinephrine stimulation of the endogenous beta(2)AR in HEK293 cells (20-30 fmol/mg) caused a rapid and transient activation of ERK1/2 with an EC(50) of 5 to 6 nM. In contrast, the potency of epinephrine stimulation of ERK1/2 in cells stably overexpressing WTbeta(2)AR and PKA(-) (2-4 pmol of beta(2)AR/mg) was increased by over 100-fold relative to HEK293 cells, the EC(50) values being 20 to 60 pM. The nearly identical 100-fold shift in EC(50) for ERK1/2 activation in the PKA(-) and WTbeta(2)AR relative to that in the HEK293 showed that the PKA(-) are fully capable of activating ERK1/2. We also found maximal activation of ERK1/2 in the overexpressing cell lines at concentrations of epinephrine that cause no internalization (i.e., the EC(50) for internalization was 75 nM). Pertussis toxin pretreatment caused only a weak inhibition of epinephrine activation of ERK1/2 in the HEK293 (7-16%) and no inhibition in the PKA(-) cells. Finally we found that the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (10 microM) caused a >90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of beta(2)AR activation of ERK1/2 does not require PKA phosphorylation of the beta(2)AR, receptor internalization or switching from activation of G(s) to G(i) but clearly requires activation of a Src family member that may be downstream of PKA.

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