Rational thresholding of circulating tumor DNA concentration for improved surveillance of metastatic breast cancer.

BACKGROUND: The use of circulating tumor DNA (ctDNA) concentration for metastatic cancer surveillance is promising, but uncertainty remains about cut-offs with clinical validity. MATERIALS AND METHODS: This observational study recruited 136 subjects with advanced metastatic breast cancer (irrespective of ERBB2/hormone receptor status) for sequencing of their primary tumor in search for PIK3CA hotspot variants amenable for monitoring by droplet digital PCR (ddPCR). The study analyzed 341 on-treatment samples from 19 patients with PIK3CA variants H1047R or E545K enrolled for long-term (median 85 weeks, range 13-125 weeks), frequent (every 3-5 weeks, median of 14 time points per subject, range 2-29) blood sampling for ctDNA quantification by ddPCR, orthogonally validated by deep sequencing. The diagnostic accuracy of ctDNA versus cancer antigen 15-3 (CA15-3) concentrations to predict disease progression within 12 weeks was investigated using receiver operating characteristic (ROC) analysis. Likelihood ratios were used for rational selection of ctDNA result intervals. RESULTS: ctDNA [area under the ROC curve (AUC) 0.848, 95% confidence interval (CI) 0.791-0.895] showed superior diagnostic performance than CA15-3 (AUC 0.670, 95% CI 0.601-0.735, P < 0.001) to predict clinical progression within 12 weeks. ctDNA levels below 10 mutant allele copies/ml had high negative predictive value (88%), while levels above 100 copies/ml detected 64% of progressions 10 weeks earlier versus standard of care. Logistic regression analysis indicated complementary value of ctDNA and the presence of two consecutive CA15-3 rises, resulting in a model with 86% (95% CI 74% to 93%) positive predictive value and a clinically meaningful result in 89% of blood draws. CONCLUSIONS: Intensive ctDNA quantification improves metastatic breast cancer surveillance and enables individualized risk-based scheduling of clinical care.

Endothelium-dependent hyperpolarization in small gastric arteries.

OBJECTIVE: In many blood vessels, stimulation of the endothelium with various vasoactive substances induces, besides the nitric oxide (NO) and prostacyclin pathways, a third mechanism evoking dilatation. It is based on hyperpolarization of the vascular smooth muscle cell membrane. In the present study, we investigated the existence of endothelium-dependent hyperpolarization in small gastric arteries of the rat and explored its underlying mechanism. METHODS: Membrane potentials were recorded by conventional microelectrode techniques in isolated segments of small gastric arteries, the normalized diameter of which was determined from the passive wall tension-internal circumference characteristics as measured with a myograph. RESULTS: After blocking NO and prostaglandin synthesis, application of acetylcholine (3 x 10(-7) M) resulted in a membrane hyperpolarization in endothelium intact but not in endothelium-denuded arteries. This membrane potential change was increased by pre-exposure to a low concentration (30 microM) of Ba2+, which selectively inhibits inward rectifying potassium channels. Moreover, the acetylcholine-induced hyperpolarization was unaffected by additional pre-exposure to high concentrations (0.5 mM) of the Na/K-ATPase inhibitor ouabain, which by itself caused a secondary slow endothelium-independent hyperpolarization after an initial peak depolarization. CONCLUSIONS: We conclude that acetylcholine produces endothelium-dependent hyperpolarization in gastric small arteries, which does not rely on activation of smooth muscle cell inward rectifying K+ channels or Na/K pumps, and might prove to be another important regulator of gastric mucosal blood flow.

Three arginine residues in apolipoprotein A-I are critical for activation of lecithin:cholesterol acyltransferase.

Previous studies have suggested that the helical repeat formed by residues 143;-164 of apolipoprotein A-I (apoA-I) contributes to lecithin:cholesterol acyltransferase (LCAT) activation. To identify specific polar residues involved in this process, we examined residue conservation and topology of apoA-I from all known species. We observed that the hydrophobic/hydrophilic interface of helix 143;-164 contains a cluster of three strictly conserved arginine residues (R149, R153, and R160), and that these residues create the only significant positive electrostatic potential around apoA-I. To test the importance of R149, R153, and R160 in LCAT activation, we generated a series of mutant proteins. These had fluorescence emission, secondary structure, and lipid-binding properties comparable to those of wild-type apoA-I. Mutation of conserved residues R149, R153, and R160 drastically decreased LCAT activity on lipid-protein complexes, whereas control mutations (E146Q, D150N, D157N, R171Q, and A175R) did not decrease LCAT activity by more than 55%. The markedly decreased activities of mutants R149, R153, and R160 resulted from a decrease in the maximal reaction velocity V(max) because the apparent Michaelis-Menten constant K(m) values were similar for the mutant and wild-type apoA-I proteins. These data suggest that R149, R153, and R160 participate in apoA-I-mediated activation of LCAT, and support the "belt" model for discoidal rHDL. In this model, residues R149, R153, and R160 do not form salt bridges with the antiparallel apoA-I monomer, but instead are pointing toward the surface of the disc, enabling interactions with LCAT. - Roosbeek, S., B. Vanloo, N. Duverger, H. Caster, J. Breyne, I. De Beun, H. Patel, J. Vandekerckhove, C. Shoulders, M. Rosseneu, and F. Peelman. Three arginine residues in apolipoportein A-I are critical for activation of lecithin:cholesterol acyltransferase J. Lipid Res. 2001. 42: 31;-40.