Vasostatin-1: A novel circulating biomarker for ileal and pancreatic neuroendocrine neoplasms

<div><p>Background</p><p>Chromogranin A (CgA) is a plasma biomarker widely used in the follow-up of patients with neuroendocrine neoplasms (NENs). However, its accuracy as a tumor biomarker is relatively low because plasma CgA can increase also in patients with other diseases or in subjects treated with proton-pump inhibitors (PPIs), a class of widely-used drugs.</p><p>Methods</p><p>In the attempt to identify a more reliable biomarker for NENs, we investigated, by ELISA, the circulating levels of full-length CgA (CgA_1-439) and of various CgA-derived fragments in 17 patients with ileal or pancreatic NENs, 10 healthy controls, and 21 healthy volunteers before and after treatment with PPIs.</p><p>Results</p><p>Patients with ileal or pancreatic NENs showed increased plasma levels of total-CgA and CgA_1-76 fragment (vasostatin-1, VS-1) compared to controls [median (25^th-75^th-percentiles); total-CgA: 1.85 nM (1.01–4.28) <i>vs</i> 0.75 nM (0.52–0.89), <i>p</i> = 0.004; VS-1: 2.76 nM (1.09–7.10) <i>vs</i> 0.29 nM (0.26–0.32), <i>p</i><0.001, respectively], but not of CgA_1-439 or CgA_1-373 fragment. VS-1 positively correlated with total-CgA (r = 0.65, <i>p</i><0.001). The Receiver Operating Characteristic area under the curve was 0.9935 for VS-1 and 0.8824 for total-CgA (<i>p</i> = 0.067). Treatment of patients with somatostatin analogues decreased both total-CgA and VS-1. In contrast, administration of PPIs increased the plasma levels of total-CgA, but not of VS-1.</p><p>Conclusion</p><p>These findings suggest that plasma VS-1 is a novel biomarker for ileal and pancreatic NENs. Considering that VS-1 is a well-defined fragment not induced by proton-pump inhibitors, this polypeptide might represent a biomarker for NENs diagnosis and follow-up more accurate and easier to standardize than CgA.</p></div

Effect of proton pump inhibitors on the plasma levels of total-CgA, VS-1, CgA_1-439 and CgA_1-373.

<p>**(<i>p</i> < 0.01), by two-tailed paired t-test.</p

Plasma levels of VS-1 and total-CgA, VS-1/total-CgA, CgA_1-373/total-CgA, and CgA_1-439/total-CgA ratios (relative levels) in healthy subjects and patients with ileal and pancreatic NENs (case-control analysis by site).

<p>Box-plots with median (middle line), 75^th percentile (top line) and 25^th percentile (bottom line). The top and bottom whiskers represent the upper and lower adjacent values, respectively. Values outside the whiskers are plotted individually (circles). Ileal NENs (n = 10); pancreatic NENs (n = 7); controls (n = 10). * (<i>p</i> < 0.05), **(<i>p</i> < 0.01), by analysis of covariance.</p

Schematic representation of the ELISAs used to detect CgA and its fragments.

<p>Upper panel—Schematic representation of human full-length CgA (439-residues long, CgA_1-439): Q₇₆ and R₃₇₃ cleavage sites are indicated; vasostatin-1 (CgA_1-76, VS-1), catestatin (CgA_352-372) and serpinin regions (CgA _410–436) are boxed. Lower panel—Schematic representation of the *439-, *373-, *76-, and *total-CgA-ELISAs (sandwich ELISA). The epitope location of mAb B4E11 and 5A8 (capturing antibodies), and α439, α373, α76 and αFRs (detecting antibodies) is shown. The rational for using different capturing antibodies (mAb B4E11 and mAb 5A8) in the different assays relies on the fact that, in the case of *76-ELISA, mAb B4E11 and α76 cannot form molecular sandwiches, because of steric hindrance. *439-ELISA can detect molecules with N-terminal domain and intact C-terminal region (i.e. “full-length CgA”), but not fragments; *373-ELISA can selectively detect CgA_1-373, but not CgA_1-439; *76-ELISA can selectively detect CgA_1-76, but not the other fragments; total-CgA-ELISA can detect full-length CgA plus fragments containing the N-terminal and all or part of the central and C-terminal regions (FRs) including CgA_1-373, but not CgA_1-76.</p

Correlation and receiver operating characteristic (ROC) curve of VS-1 and total-CgA.

<p>A) Correlation between the plasma levels of VS-1 and total-CgA in patients with NENs (solid circles) and healthy subjects (hollow circles). B) ROC curve of VS-1 and total-CgA.</p

Sample description (NENs patient characteristics).

<p>Sample description (NENs patient characteristics).</p

Levels of VS-1, CgA_1-373 and CgA_1-439 relative to the total-CgA in the plasma of patients with ileal and pancreatic NENs (cases) and healthy subjects (controls).

<p>Box-plots with median (middle line), 75^th percentile (top line) and 25^th percentile (bottom line). The top and bottom whiskers represent the upper and lower adjacent values, respectively. Values outside the whiskers are plotted individually (circles). Cases (n = 17); controls (n = 10). * (<i>p</i> < 0.05), **(<i>p</i> < 0.01), by analysis of covariance. As total-CgA does not include VS-1, VS-1/total-CgA ratio can be also greater than 1.</p

Effect of therapy with somatostatin analogues (SSAs) on the plasma levels of total-CgA and VS-1 in patients with ileal and pancreatic NENs.

<p>(A, B) Plasma levels of total-CgA and VS-1 in NENs cases (ileal plus pancreatic NENs) before and after the administration of octreotide LAR. ** (<i>p</i> < 0.01), by Wilcoxon signed-rank test. (C) Correlation between the decrease (%) of the plasma levels of VS-1 and total-CgA after therapy.</p