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Letters |
1
Department of Biochemistry, School of Medicine, Fukuoka University, Fukuoka 814-0180 Japan
2
Gastroenterology Research Unit, Mayo Clinic, Rochester, MN 55905
3
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905
aAuthor for correspondence. Fax 81-928013600; e-mail tateishi{at}fukuoka-u.ac.jp.
To the Editor:
Currently there is no simple test to detect pancreatic cancer (PC). Fasting plasma islet amyloid polypeptide (IAPP) was reported to be increased in patients with PC (1), and a soluble factor from PC cells was reported to stimulate IAPP secretion (2), suggesting the possibility of an increase of IAPP in the plasma of patients with PC. In a recent clinical study, however, IAPP was not a satisfactory marker for detecting PC (3).
Human IAPP is easily degraded by proteases present in serum or plasma and also degraded during the clotting process. A possible cause for the loss of utility of IAPP for PC detection could be degradation of the polypeptide before analysis. Therefore, we designed a study to reevaluate this marker using plasma samples carefully collected in tubes containing protease inhibitors, with the plasma separated immediately and stored at -80 °C before analysis. Unfortunately, this improved sample handling did not improve the utility of IAPP for detection of PC.
Blood samples were collected in tubes containing protease inhibitors (2 mg/L antipain dihydrochloride, 25 mg/L elastatinal, 0.5 mg/L leupeptin, and 0.5 g/L EDTA). Our IAPP assay procedure was a modification of the method described by Permert et al. (1). Tracer was made by iodination of synthetic human IAPP with the Iodogen method and purified by HPLC. Before RIA, plasma samples were extracted with 600 mL/L acetonitrile on Sep-Pak C18. For RIA, anti-human IAPP antibody (RAS 7321) was used. The detection limit was 1.5 pmol/L, and the intra- and interassay CVs were <10%.
IAPP was stable for 2 h at 4 °C when synthetic IAPP was incubated with human plasma in the presence of the protease inhibitors. By contrast, synthetic IAPP added to plasma samples containing aprotinin and EDTA, which are routinely used for estimation of other peptides, lost 16% of IAPP activity after 2 h at 4 °C. Therefore, samples collected freshly in such an appropriate condition were used for this study. The study protocol was approved by the Human Studies Committee of the Mayo Clinic Institutional Review Board, and all study participants gave informed consent.
As shown in Fig. 1
, the mean plasma IAPP concentration was increased in patients with PC as compared with controls (mean ± SE, 7.1 ± 0.9 vs 4.0 ± 0.3 pmol/L). In samples (n = 22) from controls, IAPP was 1.8–5.9 pmol/L. Patients with islet cell, ampullary, gallbladder, and renal cell cancer also showed high concentrations. In some patients with acute and chronic pancreatitis, high values were detected. Therefore, sensitivity and specificity of IAPP for detection of PC were 47% and 73%, respectively, when a cutoff of 6 pmol/L was used.
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To compare with IAPP immunoreactivity measured by another assay detecting different antigenic determinants, the same samples were assayed by the solid amylin enzyme immunoassay (Amylin Pharmaceuticals Inc) without extraction (4). Values obtained by the two assays correlated well, with a correlation coefficient of 0.83 and no difference in sensitivity and specificity for detection of PC. A combination of IAPP and CA19-9 showed a sensitivity of 85% with a specificity of 63% when either IAPP or CA19-9 was higher than each cutoff value. Furthermore, IAPP was not effective as a PC marker when measured by a competitive microtiter plate assay (S-1187; Peninsula Laboratory, Inc) with a modified C18 extraction with 800 mL/L acetonitrile.
These results suggest that plasma IAPP lacks sensitivity for detection of PC even when the specimen integrity is preserved. The heterogeneity of PC cells may make it difficult to find a tumor marker for PC.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and funds from the Central Research Institute of Fukuoka University.
References
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