The Clinical Features and Molecular Mechanisms of ACTH-Secreting Pancreatic Neuroendocrine Tumors

Cui Zhang; Jiabin Jin; Jing Xie; Lei Ye; Tingwei Su; Lei Jiang; Weiwei Zhou; Yiran Jiang; Luming Wu; Ting Wang; Xu Zhong; Guang Ning; Baiyong Shen; Weiqing Wang

Disclosures

J Clin Endocrinol Metab. 2020;105(11) 

In This Article

Subjects and Methods

Patients and Diagnostic Criteria

Seven patients with ACTH-secreting pNETs were referred to the Shanghai Clinical Center for Endocrine and Metabolic Diseases and Pancreatic Disease Center in Ruijin Hospital between 2001 and 2019. The diagnosis of EAS was made according to 2008 Endocrine Society clinical practice guidelines[15] with the following criteria: (1) typical clinical features of CS, such as moon face, hairiness, hypokalemia, hypertension and diabetes; (2) biochemical examinations: elevated serum ACTH and cortisol levels, 24-hour urinary free cortisol (UFC), and low-dose and high-dose (administration of 8 mg of dexamethasone for 2 days) dexamethasone suppression test (LDDST/HDDST) failed to suppress serum cortisol; (3) image examinations: pancreatic CT, magnetic resonance imaging (MRI), PET-CT scanning and/or 68Ga DOTATATE PET/CT revealed pancreatic tumors; and (4) positive ACTH staining in pathological examination by 2 experienced pathologists.[16]

In the same period, 20 patients with ACTH-secreting TNETs as mentioned and diagnosed previously[13,17,18] were compared regarding long-term survival. Meanwhile, 7 patients with nf-pNETs and 7 normal pancreatic tissues were compared regarding DNA methylation analysis.

Clinicopathological information, including the type of surgery performed, tumor stage,[19,20] and follow-up data, was collected. Informed consent was obtained from all participants. All protocols were approved by the Ruijin Hospital Ethics Committee, Shanghai Jiao-Tong University School of Medicine.

Laboratory Assays

All tests were performed in a College of American Pathologists (No. 7217913)-accredited laboratory. The plasma and UFC were examined by using an Access Immunoassay System (Beckman Coulter Inc., Fullerton, CA, USA). The ACTH levels were determined by using an ELSAACTH immunoradiometric assay (Cisbio Bioassays, France).

Immunohistochemistry

Immunohistochemical staining was performed using anti-ACTH (rabbit, Sigma-Aldrich) on formalin-fixed paraffin-embedded sections using a Chem Mate ENVISION kit (Dako). The anti-ACTH antibodies were used at 1:50 dilutions.

DNA Methylation Analysis of the POMC Promoter Region

For ACTH-secreting pNETs, nf-pNETs, and pancreatic tissues, genomic deoxyribonucleic acid (DNA) was extracted from formalin-fixed paraffin-embedded sections of the tumor. Sample preparation and pyrosequencing were performed using the PyroMark Q96 ID system (QIAGEN). Polymerase chain reaction (PCR) assays were designed to amplify parts of the promoter regions of the POMC gene. The PCR primers for CpG sites tested within the POMC promoter region are POMC-F1: 5′-GGAGGTTGTTTTTTGAGGG-3′; POMC-R1: 5′ (Biotin)-ACAATACTAATTCCAACCCCTTTC-3′; POMC Seq1: 5′-GAGGGGGGTTTTTAG-3′; POMC Seq2: 5′-GGGTTAGGGGAAGGG-3′; POMC domain IV F1: 5′-TGTTGGGGTTGGTATTTGT-3′; and POMC domain IV R1: 5′ (Biotin)-CCTCCCCTCCCCAAAATAA-3′. For ACTH-secreting TNETs, DNA extraction, bisulfite modification, and sequencing were performed as described in a previous study.[13]

Statistical Analysis

SPSS 22.0 (IBM, Chicago, IL, USA) was utilized for statistical analysis. Continuous variables are expressed as medians (interquartile range [IQR] 25, 75), and categorical variables are expressed as frequencies and percentages. The Mann–Whitney U test was used for comparisons of continuous variables. The chi-squared test was used to compare the proportions of males and staging of NETs. Patient survival was evaluated using the Kaplan–Meier method and statistically checked with the log-rank test. A P value <.05 was considered statistically significant.

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