PMC7438413_01
(A) Computerized tomography showing neuroendocrine tumor at the pancreatic tail causing liver metastases (white arrows). (B) Octreotide-single-photon emission computerized tomography (SPECT) study showing hepatic and pancreatic focal lesions with enhanced uptake (white arrows). (C) Clinical picture of the patient remarking the skin hirsutism (white arrows), and hyperpigmentation. (D) 18-Fluorodeoxyglucose (18-FDG) positron emission tomography-computerized tomography (PET-CT) 18 months after diagnosis with persistence of tumor liver metastases (black spots).
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F1
Physical examination revealed an overweight patient with a rounded "full moon" face, hirsutism (Figure 1C), mucocutaneous hyperpigmentation, a buffalo hump, upper extremity bruising, generalized weakness, and muscle wasting evident in the lower limbs. IPSS was not considered necessary in this case, as pituitary corticotroph adenoma was very unlikely. Instead, a computerized tomography study was performed, and result showed a 2.7 cm hypodense hepatic nodule, with a 4.3 cm distal pancreatic lesion, and bilateral diffuse adrenal hyperplasia (Figure 1A). This showed increased metabolic activity in both tumors (Figure 1B). The clinical picture of patient improved; however, 18-fluorodeoxyglucose (18-FDG) positron emission tomography-computerized tomography (PET-CT) confirmed the presence of inoperable liver metastases (Figure 1D).
Western blot analysis from samples of pancreatic tumor and liver metastasis. (A) Normal pancreas; (B) Pancreatic NET; (C) Normal liver; (D) Liver metastasis. Pancreatic and liver tissues were homogenized in the presence of RIPA buffer (PBS with detergents and protease inhibitors cocktail). Total protein concentration was determined using the commercial Bradford reagent assay (Bio-Rad, Hercules, CA). Whole protein (200 mug) was used for the detection of the ACTH protein. Samples were first boiled in sample buffer (125 mM Tris-HCl, pH 6.8, 1% v/w SDS, 10% v/v glycerol, 0.1% bromophenol blue, 2% v/v 2 alfa-mercaptoethanol) for 5 min and separated by 12% SDS-PAGE. Then, the gels were transferred to PVDF membranes (Merck Millipore Corporation, Darmstadt Germany) using a Trans-Blot Cell system (Bio-Rad, Hercules, CA) in transfer buffer (25 mM Tris, 190 mM glycine, and 10% methanol) at 40 V overnight. The following day, the membranes were blocked with non-fat dried milk (5%) dissolved in TBS buffer (150 mM NaCl, 20 mM Tris, and 0.1% Tween) and probed overnight at 4  C with rabbit monoclonal anti-ACTH antibody (Abcam, Cambridge UK) diluted 1:2,000 in TBS buffer with BSA (150 mM NaCl, 20 mM Tris, 0.1% Tween, and 1% BSA at pH 7.5). After washing, the membranes were incubated for 1 h with anti-rabbit immunoglobulin-HRP (Thermo Scientific, Rockford, IL USA). The signals were detected by enhanced chemiluminescence using the SuperSignalTM system (Thermo Scientific, Rockford, IL USA) on X-ray film (Kodak, USA). As the control loaded, actin was simultaneously detected, using mouse anti-human beta-actin antibody (Santa Cruz Biotechnology, Santa Cruz CA, USA). The signal was developed using anti-mouse immunoglobulin-HRP (Thermo Scientific, Rockford, IL) and chemiluminescence system.
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F2
Results showed a 30 kDa protein in both pancreatic and liver tumors, but negative on normal pancreas and liver as control (Figure 2). Taking all together, WB results suggested the presence of a high molecular weight ACTH-precursor such as POMC (Figure 2).
Timeline of the case presented.
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F3
Her survival following the initial diagnosis was no >36 months (Figure 3).