Chromogranin a

Formalin-fixed and paraffin-embedded resection specimens were sectioned to 4 μm in thickness and deparaffinized, rehydrated, and processed for antigen retrieval. The slides were further incubated with appropriate dilutions of the following antibodies at room temperature for 1 h. After incubation, the slides were washed three times in phosphate-buffered saline (PBS), incubated with a horse reddish peroxidase conjugated antibody polymer (Zymed) at room temperature for 10 min, and were then developed by treatment with 3,3′-diaminobenzidine (Roche) at room temperature for 10 min. The used monoclonal antibody for Immunohistochemistry (IHC) were against Ki-67 (1:1000, Dako) and chromogranin A (1:500, Dako). For gastrin staining we used polyclonal antibody against gastrin (LEICA biosystems) without antigen retrieval. Ki-67 staining and mitotic counts per 10 high power field were used for tumor grading [25 ]. In brief, more than 20 in mitotic index were classified as G3. Independent experienced pathologists blinded of patient characteristics and outcomes studied the results of immunohistochemical staining for diagnosis and classification [25 ].

FFPE blocks were sectioned and mounted on coated slides (Dako, Glostrup, Denmark) and stained with H&E according to standard protocols. Tumor sections were deparaffinized using EZ-prep (Ventana Medical Systems^®, Tucson, AZ, USA) and immunohistochemistry was performed using the Ventana Benchmark Ultra platform (Ventana Medical Systems) as previously described [33 (link)]. The following primary antibodies were employed: CD117 (polyclonal, 1:100, Dako (Glostrup, Denmark)), CD56 (clone 1B6, 1:50, Novocastra (Newcastle, UK)), chromogranin A (polyclonal, 1:2000, Dako), CK7 (clone OV-TL 12/30),1:1000, Dako), CK20 (clone KS20.8, 1:400, Dako), ki-67 (clone MIB1, 1:100, Dako), MYB (clone EP769Y 1:150 (AbCam, Cambridge, UK), napsin A (clone IP64, 1:400, Novocastra), and TTF-1(clone SPT24, 1:100, Novocastra). MYB was considered positive when nuclear staining was observed in at least 5% of tumor cells [31 (link)]. Positive controls as suggested on datasheets were used. Negative controls omitting the primary antibody were performed for all antibodies.

HE staining: All specimens were fixed by 10% neutral formalin, regularly dehydrated, paraffin-embedded, sectioned into 3-μm sections, and processed for HE staining. IHC staining: IHC staining was performed using the EnVision two-step strategy. Primary antibody information: CD117, HMB45, Desmin, synaptophysin, chromograninA, S100 protein, wide-spectrum cytokeratin, Ki67, and vimentin (DAKO); Bcl2, P53, CD56, CD57 (LEICA); muscle specific actin (MSA), CD34, H-caldesmon (LongIsland); SOX10, INI1, DOG-1 (Gene Tech); somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5) (Abcam); α-smooth muscle actin (α-SMA) (Thermo); ATRX (Sigma); calponin, Collagen type IV (MXB Biotechnologies); BRAF V600E (ZSGB Biotechnology). The dilutions, clone and sources of these antibodies were listed in Table 1.

All tumors were examined pathologically and classified according to the World Health Organization (WHO) classification²⁰ , UICC TNM classification⁴² , and the Japanese Pancreas Society classification of pancreatic carcinoma⁴³ .
Immunohistochemistry was performed on cryostat sections as described previously^{44 (link)}. We used antibodies against the following: Chromogranin A (1:100), CD45 Leucocyte common antigen (1:100), CD68 (1:250), epithelial membrane antigen (EMA) (1:200) and cytokeratins AE1/AE3 (1:200) from DAKO (Glostrup, Denmark), and Bcl-10 (331.3; 1:100) from Santa Cruz Biotechnology (Santa Cruz, CA). Immunohistochemistry without the primary antibody was considered as negative control. Aniline blue staining and Sudan-III staining (Muto pure chemicals, Tokyo, Japan) were performed as instructed. After immunohistochemistry or staining, the microscopic images were imported as digital photo files using a NanoZoomer Digital Pathology system (Hamamatsu Photonics, Hamamatsu, Japan), and the density of the immunolabeled cells or stained area was analyzed using the image analysis software, Tissue Studio (Definiens, Munich, Germany).