Despite such limits, the current grading proved essentially effective for death prediction in multiple independent studies [15, 17, 25, 41, 42]. For pancreas, it was demonstrated that Ki67 5 % is the value above which the death risk increases in a statistically significant fashion as previously suggested [15, 25]. This value needs to be taken into adequate account for any specific decision for NET G2 patients.

G1-G2 NETs may display most types of the structures described originally by Soga and Tazawa in 1971 for carcinoids [43], namely, solid islet (type A), trabecular (type B), and glandular (type C) (Figs. 6.2 and 6.3). Usually, a pancreatic NET may display a mixture of types. The margins are usually expansive, with a delicate stroma rich in vessels, a feature typical of neuroendocrine cancer. Often, the stroma may become more solid with desmoplastic, parenchyma-substituting aspects reminiscent of the more aggressive adenocarcinoma. This latter feature may associate with deposits of amorphous eosinophilic material defined as amyloid and displaying metachromasia at Congo red stain. Amyloid deposits have been classically described in insulinomas and are composed by fragments of the hormonal peptide islet amyloid polypeptide (IAPP) as produced by the tumor beta cells [44]. NET cells are usually rather monomorphic, with abundant eosinophilic cytoplasm, round, monomorphic nuclei with spotted chromatin and rather rare mitoses, only exceptionally atypical. The degree of atypia does however increase in G2 cases, with prevalent solid structure, polymorphic cytological features including irregular cell size, pronounced atypia with increased nuclear/cytoplasmic ratio, evident nucleoli, and mitoses, sometimes atypical. Necrosis is absent and when present usually heralds the more severe cytological-histological features of NEC (Fig. 6.4). Angioinvasion and perineural space invasion may also be observed and, though of potential prognostic significance, are not included in the current classification criteria.

G1-G2 NETs stain positive for all markers of neuroendocrine differentiation, including chromogranin A and synaptophysin, the two minimum stains recommended for diagnosis by ENETS (Fig. 6.5) [16]. It is usually considered good practice that at least two neuroendocrine- specific stains should be positive in the large majority of tumor cells to set the diagnosis of NET. The degree of specificity of neuroendocrine stains is the highest for chromogranin A and decreases for synaptophysin, NSE, PGP 9.5, and CD56 N-CAM, all of which may stain non-neuroendocrine neoplasms. By converse, the search for cell-type-specific markers, namely, the peptide hormones like insulin, glucagon, pancreatic polypeptide, somatostatin, and ghrelin, is not required. When performed, usually it may show only a variable fraction of positive NET cells or, more rarely, a diffuse pattern of expression (Fig. 6.6a) [6]. The search for specific hormones is not mandatory for the diagnosis of NET but may be required for confirmation of a clinically functional condition. Other immunohistochemical stains may be required to answer specific clinical query or in the metastatic setting. In particular, the assessment of the somatostatin receptor subtype 2A (SSTR2A) may be requested on tissue when the in vivo assessment of lesions expressing the somatostatin receptors by nuclear medicine methods (e.g., Octreoscan® or Ga68-DOTA-NOC/TOC) is not available (Fig. 6.6b). Other markers including the nuclear transcription factors like the insulin gene enhancer protein islet-1 (ISL-1), the homeobox CDX2, and the NK2 homeobox 1 (NKX2-1), also known as thyroid transcription factor 1 (TTF-1), may be useful to determine the origin of a neuroendocrine cancer metastasis of potential pancreatic origin. Their use and significance need to be carefully weighed according to their known tissue-specificity limits and to the clinical evidence of the specific case under study.

NECs by definition display a high proliferative fraction, though histology may reveal different features. A solid structure with some trabecular packed areas – a structure generally defined as “organoid” – is often observed, together with moderate to severe cytological atypia, sometimes with relatively abundant eosinophilic cytoplasm, polymorphic large nuclei, increased nuclear/cytoplasmic ratio, evident nucleoli, and multiple mitoses often atypical (Fig. 6.4). Such aspects may well refer to the “structures of lower or atypical differentiations” defined as Type D by Soga and Tazawa [43]. Necrosis in such cases is usually scant and spotty; however, it becomes more evident with increased large solid sheet structure and increased cell atypia with large nuclei and reduced cytoplasm, high-grade features more classically associated with large cell NECs. Not infrequently, cases showing areas with different histology of low and high grade may be observed [45]. In such cases, the highest-grade areas drive the diagnosis, according to the WHO 2010 grading rules. At immunohistochemistry, both chromogranin A and synaptophysin are widely expressed, though, while synaptophysin is usually intense, chromogranin A may be only scant and presenting as a thin rim of positive stain at cell periphery or as dots at Golgi cell site. Such features reflect the relative absence of large dense-core vesicles (LDCV) containing chromogranin A as observed at ultrastructure in NECs. Hormones in general are only rarely observed similar to SSTR2A, which however may be present in about 25–30 % of NECs [46]. Other neuroendocrine markers like NSE and CD56 N-CAM may be useful since they do not associate with neuroendocrine granules [13].