Macroscopically, PanNENs are usually solitary, solid masses, from 1 to 5 cm in diameter, with rounded borders. The expansive pattern of growth determines compression and deviation of the main pancreatic and biliary ducts and of adjacent structures when extending outside the pancreas. The usual PanNEN is rich in small vessels and has scant fibrotic stroma. Necrotic yellowish foci can be observed in larger masses. Features of malignancy evident at macroscopic examination include involvement of the perivisceral fat and invasion of the duodenal wall or adjacent organs. PanNENs may have unusual features, including cystic aspects, and may lead to be misinterpreted as cystic neoplasia; more rarely, they show considerable fibrosis, mimicking ductal adenocarcinoma.

Microscopically, the majority of PanNENs are well-differentiated tumors that grow as solid nests or with trabecular patterns (Fig. 8.1), although glandular, acinar, and cribriform features are observed as well. A rich vascularization is typical. Necrosis can be present as either confluent areas (“infarct-like”) in large tumors, or as punctate foci. Cytologically, PanNENs are composed of small to medium-sized cells with a finely granular cytoplasm and round or oval nuclei with salt-and-pepper chromatin. Tumoral infiltration of the duodenum and/or the biliary duct wall together with lymph node metastases identify the malignant forms, as does the involvement of the peripancreatic fat.

Immunohistochemistry serves to confirm the endocrine nature of the neoplasia and thus to differentiate PanNENs from other neoplasms, based on the use of antibodies to at least one general endocrine marker, either synaptophysin [11] or chromogranin A (CgA) [12]. The cytosolic neuron-specific enolase (NSE) [13] and protein gene product 9.5 (PGP 9.5) [14] are less specific and their diagnostic utility is limited. PanNENs may express the normally produced pancreatic hormones (insulin, glucagon, somatostatin, and pancreatic polypeptide), or hormones of ectopic origin (gastrin, vasoactive intestinal polypeptide, adrenocorticotrophic hormone), or bioamines (serotonin). While any of these may be demonstrated by immunohistochemistry, the information has no clinical application.

Proliferative activity has a recognized prognostic value [10, 15], and its assessment by Ki67 immunostaining is a routine practice in several institutions, including ours (Fig. 8.2).

Poorly differentiated NECs are solid masses with extensive necrosis. Histologically, they resemble small-cell carcinomas or large-cell endocrine carcinomas of others organs, with a high mitotic rate, a proliferative activity of > 20%, and abnormal immunostaining for p53 that correlates with intragenic mutations in the TP53 gene [8, 10, 16]. NECs are usually negative for CgA while synaptophysin persists in the neoplastic cells.

The European Neuroendocrine Tumor Society (ENETS) has proposed a tumornode-metastasis (TNM)-based staging system for PanNEN [17] to which subsequent modifications have been proposed [18]. The TNM system is based on the evaluation of the following parameters: size, extrapancreatic invasion, and lymph node and liver metastasis. The clinical need to differentiate between carcinomas at the same stage is facilitated by the use of a grading system based on the measurement of the proliferative activity, by counting mitosis or assessing the immunohistochemical Ki-67 index. Both the TNM staging and the tumor grading systems have been shown to be valid tools for prognostic stratification of PanNENs in clinical practice [19, 20].

Most PanNENs occur sporadically (90%), but they may be part of four hereditary cancer syndromes [9]: Multiple endocrine neoplasia type 1 (MEN1), von Hippel-Lindau disease (VHL), neurofibromatosis type 1 (NF1), and tuberous sclerosis complex (TSC). Of note, studies concerning these familial syndromes have furnished clues as to the molecular mechanisms involved in sporadic PanNEN tumorigenesis. The genetic aberrations associated with PanNENs include chromosomal alterations, epigenetic changes such as methylation, and mutations in single genes. The functional genomic alterations are found at the RNA level and include protein-coding mRNAs and regulatory small RNAs known as “non-coding RNAs” [21].

Accumulating evidence points towards a tumor suppressor pathway and chromatin remodeling as the most important mechanisms associated with PanNENs. Genome-wide analyses by comparative genomic hybridization have shown that virtually all PanNENs display chromosomal alterations [22–24]. Chromosomal losses are slightly more frequent than gains, whereas amplification events are uncommon. The presence of numerous regions of chromosomal losses and gains suggests the existence of two molecular subgroups: one showing frequent allelic imbalances (AI) and another showing low AI [25, 26]. These two subgroups have been shown to correspond to aneuploid and near-diploid tumors, respectively, and their identification was suggested to have prognostic value [26]. The total number of genomic changes per tumor appears to be associated with both tumor burden and disease stage, suggesting the accumulation of genetic alterations during tumor progression. A strong correlation has been found between sex-chromosome loss and an aggressive behavior of PanNENs, namely, the presence of local invasion or metastasis [27]. Retinoblastoma and TP53 gene defects have never been observed in PanNENs but are consistently present in NECs [16]. DNA methylation of the RASSF1A gene has been suggested as a major event in PanNENs [28, 29], possibly leading to gene inactivation. However, a very recent study demonstrated RASSF1A expression in the presence of promoter methylation [30]. Mutations in oncogenes are never or rarely observed in PanNENs [31, 32]. Instead, mutations in tumor suppressor genes represent the major genetic anomaly encountered in this type of tumor. Indeed, mutations in the tumor suppressor gene MEN1 are the most common anomaly associated with PanNENs [33, 34]. MEN1 encodes for the scaffold protein menin, which is known to interact with several proteins involved in, for example, transcription regulation, maintaince of genome stability, and histone modification.

A recent systematic whole-exome analysis exploiting next-generation sequencing technologies confirmed that MEN1 gene mutations are the most relevant anomalies in PanNEN [32]. More interestingly, mutually exclusive mutations were found in genes involved in chromatin remodeling (ATRX and DAXX). The majority of the mutations in these genes were associated with the loss of corresponding proteins that normally associate to form a macromolecular complex. This complex is involved in the deposition of histone H3 family member H3.3 at transcriptionally silent regions of the genome, including telomeres, and therefore is responsible for correct nucleosome assembly, the dysfunction of which likely leads to increased DNA damage and genome instability. Furthermore, the loss of ATRX/DAXX seems to be associated with ALT (alternative telomere lengthening), a crucial mechanism by which PanNENs maintain telomere length [35].

In addition to alterations in chromatin-associated genes, other tumor suppressor genes that have been found mutated in PanNENs are PTEN and TSC2, which are negative regulators of the mTOR pathway. Activation of mTOR pathways in primitive PanNENs was already demonstrated in analyses of expression profiles, which revealed the down-regulation of the TSC2 gene and alteration of TSC2 and PTEN protein expression in the vast majority of tumors analyzed [36].

Finally, global microRNA expression analysis revealed that the overexpression of a specific microRNA (miR-21) is strongly associated with an aggressive clinical behavior of PanNENs [37]. MicroRNAs are small non-coding RNAs that regulate gene expression by targeting specific mRNAs for degradation or translation inhibition. MiR-21 has several targets, including PTEN, whose expression is therefore reduced following up-regulation of the microRNA, leading to mTOR activation as well.

In patients with PanNENs, the pathology report must include information permitting disease classification, staging, and grading in order to obtain a prognostic evaluation. Genes involved in sporadic PanNEN tumorigenesis mainly belong to tumor suppressor pathways that are responsible for chromatin remodeling and the maintenance of genome stability. The direct consequences of these defective pathways are consistent chromosomal alterations that are a hallmark of PanNENs. Finally, global expression profiling analysis has furnished a strong rationale for the use of targeted therapy in the treatment of advanced-stage disease.