Small biopsies obtained by PUS or EUS investigation allow primary or metastatic GEP-NETs to be preoperatively diagnosed through the adoption of larger needles with cutting edges, which collect adequate tissue fragments. Another source of diagnostic material are endoscopic biopsies from the stomach or large bowel in the case of mass-forming lesions. Core tissue and endoscopic biopsies with different dimensions are optimal materials for histologic interpretation, tissue staining and molecular investigation. Core needle biopsies (CNB) using 25–20-gauge needles, endoscopic sampling and Tru-Cut needle biopsies with 18–19-gauge needles are the technical instruments most often used in clinical practice (Fig. 9.2a–d).

Despite small size, CNB is advantageous because tissue architecture is well preserved for tumor diagnosis and biological assessment in paraffin embedded material [20–22]. Upon histology, tissue fragments lie often intermingled with blood clots due to the high vascularization of most NETs, in which isolated neoplastic cells are preserved enabling a definitive tumor categorization.

There is a great debate in the reported utilization of CNB in NETs patients, with either a preferential use of CNB over FNAC or even limitation of CNB to only indeterminate cases on FNAC being reported on literature [23, 24]. CNB specificity ranges from 96% to 100% for diagnosing primary and secondary NETs, with sensitivity ranging between 60% and 90% [23, 24]. It is accepted that CNB samples provide more accurate subtyping on some tumors than do FNAC samples, with an incremental value of histology over cytology in the specific characterization of pancreatic neoplasms other than adenocarcinoma [25, 26]. On the other hand, it has been proposed to carry out the on-site evaluation of biopsy adequacy by rolling the cores across slides to obtain cytological specimens for immediate assessment [27, 28]. This procedure on CNB can improve the overall diagnostic sensitivity by 8% [29], supporting the view that the combination of cytology and histology is by far superior to either one individually [23, 30, 31]. Overall, tissue fragments of GEP-NETs make up a precious reservoir of material for differential diagnosis, immunohistochemistry characterization and molecular testing by providing the opportunity for multiple tissue sections [20–22]. This is particularly crucial in liver metastases of unproven origin NETs, where a combination of different markers (TTF-1, CDX-2, PAX-8, Islet 1, serotonin, PSAP) may help characterize the cell lineages of tumor growths [5–7, 32].

Whether biopsy samples of primary or metastatic NETs are optimal material also to grade tumors is still a debated but undeniable issue for its important clinical implications. Recent prospective studies have demonstrated that either FNAC [20] or biopsy [33, 34] was adequate for histological diagnosis and Ki-67 LI assessment in patients with non-functioning pancreatic NETs, but the crucial question remains as to whether this recruited material is really representative of the whole lesion due to intratumor heterogeneity [35]. For example, pancreatic NETs larger than 1.8–2.0 cm sampled by CNB showed a lower concordance between Ki-67 LI and tumor grading than smaller tumors [34], indicating that the intratumor heterogeneity accounted for such a discrepancy [20, 33–35].

Some studies have indicated that biopsies from liver metastases of the GEP NETs failed to reliably separate G1 from G2 as compared to the same assessment on the whole tissue sections [35], thus confirming a major role for intratumor heterogeneous distribution of cell clones with different proliferation activity. This phenomenon can even become grueling, when diversely and unpredictably distributed neuroendocrine components with well-differentiated and poorly differentiated features coexist in the same tumor mass as suggested for NETs arising in the GEP tract [36], lung and thymus [37]. However, a unique study has thus far demonstrated that Ki-67 LI assessment may be concordant between biopsy samples and resection specimens of lung NETs when strict counting guidelines were applied [38]. These included the identification of hot spot regions, which remains a matter of perception, in either type of material and the KI-67 LI assessment on 2,000 cells, 2 mm² or the entire biopsy fragments to account for tumor sampling, tissue fragment sizing and intratumor heterogeneous distribution of defining criteria [38]. This innovative approach paves also the way to using Ki-67 LI for better grading of these tumors in close combination with morphologic classification [38].

A crucial issue regards the management of biopsies or FNAC of liver metastases from NETs originating in extra-GEP anatomical sites, such as the lung, where terminology (typical and atypical carcinoid instead of NETs) and defining criteria (grading based on morphology rather than Ki-67 LI) are quite different [39]. In these cases, clinical correlation is always fundamental and the relevant terminology should be adapted and commented to avoid an improper generalization of classification criteria.

Surgical specimens represent the ideal and irreplaceable material of investigational studies, where most features of the GEP-NETs can be elucidated, although representative only of a fraction of lesions, predominantly NETs, amenable to surgical resection [40].

They are the basis for the deepest knowledge on the pathobiology of these neoplasms, inasmuch as many biological, diagnostic, prognostic and predictive characteristics can be here developed [1]. Therefore, surgical specimens should be optimally fixed, processed and stored to avoid technical artifacts and then accurately evaluated on gross and microscopic pathology [1]. Furthermore, storing frozen material and blood samples from paired patients is a powerful tool for many research activities on genomics, metabolomics or proteomics of NETs. In general, gross features do not help differentiate GEP-NETs from each other and from the more common non-neuroendocrine tumors. However, the distribution and number of lesions may provide helpful hints to recognize varying subtypes of NETs.

Most gastrinomas (74%) and somatostatinomas (84%) arise on the right of the superior mesenteric artery in the pancreatic head, whereas most insulinomas (74%) and glucagonomas (77%) are positioned on the left of this artery in the body and tail of the pancreas [1]. Inside the pancreas VIPomas are more prevalent in the distal pancreas (77%), while non-functioning NETs have been reported to be either uniformly distributed within the whole organ or preferentially restricted to the cephalic region. Gastrinomas and somatostatinomas may frequently arise in extrapancreatic locations, such as duodenum or proximal jejunum. Ninety per cent of gastrinomas occur within the so-called “gastrinoma triangle”, a virtual anatomic area of triangular shape bounded by the junction of the cystic and common bile ducts, the junction of the second and the third portions of the duodenum, and the junction of the head and the body of the pancreas. The most common site of sporadic gastrinomas is the head of the pancreas, followed by the first and second portions of the duodenum and the periduodenopancreatic lymph nodes as metastatic deposits of unrecognized duodenal tumors. In turn, the localization in bile ducts or stomach or in other anatomical sites including the jejunum [1], liver, kidney, ovary, and parathyroid is very rare, in opposition to the normal distribution of gastrin-producing neuroendocrine cells in the gastric antrum and the duodenal wall [4].

In MEN1 patients, gastrinomas most commonly originate in the duodenum, and, less frequently, in the pancreas. Duodenal somatostatinomas are more frequent than those arising in the pancreas, and are generally located in the second portion of the duodenum, often in the ampullary-periampullary region. Unlike gastrinomas and somatostatinomas of the pancreas, which are nearly always functionally active, the same duodenal neuroendocrine tumors are associated with hormonal symptoms in only 40–45% of the patients. GEP-NETs may present as either sporadic or familial tumors. Sporadic lesions are generally single, although multifocal tumors, either synchronous or metachronous, have been described in 2–13% of pancreatic insulinomas, 40% of duodenal gastrinomas, 30% of duodenal somatostatinomas and gastric NETs associated with chronic atrophic gastritis [1]. Multifocal pancreatic, duodenal or gastric NETs are instead the rule in MEN1 patients.