**2.1. Tumor sites and staining**

Although they may appear benign tumors for a certain period of time, they can originate tissue infiltration and give metastasis. All these pathophysiological attributes of neuroendocrine tumors, which may occur in varying degrees of intensity in different patients, arouse general interest, and so they are studied by professionals from different expertise. In this chapter, we will turn to the study of some relevant aspects of these tumors that originate in the stomach. However, since these neoplasias are part of a large family of gastrointestinal tumors, we will carry out a summary review of the main attributes of this family which should be useful to

The terms "neuroendocrine tumors," "carcinoid tumors," and "endocrine tumors" are widely used when referring to tumors of the digestive tract. These designations can be found simultaneously even as part of the same classification. Neuroendocrine tumors were firstly called as "carcinoid tumors" by the German pathologist Oberndorfer, in 1907 [1]. And until now, the term carcinoid has been used ostensibly, colloquially, and even in almost all current classifications [2, 3]. The term "apudoma" may also be considered as being a synonym of carcinoid tumor. The denomination of "APUD cells" was proposed by Pearse in the late 1960s as an acronym of *amine precursor uptake and decarboxylation* [4]. The term APUD summarizes some of the most important characteristics of these cells, which are (a) a high amine content and/or amine precursor uptake, (b) amino acid decarboxylase activity, and (c) characteristic ultrastructural pattern. Initially, the term "apudoma" was used mainly from the clinical point of view to designate those tumors of symptomatic patients due to the pathological secretion of bioactive products and afterward also for clinically asymptomatic tumors originating from APUD cells [5–7]. As we can see, unlike the roots of the other designations for these tumors, the term

better understand some nuances of these tumors which originate in the stomach.

"apudoma" has been derived from consistent morphologic and biochemical basis.

medical literature indexing.

30 Gastric Cancer

Apudomas are derived from the APUD cell line and so they have characteristic ultrastructural pattern recognized due the presence in the tumor cells of secretory granules where the regulatory peptides are located as well as the biogenic amines that they produce [8, 9]. Normally, the APUD cells are rich in amino acid decarboxylase, which gives them the ability to capture 5-hydroxytryptophan and dihydroxyphenylalanine and produce, respectively, serotonin and dopamine. Although this property has not been demonstrated in all the cells morphologically characteristic of this system, this biochemical link may occur in all of them whatever their specific function is. This gives these cells a familial, morphological, and biochemical bond, which extends to a greater or lesser degree to the apudomas. In addition, these morphological and biochemical characteristics give to the term "APUD" a biologically more specific designation compared to the other denominations. Thus, the term "apudoma" is less vulnerable to temporal changes than those currently used, namely, "carcinoid," "endocrine tumor," and "neuroendocrine tumor." The latter denomination is being adopted in this article because of its extensive use in modern classifications and also throughout the

**2. General aspects of neuroendocrine gastrointestinal system**

One of the first steps, both in pathology and clinics, when we are faced with the possibility to do the diagnosis of a gastrointestinal apudoma, is the knowledge of its possible primary location (**Table 1**). It may seem like an unimportant detail, but this can be very valuable as a first step to better understand the biology of the tumor [10, 11].

Sometimes, we deal with an unknown primary site metastasis. But knowing which segment of the digestive tract derives the tumor may be helpful because this information gives an idea on the possible evolution of the apudoma. Regarding the gastric apudomas, this is a crucial knowledge as we will see later on. In addition, the pathologist gains confidence in the study of the diagnostic possibilities, including for the evaluation which stain techniques or which neuroendocrine marker should be used [12]. Most of the gastric apudomas do not produce serotonin and are negative for argentaffin staining; on the other hand, they usually are positive for argyrophilic and chromogranin staining (**Figure 1**). These properties of apudoma, in relation to certain staining techniques, often depend on their place of origin (**Table 2**) [13–15].

Currently, immunohistochemistry techniques have been used as tools of choice for the specific histologic diagnosis of apudomas. Through this methodology, it is sought to mark the presence of antigenic products typical of APUD cells and also frequently present in apudomas (**Figures 2** and **3**). Neuron-specific enolase (NSE) was the first immunohistochemical marker for histological diagnosis of apudomas [16]. Before the discovery of this marker, the most commonly available methods were silver staining. Synaptophysin is also a neuroendocrine marker, located on the membrane of the synaptic vesicles, and present in neurons, neuroendocrine cells and in many neuroendocrine tumors [17, 18]. Chromogranins comprise a group of acidic polypeptides that make up about 40–50% of the soluble protein content of the suprarenal medullar granules. The chromogranin A is the most widespread; it is present in varying amounts in the secretion granules of neuroendocrine cells and in neuroendocrine tumors [15, 18]. If the tumor is less differentiated and with fewer secretory granules in the cytoplasm, the staining can give doubtful results or even a false-negative reaction. Chromogranin is one of the main markers used for the histological diagnosis of gastric apudomas presenting high index of sensitivity and specificity. It is also tentatively used as a serologic marker of apudoma evolution [19]. It is always appropriate to remember that the antibodies used against apudoma antigens are derived from different clones and are provided by different companies. This may result in differences in sensitivity and specificity of each set of reagents, and these different properties must be under the control of each laboratory.


Apudomas can arise in any region of the gastrointestinal tract. These regions are known as foregut, midgut, and hindgut. Apudomas from these different sites frequently present different clinical behaviors.

**Table 1.** Primary sites of the gastrointestinal apudomas.

**Figure 1.** Although it is nonspecific staining for APUD cells and apudomas, the argentaffin and argyrophilic stains may be useful for the demonstration of these elements [13].


+, seldomly positive; ++, sometimes negative; +++, much frequently positive

**Table 2.** Almost all the gastric apudomas are demonstrated by argyrophilic staining and/or by argentaffin methods.

**Figure 2.** Some of the neuroendocrine markers are scattered in the cytoplasm of apudoma cells such as PGP 9.5, synaptophysin, and neuron specific enolase. Chromogranin and the regulatory peptides are stored in secretory granules. The amount of these granules in the neoplastic cells will determine weaker or stronger staining of the tumor.

#### **2.2. General view on pathology of gastrointestinal apudomas**

We have now reached a point which concerns more directly the attending physician. Since the apudomas frequently present unpredictable clinical evolution, how could the histopathology help on this matter? In fact, all the factors shown in **Table 3** are important to evaluate the possible clinical behavior of a given gastrointestinal apudoma. However, the two first, i.e., degree

**Figure 3.** (A) G cell of the gastric mucosa stained specifically with antibody against gastrin. The staining is very specific by observing that the background elements, i.e., other cells and surrounding tissues, are completely negative. The very strong contrast between the positive and negative elements of tissue confers confidence of the final result in relation to the specificity of the staining reaction. (B) The staining contrast is not always very clear in regard to apudomas which may exhibit cells with various degrees of differentiation. A variable number of neoplastic cells with low secretory granule density could present a dubious or completely negative reaction. As a result, a given gastrinoma may exhibit cells strongly reactive to the gastrin antibody alongside others completely negative.

of histologic tumor differentiation and the proliferative activity of the neoplastic cells, depend exclusively on the pathologist interpretation. And unlikely, the last four parameters shown in **Table 3** depend on the opinion of different expert professionals to reach more reliable conclusions about the clinical course of the tumor.

In general pathology, the degree of differentiation of a given epithelial tumor, evaluated by histology, has always been, and still is, a criterion that together with other ones, gives an idea about the grade of malignancy that a particular carcinoma must have: a less aggressive evolution, more common among the well-differentiated ones, or a more aggressive evolution more common in those poorly differentiated (**Figure 4**). This histopathological criterion continues to be applied

**Table 3.** Main criteria to determine the malignant potential of the apudoma.

**2.2. General view on pathology of gastrointestinal apudomas**

**Gastrointestinal apudomas and silver staining properties**

be useful for the demonstration of these elements [13].

32 Gastric Cancer

Argyrophilic tumors +++ +++ ++ Argentaffin tumors + +++ +

+, seldomly positive; ++, sometimes negative; +++, much frequently positive

We have now reached a point which concerns more directly the attending physician. Since the apudomas frequently present unpredictable clinical evolution, how could the histopathology help on this matter? In fact, all the factors shown in **Table 3** are important to evaluate the possible clinical behavior of a given gastrointestinal apudoma. However, the two first, i.e., degree

**Figure 2.** Some of the neuroendocrine markers are scattered in the cytoplasm of apudoma cells such as PGP 9.5, synaptophysin, and neuron specific enolase. Chromogranin and the regulatory peptides are stored in secretory granules.

**Foregut Midgut Hindgut**

**Table 2.** Almost all the gastric apudomas are demonstrated by argyrophilic staining and/or by argentaffin methods.

**Figure 1.** Although it is nonspecific staining for APUD cells and apudomas, the argentaffin and argyrophilic stains may

The amount of these granules in the neoplastic cells will determine weaker or stronger staining of the tumor.

**Figure 4.** Well-differentiated type 1 (A) and poorly differentiated type 2 (B) gastric apudomas. Although the degree of tumor differentiated could have some importance regarding the degree of malignancy, the proliferative activity remains as a better histopathological indicator for this purpose.

mainly in relation to the nonendocrine carcinomas. Concerning the apudomas, this parameter, as an isolated element, has little value. Differently, the well-differentiated apudoma can present unpredictable clinical behavior. However, when the tumor is histologically poorly differentiated, the prognosis is often worse. In this respect, this criterion of cellular differentiation may be helpful. In addition, apudomas present varying densities of membrane receptors for different regulatory peptides, including somatostatin. And, a high density of these receptors in apudoma cells acquires current medical importance for the patient treatment. Some data indicate that the somatostatin receptor density would be dependent on the degree of tumor grade [20].

Under the view of histopathological diagnosis, the malignancy potential of a particular apudoma rests mainly on the degree of tumor cell proliferation. This criterion can be applied to all apudomas regardless of their origin. There are two histopathological tools to assess the degree of cell proliferation: (i) the number of observable mitoses in the routine preparations stained by *hematoxylin* and *eosin* and (ii) the index of cell immunoreactivity for Ki-67 antibody. The Ki-67 reactive protein is only expressed in the nucleus of cells that are in the various phases of the active cell replication cycle. This protein is not expressed in resting cells, that is, the cells that have not yet entered the active mitotic cycle. Moreover, there is not always any correspondence between the degree of differentiation of a given apudoma and the degree of cell proliferation [21, 22].

Concerning proliferative activity, the low-grade (G1) tumors present very few numbers of mitosis in routine HE preparation or otherwise less than 3% of neoplastic cells stained by Ki-67 antibody. The high-grade (G3) tumors should present more than 20 mitoses per 10 microscopic high-power field (hpf) or more than 20% of the neoplastic cells positive to Ki-67 antibody. Finally, tumors with intermediate degree of cell proliferation that lie between these two extremes are considered to be G2.

#### **2.3. Gastric apudomas**

Almost all apudomas of the stomach are derived from the endocrine cells of the body mucosa and rarely from the endocrine cells of the antral mucosa. They represent approximately 2–4% of all gastric neoplasias and 7.2% of all apudomas of the gastrointestinal tract [10].

In recent decades, there has been evidence that favors believing in an increase in the incidence of gastric apudomas. To a great extent, this increase must be occurring due to the technological evolution of the instruments connected to endoscopy of the upper digestive tract because these tumors are mostly often discovered incidentally during endoscopy. However, a real increase in the prevalence of these tumors cannot be ruled out [11, 23, 24].

The apudomas of the stomach are generally divided into three different groups based on their clinical and physiopathological characteristics: type 1, apudomas of the stomach associated with atrophic body gastritis (ABG), with or without pernicious anemia; type 2, apudomas of the stomach associated with Zollinger-Ellison syndrome, sporadic or familial; and type 3, sporadic apudoma not associated with known predisposing disease [25].
