Insights into gastric neuroendocrine tumors burden.

Type 1 gastric neuroendocrine tumors (gNETs) are usually small lesions, restricted to mucosal and sub-mucosal layers of corpus and fundus, with low aggressive behavior, for the majority of cases. Nevertheless, some cases present aggressive behavior. The increasing incidence of gNETs brings together a new relevant problem: how to identify potentially aggressive type 1 gNETs. The challenging problem seems to be finding out signs or features able to predict potentially aggressive cases, allowing a tailored approach, since the involved societies dedicated to provide guidelines for management of these neoplasms apparently failed in producing staging systems able to accurately predict prognosis of these tumors. Additionally, it is also important to try to find out explanations for increasing incidence, as well as to identify potential targets aiming to reach better control of this neoplasia. Here, we discuss potential pathways implicated in aggressive behavior, as well as new strategies to improve clinical management of these tumors.

Introduction

Gastric neuroendocrine tumors (gNETs) are rare, occurring in 1 to 2 cases/1,000,000 persons per year, and accounting for 8.7% of all gastrointestinal neuroendocrine tumors (1), less than 2% of all neuroendocrine tumors and less than 1% of all gastric cancers (1-3). However, incidence of gNETs has increased in most countries over the past decades, in part because of better awareness of the disease among physicians, improved diagnostic techniques and more widespread use of upper gastrointestinal endoscopy (4). In many countries, including Brazil, epidemiological data on gNETs are scarce (5).

The increasing incidence of gNETs (6,7) brings together a new relevant problem: how to identify potentially aggressive type 1 gNETs.

Sporadic type 1 gNETs occur secondary to autoimmune gastritis, since antibodies against parietal cell from the gastric corpus cause atrophy of these cells and therefore reduce acid secretion (8). The resultant absence of acid implies in hyperplasia of G cells from the normal antrum, producing hypergastrinemia in an effort to stimulate the corpus parietal cells to start producing acid and re-establishing normal stomach pH. Since the atrophic parietal cells will not respond to gastrin stimulus, the hypergastrinemia is maintained and finally provokes enterochromaffin-like cell (ECLC) hyperplasia. As the stimulations persist, the ECLC suffers dysplasia and finally gives origin to type 1 gNETs (9).

Some hypotheses try to explain the increment in type 1 gNETs: 1) there is an ongoing global shift characterized by decrease of infection diseases and rise of immune diseases, and this includes reduction of gastricHelicobacter pylori (H. pylori) infection and intensification of autoimmune gastritis (10-13); 2) the excessive and liberal utilization of proton pump inhibitors (PPIs) provokes elevation of gastrin levels and this could contribute to higher indices of type 1 gNETs (14); 3) the incidence of type 1 gNETs remains the same, but the higher number of endoscopy and improvement on quality of the exams and pathology analyses allowed the discovery of tumors that already existed (15); and 4) there are new yet unknown driven forces implicating in the actually increasing incidence.

None of the cited hypotheses seems to fulfill the gaps on the mechanism of this epidemiologic phenomenon. Moreover, management of type 1 gNETs remains a challenge for clinicians, surgeons and oncologists, as will be discussed below.

Clinical behavior and management

Type 1 gNETs are usually small lesions, restricted to mucosal and sub-mucosal layers of corpus and fundus, with low aggressive behavior, for the majority of cases, although being multiple and recurrent. Lymph nodes and distant metastasis are rare, but do occur (16,17).

Since this low metastatic potential and indolent behavior is the rule, recommendations from the majority of medical societies dedicated to NET management include surveillance, endoscopic resection of prominent lesions and, less frequently, surgical approach (18).

For multiple and more prominent lesions, the National Comprehensive Cancer Network (NCCN) guidelines consider antrectomy as a possible treatment strategy. Antrectomy should eliminate G cells and control hypergastrinemia, ending the stimulus to ECLCs (18). Although there are some followers of this approach, especially among United States surgeons (19), it remains an issue of intensive debate.

If antrectomy is reserved for patients at higher risk, and risk could be understood as local tumor aggressiveness including lymph nodes metastasis, as well as distance metastasis dissemination, antrectomy would not eliminate these possibilities, since the tumors are located at corpus and fundus, away from the resected antrum. Additionally, antrectomy does not include lymphadenectomy, neither as a staging process nor as a therapeutic measure (20-23).

In other words, if the supposed high-risk lesion is already an aggressive tumor, it might harbor local malignant features, as local invasion and lymph nodes metastasis, not reached by the antrectomy procedure. Additionally, antrectomy brings risk of complications and even mortality (24). Moreover, the recurrence rate is not completely abolished, since in some cases G cells are not completely removed (25).

By the other side, the conservative management is also unable to treat the aggressive cases, as extended disease and lymph nodes metastasis are not addressed by this approach.

Although the great majority of type 1 gNETs present indolent clinical course, with very low disease related mortality rates, so the lethality is currently considered low, the rising incidence causes a possible shift in mortality index.

The challenging problem seems to be finding out signs or features able to predict the potentially aggressive cases, allowing a tailored approach. The involved societies dedicated to provide guidelines for management of these neoplasms apparently failed in producing staging systems able to accurately predict prognosis of these tumors. Neither the World Health Organization (WHO) classification, nor European Neuroendocrine Tumor Society (ENETS) staging or even the American Joint Committee on Cancer (AJCC) staging for low-grade gNETs had high accuracy in prognosis prediction (26-28).

Tumor behavior is supposed to be a consequence of the molecular profile, as it was demonstrated for many tumor types. Therefore, a joint effort to provide data on molecular signatures of type 1 gNETs is urgent to fight against this growing medical problem.

Growing incidence hypotheses

As mentioned before, the increase in incidence of type 1 gNETs is not well explained. The main characteristics of these tumors are: hypergastrinemia and elevated stomach pH. Even though autoimmune gastritis is currently recognized as the unique origin of the disease, by causing corpus atrophy (9), several hypotheses have been considered to explain the occurrence of so many new cases, and some of that will be briefly discussed.

Autoimmune gastritis augmentation

The augmentation of immune diseases and diminishing of infections are a reality affecting diverse organs and systems, and might partially explain the growing incidence of autoimmune gastritis, taking in mind only the epidemiologic evidences (11,12). However, the mechanism of this shift in gastric mucosa requires further investigation.

The hypothesis ofH. pylori antigens mimicking parietal cell antigens causing immune reaction to self gastric cells could explain an extra etiology and by so, contribute to new cases of type 1 gTNEs (29). Nevertheless, this new contributing factor —H. pylori infection, is decreasing in recent years, bringing doubt to real impact of this mechanism on rising incidence of these tumors. Additionally, the ongoing shift on infection versus immune disease incidence implies in decreasing of the first impacting on increasing the last, instead of a collaborative pattern.

PPIs role

PPIs are largely used, even without medical prescription, to treat gastroesophageal reflux disease, peptic ulcers, gastritis, and to alleviate dyspeptic symptoms (14,30,31). The main mechanism of action is through impairment of H-K pump, leading to a diminishing of acid secretion and consequence elevated gastric pH (32). In the absence of physiologic acid pH, gastrin is secreted, as discussed above, to recover acid secretion, normalizing stomach pH. If blockage of proton pump is maintained by continuous PPIs administration, hypergastrinemia could cause a stimulus to ECLC and eventually favor type 1 gNETs occurrence (14,33).

This hypothesis is not proved, neither completely rejected, since there are few well-controlled trials addressing this evidence.

Aiming at shedding light on this issue, Calveteet al. (34) recently described a family with consanguineous parents and ten children, five of whom are affected by type 1 gNETs. They had atypical clinical behavior including: an earlier age of onset (around 30 years), high aggressiveness (3 with lymph node infiltration, and one with a synchronous focus of adenocarcinoma); iron-deficiency, rather than megaloblastic anemia (34). They identified a homozygous missense mutation in the 14th exon ofATP4A gene (c.2107C>T), which encodes the proton pump, and is responsible for acid secretion by gastric parietal cells. This mutation originates from a change in one of the transmembrane domains that avoid the liberation of protons from cells to stomach lumen, causing the achlorhydria observed in the affected individuals. Interestingly, no germline or somatic mutations inATP4A gene were found in sporadic gastric NET patients (34). Then the group described a mouse model for theATP4A mutation. Homozygous mice developed premalignant condition with severe hyperplasia, dysplasia and glandular metaplasia in the stomach. Furthermore, when the homozygous mice were treated with 3% HCl acid in the drinking water, the development of glandular metaplasia and dysplasia were prevented (if treated from birth) or partially reverted (if treated during adulthood) (35).

Although this model did not reproduce typical human disease, it represents a new perspective in understanding molecular pathways leading to more aggressive behaviors, as well as, novel approaches to control the disease.

Cholecystokinin B receptor (CCK2R)

Gastrin is a peptide secreted by neuroendocrine G cells that triggers the release of hydrochloric acid by parietal cells, and binds to cholecystokinin B receptors, known as CCK2R and CCKBR, to produce its effects (36,37).

Besides its role in gastric acid secretion, some studies have revealed relevant cellular functions of gastrin, including regulation of proliferation, migration, invasion, differentiation, angiogenesis and apoptosis (38-40). These effects are also achieved by its binding to CCK2R, which in turn triggers downstream signaling involving many important pathways, such as protein kinase C (PKC), phosphatidyl inositol 3’-kinase (PI3K) and mitogen activated protein kinase (MAPK) (38,40).

Gastrin is also known to play an important role in the development of gastric adenocarcinoma (41,42) in addition to its participation in gNETs development (38).

To investigate the molecular mechanisms by which gastrin promotes tumor development, many studies using CCK2R expressing gastric cancer cell lines were performed. Sunet al. (43) observed that the proliferation of MKN-45 cells decreased when treated with CCK2R antagonist. Accordingly, the AGS-B cell line transfected with human CCK2R was found to proliferate more rapidly in the presence of gastrin, and it was correlated with the upregulation of cyclin D1 (44).

Sunet al. (43) also demonstrated that MKN-45 has been shown to be more susceptible to apoptosis when treated with a CCK2R antagonist, and this was associated with upregulation of Bax (proapototic protein) and downregulation of Bcl-2 (antiapoptotic protein). Similarly, Pritchardet al. (45) observed that gastrin increases mcl-1 (antiapoptotic member of the bcl-2 family of proteins) expression in type 1 gNETs and in a gastric epithelial cell line that expresses the CCK2R.

Furthermore, gastrin has been shown to increase cyclooxygenase-2 (COX-2) secretion in AGS-E cells transfected with human CCK2R (AGS-GR) via an Akt-dependent mechanism (46-49). Importantly, Xuet al. (50) demonstrated that antagonizing or silencing CCK2R blocked activation of signal transducers and activators of transcription 3 (STAT3) and Akt induced by gastrin in gastric cancer cell lines. Moreover, they stated that gastrin-induced COX-2 overexpression and cell proliferation were blocked by antagonizing CCK2R and inhibiting PI3K and Janus kinase 2 (JAK2). In addition, STAT3 silencing significantly attenuated COX-2 expression, and PI3K/Akt activation, as well as cell proliferation stimulated by gastrin. These data strongly suggest that CCK2R has a key role in the proliferative effect of gastrin on human gastric cancer cells, by inducing overexpression of COX-2 through JAK2/STAT3/PI3K/Akt pathway (50).

AGS-GR cells have also been used to elucidate the mechanisms responsible for the effects of gastrin on cellular migration and invasion. In the presence of gastrin, AGS-GR morphology was modified acquiring a branched shape, remodeling the cytoskeleton. These effects were not observed when cells were treated with a CCK-2 receptor antagonist (51).

One of the mechanisms by which gastrin leads to an increase in cell migration was recently described by Lloydet al. (52). They observed that overexpression of miR-222 induced by gastrin is followed by a decrease in the expression of p27in vitro andin vivo, via activation of CCK2R and subsequent PKC and PI3K pathways. Reduced expression of p27, therefore, triggered actin remodeling and increased migration in AGS-GR cells. Interestingly, miR-222 expression is increased in the serum and gastric corpus of patients with hypergastrinemia and type 1 gNETs, and is significantly reduced when patients are treated with a CCK2R antagonist. Since intervention on miRNAs expressions represent an important perspective in many human’s disease, exploring the role of miR-222 and its interactions on regulation of gNETs mechanism and pathways might be relevant to future molecular approaches aiming to block these tumors development.

CCK2R polymorphisms

One hypothesis to explain the occurrence of gNETs or even differences in disease behavior could be the presence of polymorphisms inCCK2R gene, modifying the quantity or structure of the encoded protein, resulting in the amplification of the downstream response, which is known to be associated with tumor development. Although there are many single nucleotide polymorphisms (SNPs) described in genomic databanks [353 in National Center of Biotechnology Information (NCBI), of which 253 are missense], none was associated with the development of type 1 gNETs. However, one in particular (C>A; rs1800843) has been correlated with risk for pancreatic cancer (53,54).

This SNP occurs in position 32 of the 4th intron ofCCK2R gene and originates from a novel splice variant of this gene with retention of intron 4, resulting in 69 additional amino acids ate portion of the receptor involved in signal transduction and cell proliferation (54,55).

Interestingly, the presence of A allele significantly increases aggressiveness and shortens survival of patients with pancreatic cancer. Although few patients with the AA genotype presented advanced stage of the disease compared to patients with the wild genotype CC, the survival of these patients was shorter (53).

These evidences highlight the need to investigate polymorphisms inCCK2R gene, which can bring extremely important information to understand the occurrence of type 1 gNETs, and also shed light on mechanism implicated in disease aggressiveness. Access to new gene sequencing technologies might improve the discovery of new SNPs eventually associated with disease behavior, accelerating the translation of the knowledge to clinical practice.

Targeting CCK2R

In non-clinical studies, netazepide is a potent and highly selective antagonist for CCK2R that has good oral bioavailability and effectively suppresses gastric acid secretion (56).

In healthy subjects, netazepide and the PPI rabeprazole were similarly effective in suppressing pentagastrin-stimulated gastric acid secretion and increasing serum gastrin level. Rabeprazole increased plasma chromogranin A (CgA), a sign of ECLC hyperactivity, whereas netazepide reduced plasma CgA, a sign of ECLC hypoactivity. Netazepide also prevented the increase in CgA resulted from rabeprazole-induced hypergastrinemia, probably by blocking CCK2R on ECLC (57). A clinical trial in patients with type 1 gNETs and autoimmune chronic atrophic gastritis had showed that netazepide can eradicate type 1 gNETs and is an alternative to regular gastroscopy management or even surgery (58).

There is accumulating evidence that gastrin influences tumor development by binding to CCK2R, which highlights potential role of netazepide as a targeted therapy, in addition to, or as an alternative, to traditional treatments of patients with gNETs.

In this regard, it is important to mention that the current therapy seems to be unable to control more aggressive type 1 gNETs. According to the majority of epidemiological data, at least 5% of patients with type 1 gNETs will experience a high aggressive disease, with regional, and even distal metastasis, not addressed by conventional recommended approaches. Moreover, it is actually not possible to identify these aggressive tumors at early stages, before metastasis development. Since netazepide seems to block the pathways involved in the disease occurrence, it is supposed to be effective in stopping the disease, regardless of being indolent or aggressive (9,23).

Risk prediction

Since most of discussed topics are still elusive, and clinicians need references for management of these tumors, it is important to take in mind the available information on risk factors for aggressive behavior. The occurrence of tumor size over 1 cm, deep of penetration beyond mucosa layer, and early recurrence after endoscopic treatment are signals of more aggressive disease and should require rigorous follow up, clinical intervention, or even surgical approach. If confirmed by large series and multi-institutional investigations, the identification of individuals with polymorphisms inCCK2R gene should also be consider as additional risk factor for medical decision.