According to the latest World Health Organization(WHO) nomenclature for neuroendocrine neoplasms in 2019, neuroendocrine neoplasm is used to refer to all tumours of neuroendocrine cell origin, of which highly differentiated neuroendocrine tumours are named neuroendocrine tumor and poorly differentiated neuroendocrine tumours are named neuroendocrine carcinoma[5]. Gallbladder NEC is a rare and specific subtype of gallbladder cancer, accounting for less than 2% of all types of gallbladder cancer[6]. Currently, gallbladder NEC is classified using the same American Joint Committee on Cancer (AJCC) staging system as gallbladder adenocarcinomas, although it is known that other gastrointestinal and pancreatic NETs generally have a more indolent clinical course than adenocarcinomas. Some researchers insist that NEC is transformed from gallbladder adenocarcinoma and a mutual conversion of NET and adenocarcinoma in gastrointestinal tract has been proved[7]. Knowledge of the differences and similarities between gallbladder NEC and gallbladder carcinoma is equally limited. In this study, we summarize previous articles and use bioinformatics analysis to compare the clinicopathologic and genetic characteristics of the gallbladder NEC with gallbladder carcinoma.
Genetic Part
According to the results of targeted gene sequencing, 12 mutations types that were private to that specimen (Table 1). To better understand the biologic properties and potential therapeutic targets of this rare tumor, we utilized these potential oncogenes to further explore the biologic mechanisms of tumorigenesis based on several bioinformatics databases. Firstly, Protein-protein interaction networks (PPIs) were analyzed and visualized using the STRING[8] (version: 11.5, https://string-db.org/) database expansion. The outcome of STRING network consisted of 11 nodes and 14 edges with an average local clustering coefficient of 0.776 (PPI enrichment p-value < 0.0001) (Fig. 6A). To explore the potential biological mechanisms, our analysis using GeneMANIA[9] (http://genemania.org/) databases found that these mutated genes associated with transcription regulator complex, ATPase complex, SWI/SNF superfamily-type complex, negative regulation of mitotic cell cycle phase transition, negative regulation of cell cycle phase transition, protein-DNA complex subunit organization, and regulation of metaphase/anaphase transition of cell cycle (Fig. 6B). In addition, protein-protein interaction enrichment analysis was performed using Metascape (https://metascape.org/) database (Fig. 6C-6D).
The transcription factors are important regulators of gene expression and play an important role in the development of tumors. TRRUST[10] (version 2, https://www.grnpedia.org/trrust/) database was used to explore the human transcription factors of these mutated genes. YY1, KAT2B, PAX5, DNMT1, EZH2, FOS, GATA1, SPI1, MYC, E2F1, and TP53 are known to be a pivotal transcription factor related to these mutated genes (Table 2). Using Sangerbox 3.0 (http://vip.sangerbox.com) database, we analyzed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to explore these genes possible biological functions. In the GO analysis of biological processes (BP), we found that these genes may take part in regulation of molecular function, regulation of biological quality, and tissue development (Fig. 7A). In the GO analysis of cellular components (CC), these genes were mainly found to be located in the nucleoplasm, transcription factor complex, and chromosomal part (Fig. 7B). In GO analysis of the molecular function (MF), we found that the genes were mainly enriched in identical protein binding, transcription factor binding, and proximal promoter sequence-specific DNA binding (Fig. 7C). The KEGG enrichment analysis showed that these genes might be involved in pathways in cancer, hepatocellular carcinoma, and human papillomavirus infection (Fig. 7D). These results are similar to those of the Metascape database (Fig. 7E-7F).
Table 2
Key regulated factor of Neuroendocrine carcinoma of gallbladder-related genes.
| Key TF | Description | Overlapped genes | P-value | FDR |
1 | YY1 | YY1 transcription factor | APC, GNAS, TP53 | 1.74E-05 | 1.32E-04 |
2 | KAT2B | K(lysine) acetyltransferase 2B | RB1, SMAD4 | 2.40E-05 | 1.32E-04 |
3 | PAX5 | paired box 5 | RB1, TP53 | 6.45E-05 | 2.36E-04 |
4 | DNMT1 | DNA (cytosine-5-)-methyltransferase 1 | RB1, TP53 | 1.42E-04 | 3.91E-04 |
5 | EZH2 | enhancer of zeste homolog 2 (Drosophila) | APC, TP53 | 2.38E-04 | 5.24E-04 |
6 | FOS | FBJ murine osteosarcoma viral oncogene homolog | SMAD4, TP53 | 4.85E-04 | 7.61E-04 |
7 | GATA1 | GATA binding protein 1 (globin transcription factor 1) | GATA1, GNAS | 4.85E-04 | 7.61E-04 |
8 | SPI1 | spleen focus forming virus (SFFV) proviral integration oncogene spi1 | BTK, GATA1 | 5.73E-04 | 7.88E-04 |
9 | MYC | v-myc myelocytomatosis viral oncogene homolog (avian) | GATA1, TP53 | 1.48E-03 | 1.81E-03 |
10 | E2F1 | E2F transcription factor 1 | RB1, TP53 | 2.64E-03 | 2.90E-03 |
11 | TP53 | tumor protein p53 | RB1, TP53 | 3.92E-03 | 3.92E-03 |
TF transcription factor; FDR False discovery rate. |
Sakaki et al proposed that gallbladder NECs arise from the metamorphosis of gallbladder adenocarcinoma[11]. A recent study performed a whole exome sequencing analysis of 151 gallbladder cancer patients. The results showed that the most common mutated genes in gallbladder cancer included TP53 (27%), KMT2C (11%), SMAD4 (11%), PER3 (8%), ERBB3 (8%), ARID2 (7%), ARID1A (7%) and ERBB2 (7%), and that the most common mutated signalling pathway was that of the ErbB signalling pathway[12]. Based on our findings, the mutational profiles of gallbladder NEC are partly the same as those of gallbladder adenocarcinoma. Therefore, we further analyzed the relationship among them.
Tumorigenesis is based on genetic mutations, which make the nucleus become an ideal target for tumor suppression. The cBioPortal (https://www.cbioportal.org/) database was used to explore the gene mutation information of these mutated genes in gallbladder carcinoma (MSK, Cancer 2018). As was shown in Fig. 8A, a high mutation rate of SMAD4 and TP53 was observed in gallbladder carcinoma patients. In the 101 sequenced gallbladder carcinoma patients, the genetic alteration were found in 31, 59 gallbladder carcinoma patients and the mutation rate were 31%, 58%, respectively. Meanwhile, we found that these genes had the highest mutation rate in gallbladder carcinoma (Gene altered in 74.76% of 103 cases), followed by cholangiocarcinomas (Gene altered in 48.92% of 417 cases) and intrahepatic cholangiocarcinomas (Gene altered in 24.03% of 412 cases) (Fig. 8B). Taken together, these results introduce these mutated genes may as an important player in gallbladder carcinoma development.
KEAP1 is an important tumor suppressor gene. KEAP1 gene mutation reduces the affinity with Nrf2 in the cytoplasm, resulting in the accumulation of Nrf2 in the nucleus, and then promotes the occurrence and development of tumors[13]. Importantly, KEAP1 mutations are found in many types of cancer, including gallbladder cancer[14–16]. Genetic mutations serve as targets in precise therapy for cancer. In present sample, KEAP1 was the most dominant mutated gene with mutation frequency of 87.9%. To explore potential drugs based on KEAP1 mutation, the GSCA (http://bioinfo.life.hust.edu.cn/GSCA/), a web server, was used to explore the drug–gene interactions of mutated genes. As shown in Fig. 8C, the correlation between KEAP1 expression and the sensitivity of GDSC drugs (top 30) in pan-cancer were displayed.
Little is known about the genetic and molecular characteristics of gallbladder NEC, therefore, there is no clinical application of molecularly targeted therapy for gallbladder NEC[17]. There is an urgent clinical need to uncover the molecular markers that contribute to their pathological progression and thus develop new therapeutic modalities. It is reasonable to assume that the gallbladder NEC and the gallbladder adenocarcinoma. are different diseases but closely conected with each other Thus, treatment strategies based on gallbladder adenocarcinoma are not fully applicable to gallbladder neuroendocrine tumours.
Clinicopathologic Part
Almost existing literature on neuroendocrine carcinoma of the gallbladder is limited to case report and case series. The most common symptoms of gallbladder NEC often manifest as epigastric pain, weight loss or anorexia. The meidan age of gallbladder NEC is about 60 years. Most patients with neuroendocrine carcinoma of the gallbladder are female[18]. Gallbladder stones with cholecystitis are thought to promote the development and progression of the gallbladder neuroendocrine carcinoma. As in our case, the gallbladder neuroendocrine carcinoma was accompanied by gallbladder stones. Various imaging modalities are used in the diagnosis of gallbladder NEC, but it is difficult to distinguish it from other types of gallbladder cancer. Ultrasound is used for the initial screening examination, and further enhanced CT or MRI is required to assess the staging of the gallbladder tumour. The investigators considered that most of the gallbladder NEC originated in deep parts of the lamina propria or submucosa and thus showed on CT and MRI that the mucosal epithelium of the gallbladder surface remained partially intact with linear enhancement. Similar descriptions have been found in previous studies of NEC of the gastrointestinal tract[19, 20]. In addition, the size of metastatic lymph nodes in gallbladder NEC was larger than that of adenocarcinoma[21]. But the preoperative accurate diagnosis of gallbladder NEC is challenging to the clinician.
The definitive diagnoses of gallbladder NEC require both pathology and immunohistochemistry. Currently, the most commonly used specific biomarkers available are chromogranin A (CgA), synaptophysin (Syn) and neuro-specific enolase[22]. In a previous study involving 21 patients with gallbladder NEC, more than 80% of cases showed positive staining for CgA and Syn [23]. In another study, there were 15 patients with gallbladder NEC. CgA and Syn had positive rates of 92.3% and 100%, respectively[24]. Similarly, immunohistochemical results of the patients in this study showed chromogranin A and synaptophysin were positive. Then neuro-specific enolase was not verified in immunohistochemistry. In addition, previous study has reported that an elevated Ki-67 index and a high mitotic rate may be strongly associated with poor prognosis[23]. Unfortunately, the cases in this study had high Ki-67 index and mitotic index. Even with post-operative adjuvant chemotherapy, the tumour recurred quickly and the patient died 15 months after surgery.
There are no guidelines or consensus on the treatment options for NECs of the gallbladder worldwide. Usually, the most consistently practiced treatment for gallbladder NECs is complete resection, which is extrapolated from gallbladder adenocarcinoma management. Review of published case reports suggests that the management of gallbladder NETs is highly inconsistent, Early-stage NEC of the gallbladder can achieve better long-term survival with radical surgery. However, local infiltration and/or distant metastases have already occurred at the time of detection for gallbladder NEC, which make it unsuitable for surgical treatment. Chemotherapy is the palliative treatment modality for advanced gallbladder NEC. The effectiveness of chemotherapy for gallbladder NEC is controversial. Wang et al. studied 62 patients with gallbladder NEC, suggesting that postoperative adjuvant chemotherapy had no significant effect on overall survival in gallbladder NEC[3]. In addition, a multicenter, large cohort study in China showed no effect of postoperative platinum-based chemotherapeutic regimens as adjuvant chemotherapy on long-term survival in gallbladder NEC[18]. However, A Japanese case report exhibited that chemotherapy with cisplatin and irinotecan combined with radiotherapy can achieve a complete response to an advanced gallbladder neuroendocrine tumor, with no recurrence after 3 years of follow-up[25]. Ayabe et al [6]documented and compared the survival of patients undergoing resection for gastrointestinal NETs and gallbladder adenocarcinoma using the Large National Database. Then they demonstrated that gallbladder NETs have the worst survival of all gastrointestinal NETs, albeit superior to patients with gallbladder adenocarcinoma. In general, gallbladder NEC have the similar clinical and imaging features, but worse prognosis compared with gallbladder cancer.