Gallbladder neuroendocrine cancer (GBNEC) is a rare and aggressive disease that accounts for less than 0.2% of all gastrointestinal neuroendocrine cancers (1–3). Published medical literature reported that GBNEC had a more aggressive biological behavior and a poorer prognosis than other well-differentiated gallbladder neuroendocrine neoplasms (GBNEN) (4–8). The microscopy diagnosis of poorly differentiated neuroendocrine tumor cells serves as the cornerstone in the diagnosis of GBNEC (9, 10). Currently, for patients with resected GBNEC, surgical resection is the first-line treatment. The role of adjuvant therapy in the management of GBNEC is not well established.

Patients with gallbladder adenocarcinoma (GBADC) may have a tumor containing a GBNEC component on microscopy (11, 12). According to the 2019 World Health Organization (WHO) classification, if GBNEC components represent≥30% of the tumor mass, the mixed tumor is classified as gallbladder mixed adeno-neuroendocrine carcinoma (GBMANEC) (13). GBMANEC is a distinct subtype of gallbladder mixed neuroendocrine-non-neuroendocrine neoplasm (GBMiNEN), which is characterized by ≥30% GBNEC component of the tumor mass (9). Our institution has encountered cases where patients were diagnosed with GBADC or gallbladder squamous cell carcinoma with a small proportion of GBNEC (GBNEC<30%) within the tumor mass. These patients did not fulfill the diagnostic criteria for GBMiNEN as defined by the 2019 WHO classification (13). There is limited clinical evidence for these patients, as the disease incidence rate is very low.

The biological characteristics of the tumor determine the prognosis of the patient. Machairas N (14) and de Mestier L (15) concluded that in patients with mixed gastrointestinal neoplasms, the prognosis was related to the most aggressive neoplastic elements, regardless of their extension in the tumor. The WHO 2019 has defined GBMiNEN using a cut-off value of 30% (13). However, this threshold is considered inappropriate and lacks clinical support (9, 16). The aggressiveness of the tumor may not be directly related to the percentage of aggressive GBNEC elements. Published medical literature found that tumors with even a small proportion of primary GBNEC lesions may have a higher risk of developing deep infiltration or distant metastases than those without (9, 14, 16, 17). Unfortunately, due to low -incidence rate, clinical management for these patients were largely unknown. Therefore, we aim to investigate the clinicopathological features and prognosis of pure GBNEC (i.e.only the GBNEC component of the tumor mass), GBMANEC (i.e. GBNEC ≥30%) and GBADC or GBSCC, both mixed with a small fraction of GBNEC (GBNEC <30% of the tumor mass). The study also determined the factors associated with the long-term prognosis in these patients.

In this study, we included patients with pure GBNEC, GBMANEC, and GBC mixed with a small fraction of GBNEC (GBNEC component of the tumor mass<30%) to investigate the clinicopathological features and long-term prognosis difference of these neoplasms, and we noticed patients with GBC mixed with a small fraction of GBNEC (GBNEC<30%) showed a prognosis as dismal as patients with pure GBNEC and/or GBMANEC (GBNEC component of the tumor mass≥30%) in the present study.

Epithelial neoplasms with co-existing neuroendocrine and non-neuroendocrine histology (each representing from 1% to 99% of the tumor mass) have been described in almost all organs (21). These mixed neuroendocrine/non-neuroendocrine neoplasms have different definitions over time. In 2010, mixed neoplasms of the gastrointestinal and pancreatic tract containing neuroendocrine and non-neuroendocrine components in at least 30% of the tumor mass were classified separately by the World Health Organization and named MiNEN (19, 22, 23). In 2017, although the WHO also renamed MANEC as MiNEN which expanded the diagnostic range of mixed neuroendocrine tumors, the 30% threshold for each component has been maintained (18, 24). The rationale behind the 30% threshold is that a lesser-represented tumor component is unlikely to influence the biological behaviors of the whole neoplasm. However, the threshold was not supported by any clinical evidence of its relevance or significance to patients’ prognosis.

GBNEC have an aggressive biological behavior and inferior prognosis than other well-differentiated gallbladder neuroendocrine neoplasms or GBADC (25, 26). Lee et al. (27) included 34 cases of GBNECs and found that compared with stage-matched GBADCs both 1- and 3-year OS rates of patients with GBNEC were poorer than patients with GBADC (P < 0.001). Besides, clinical studies pointed out that the prognosis of GBMANEC was also worse than that of pure GBADC (4, 5, 17, 27–29). Thus, they suggested that the prognosis of digestive mixed neoplasm was determined by the most aggressive tumor component (9, 16). Simultaneously, gastric NECs and (or) gastric MANECs were found worse prognoses and more frequent recurrence than those with gastric ADC in a multicenter analysis (30). Unfortunately, multicenter large-sample studies comparing the long-term prognosis of pure GBNEC, GBMANEC, and GBADC are lacking due to their low incidence rate. Most analyses of GBMiNEN were case reports or series. Costa et al (31) published a literature review in 2021. They reported that there were only 24 cases of GBMiNEN. Although most research results support that the biological behavior of mixed neoplasms is determined by the most aggressive tumor component, whether the different proportions of these components will affect the prognosis of these GBMiNEN patients has not been reached consensus. In fact, some scholars of the gastric neopalsm have pointed out that tumor components’ aggressiveness may not be associated with these elements’ percentage (30). However, for gallbladder mixed neoplasm, this information is limited. After searching, we found 2 cases of GBADC mixed with a small fraction of NEC (16, 32). Both authors reported that GBADC mixed with a small fraction of GBNEC (GBNEC component ≤30%), which did not meet the criteria for GBMiNEN, showed rapid progress in a few months. Meanwhile, the accuracy of the WHO’s 30% threshold cannot be verified because we found no clinical studies presented the value of GBNEC components proportion of in the GBMiNEN tumor mass. In this study, we observed comparable survival duration for patients with pure GBNEC, GBMANEC, and those of GBADC mixed with a small fraction of GBNEC. In addition, we also found that the OS of patients with GB-SCC mixed with a small fraction of GBNEC was significantly poorer than that of GBNEC, GBMANEC, and GB-ADC mixed with a small fraction of GBNEC. Thus, our outcomes were not in favor of a 30% neuroendocrine component as a criterion to stratify GBNEC patients with different prognoses.

Which factors can be used to differentiate GBNEC patients’ prognosis other than 30% neuroendocrine component? Unfortunately, this question has not been answered in previous clinical investigations of GBNEC. However, a large cohort of clinical studies of gastrointestinal NEC has shown that NEC is a malignant component with a poor prognosis and there is no significant relationship with the proportion of NEC components (30, 33). Our results also have comparable findings. We noticed there is no significant OS differences between patients with GBNEC ≥30% and those with GBNEC <30%. Besides, patients with GB-SCC mixed with a small fraction of GBNEC (GBNEC<30% of the tumor mass) had the most inferior survival prognosis. The presence of highly malignant components of the tumor mass plays a key role in the patient’s long-term survival.

GBNEC is aggressive and may require extensive surgery resection to obtain a negative margin. Some studies supported aggressive treatment including extensive surgical resection and adjuvant radio-chemotherapy may be necessary for patients with GBNEC (11, 12, 29, 34). However, there were no clinical guidelines for patients with GBNEC or GBMiNEN, most treatment methods followed that of GBADC. In a recent study, Liu (3) and colleagues identified the mutation landscape associated with GBNEC, and their findings supported specific pathogenesis features of GBNEC. Thus, we hypothesize that the treatment of GBNEC should be different from GBADC. Previous studies have reported different findings. Lin (34) et al. found a GBMANEC case and suggested that a combination of surgery, ACT, and somatostatin treatment could lead to improved survival. However, Zhang et al. (29) concluded that extensive surgical resection remains the only key treatment for patients with GBNEC. In other studies, the benefits of curative resection for GBNEC were not clarified. We hypothesize this may be due to the aggressive nature of GBNEC that most patients were diagnosed in late stages, thus the patients may experience recurrence soon after surgical resection.

The study had several limitations. First, our patient data were collected from a referral medical center and thus may have introduced selection and information loss biases. Some late-stage patients were not included in the queue due to losing the opportunity for surgery. Additionally, the long follow-up span may result in poor compliance and follow-up biases, which could have affected the timely recording of specific survival outcomes for the included patients. Third, differences in patients’ living standards could have also introduced biases related to the prognosis. Fourth, the use of different immunohistochemistry (IHC) panels over time may lead to inconsistent immunohistochemistry markers for GBNEC diagnosis.

The presence of 30% GBNEC may not necessarily be the best tool for the differentiation of GBNEC and GBMiNEN. The presence of malignant tumor components may be more important in providing valuable prognostic information on survival for patients with mixed tumors. Patients with either pure or mixed GBNEC, displaying advanced tumor stages and lymphovascular invasion were at an independently increased risk of worse OS and RFS. Due to the small sample size of the study, the conclusions drawn above need to be validated by multicenter large-sample studies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

This study was conducted in accordance with the Declaration of Helsinki and in compliance with the study protocol and ethical guidelines for medical and health research involving human subjects. The study was approved by the institutional review committee of West China Hospital, Sichuan University (approval code 2021-445). A summary of the protocol of the study is available on the website of the hospital. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

Y-FH: Writing – original draft. J-KW: Writing – original draft, Resources, Writing – review & editing. W-JM: Resources, Writing – review & editing. H-FG: Resources, Writing – review & editing. H-JH: Resources, Writing – review & editing. FL: Resources, Writing – review & editing. T-RL: Resources, Writing – review & editing. S-QY: Resources, Writing – review & editing. Y-SD: Resources, Writing – review & editing. R-QZ: Resources, Writing – review & editing. F-YL and Y-WJ: Writing – review & editing.