Clinicopathological heterogeneity between primary and metastatic sites of gastroenteropancreatic neuroendocrine neoplasm

This study retrospectively collected and analyzed data from 779 patients diagnosed with GEP-NENs at Wuhan Union Hospital from 2011 to 2019; specimens of both primary and metastatic sites were available for 35 patients. The diagnosis of NENs mainly depended on the histological morphology as well as the immunohistochemical staining results of various neuroendocrine markers like CgA and Syn. NENs could also be diagnosed by CD56 or neuron-specific enolase when the immunostaining result of CgA or Syn was absent [13]. All specimens were obtained by surgical excision, fine needle aspiration, and/or core biopsy. Patients for whom complete clinical data were not available for extraction were excluded. The following demographic and clinical characteristics of the patients were collected: age, sex, primary and metastatic tumor sites, immunohistochemical results (CgA, Syn and Ki-67 index), and survival time. The grading system used in this study was based on the WHO 5th edition classification (2019) of digestive system tumors, in which NENs are classified as well-differentiated neuroendocrine tumors (NETs), poorly differentiated neuroendocrine carcinomas (NECs), and mixed neuroendocrine–non-neuroendocrine neoplasm (MiNEN). NETs were further divided into three grades based on the Ki-67-positive index: G1: < 3%, G2: 3–20%, G3: > 20% [12]. According to the AJCC cancer staging manual, if the metastasis occurred simultaneously with the primary tumor or within 4 months after the initial resection of the primary tumor, the metastasis was considered to be synchronous; if the metastasis occurred more than 4 months after the initial resection of the primary tumor, it was considered as metachronous [14].

The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IORG number: IORG0003571) and was conducted in accordance with the Declaration of Helsinki.

We performed immunohistochemical staining for CgA, Syn and Ki-67. The primary and metastatic tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks. Each block was cut into 4 μm-thick section, and deparaffinized with xylene and rehydrated with ethanol. Then the sections were incubated with hydrogen peroxide at the room temperature, then antigen retrieval and serum blocking were performed. After these steps, they were incubated with the CgA (M0869, Dako, Glostrup, Denmark), Syn (M7315, Dako) and Ki-67 antibodies (MIB-1, DAKO, Agilent Technologies, Santa Clara, CA, USA) at 37°Cfor 1 h. Then they were incubated with the secondary antibody containing the biotin. Last, slides were visualized by using the DAB (AR1022, BOSTER Biological Technology), stained with hematoxylin, dehydrated with ethanol, transparentized with xylene, and counted.

The staining results for CgA and Syn were graded according to the extent of positive cells as follows: grade I (negative, less than 5% positive tumor cells), grade II (focally positive, 5 to 50% positive tumor cells), grade III (positive, more than 50% positive tumor cells) [15]. For Ki-67 staining, the areas with abundant and more positive tumor cells were selected. The Ki-67 labeling index was determined by calculating the percentage of positive nuclei of 2000 tumor cells in the densest area of each slide [16]. Each slide was reexamined by the pathologist (Q.Z.).

A total of 779 patients with GEP-NENs were included in the analysis; their clinicopathological characteristics were shown in Table 1. There were 460 (59.1%) males and 319 (40.9%) females (male-female ratio of 1.44). The median age at diagnosis was 55 y (range: 13–87 y). Among these 779 patients, the three most common sites of NENs in the digestive tract were the pancreas, rectum and stomach. Pathological diagnosis showed that, among the 779 patients, NET-G1 accounted for 40.2%, NET-G2 18.5%, NET-G3 0.5%, NEC 34.7% and MiNEN 5.1%. Metastasis occurred in 39.9% of the patients (311/779). The positive rates of immunohistochemical staining for CgA and Syn were 57.5 and 85.4%, respectively, and the double-positive rate was 56.6% (441/779). For 35 patients with metastatic specimens, 25 (71.4%) had metastatic tumors simultaneously, and 10 (28.6%) had metachronous metastases.

For 35 patients with both primary and metastatic specimens, the clinicopathological differences between the primary and metastatic sites were analyzed. There was no significant difference in the overall positive rates of CgA and Syn, or in the grading or classification of tumors between the primary and metastatic sites (Supplementary Table 1). However, the variation of CgA, Syn and the Ki-67 index existed between the primary and metastatic sites. Examples of CgA, Syn and the Ki-67 index variation between the primary tumor sites and metastatic sites were shown in Fig. 1.

Of the 35 patients, while 18 (51.4%) had no difference in CgA between the primary and metastatic sites, 13 (37.1%) showed a variation in CgA (Table 2). The CgA variation rate was 42.9% (3/7) for rectal NENs, 42.9% (6/14) for pancreatic NENs, and 50% (3/6) for gastric NENs (Fig. 2a). The CgA variation rate was 33.3% (2/6) for NET-G1, 42.9% (3/7) for NET-G2, 0% for NET-G3, and 40% (8/20) for NEC (Fig. 2b). The CgA variation rate was 16% (4/25) for synchronous metastases and 90% (9/10) for metachronous metastases (Fig. 2c).

With regard to Syn variation between the primary and metastatic sites, while 31/35 (88.6%) patients had no change, 4/35 (11.4%) patients had variation (Table 2). The Syn variation rate was 28.6% (2/7) for rectal NENs, 7.1% (1/14) for pancreatic NENs, and 16.7% (1/6) for gastric NENs (Fig. 3a). With regard to the WHO classification, Syn variation was observed in 20% (4/20) of NEC, but not in NET-G1, NET-G2, or NET-G3 (Fig. 3b). The Syn variation rate was 12% (3/25) for synchronous metastases and 10% (1/10) for metachronous metastases (Fig. 3c).

Our results showed that the mean number and standard deviation of Ki-67-positive cells in primary tumor was 37.89 ± 33.04 (%) and the metastatic lesion was 39 ± 27.48 (%). Ki-67 index variation was observed in 19 patients (54.3%); 8 (42.1%) showed up-regulation (from primary to metastasis) and 11 (57.9%) showed down-regulation. In 16 of these 35 patients (45.7%), despite this difference in Ki-67 index between primary and metastatic sites, there was no difference in classification. Notably, for 3/35 patients (8.6%), the classification changed: 1 patient increased from NET-G1 to NET-G2, 1 increased from NET-G1 to NET-G3, and 1 decreased from NET-G2 to NET-G1 (Table 2). The Ki-67 index variation was seen in 85.7% (6/7) of rectal NENs, 50% (3/6) of gastric NENs, and 35.7% (5/14) of pancreatic NENs (Fig. 4a). Moreover, Ki-67 index variation was as high as 60% (12/20) in NEC, 50% (3/6) in NET-G1, 42.9% (3/7) in NET-G2, and 100% in NET-G3 (Fig. 4b). The Ki-67 index variation was observed in 44% (11/25) of synchronous metastases and 80% (8/10) of metachronous metastases (Fig. 4c).

The Kaplan-Meier survival analysis included sex, CgA variation, Syn variation, Ki-67 index variation, primary tumor site and metastatic tumor site and treatment methods. The results showed that the Ki-67 index variation group had a poorer prognosis than patients in the Ki-67 index non-variation group [hazard ratio 6.800, 95% confidence interval 1.833–25.230; p = 0.0346]. For Ki-67 variation group, there was no significant difference in survival time between NET and NEC groups, between synchronous and metachronous metastatic tumor groups, between Ki-67 index up-regulated and down-regulated groups (Fig. 5g-i). To explore the different treatment methods on patient survival, patients were grouped into four groups including surgery, adjuvant therapy, surgery and adjuvant therapy, and no treatment. The results showed that the prognosis of patients receiving surgery or both surgery and adjuvant therapy was better than that of patients who received adjuvant therapy only (p = 0.0350, p = 0.0109) (Fig. 6).