We identified 160 NENs and 53 non-NENs to examine the expression of DLL3 and SEZ6 using immunohistochemistry. The staining in positive cases varied from strongly and diffusely positive to focal strong or weak mixed cytoplasmic and membranous staining, and a few cases had diffuse weak positivity. The results of stains in the 160 NENs and 53 non-NENs are summarized in Table 1.

Table 1 Expression of DLL3 and SEZ6 in neuroendocrine neoplasmsPituitary Neuroendocrine Tumors

Among the 18 pituitary neuroendocrine tumors examined, DLL3 expression was observed in eight tumors (44.44%) with average H-score 50, range 10–180, and SEZ6 expression was noted in 11 cases (61%), with average H-score 65, range 10–200. We examined tumors of the various cell lineages and subtypes (Table 2). Among six corticotroph tumors that included two densely granulated, three sparsely granulated, and one Crooke cell tumor, two were positive for DLL3 and three for SEZ6. The strongest expression of DLL3 was observed in a recurrent sparsely granulated silent corticotroph tumor that also had loss of ATRX (Fig. 1). The Crooke cell tumor was negative for both DLL3 and SEZ6. The PIT1-lineage tumors included two densely granulated and one sparsely granulated somatotroph tumors, two sparsely granulated lactotroph tumors, two mammosomatotroph tumors, and one immature PIT1-lineage tumor. Among these eight tumors, six expressed DLL3 focally and weakly and seven expressed SEZ6; interestingly, the aggressive immature PIT1-lineage tumor was negative for DLL3 but expressed SEX6 diffusely (H-score 200). Gonadotroph tumors (n = 4) were negative for DLL3 and only one expresses SEZ6 (H-score 160).

Table 2 DLL3 and SEZ6 immunoreactivity in pituitary neuroendocrine tumorsFig. 1

Immunohistochemical localization of DLL3 and SEZ6 in pituitary neuroendocrine tumors. A recurrent sparsely granulated silent corticotroph tumor stains strongly for DLL3 in about 60% of tumor cells (H-score 180) and has variable positivity for SEZ6 (H-score illustrated 120)

Thymic Tumors

Two well-differentiated grade 2 thymic neuroendocrine tumors had positive staining for both DLL3 and SEZ6 (Fig. 2), with moderate intensity in about 10% of cells for DLL3 (H-score 20 for both), and 20% strong and 40% moderate positivity of cells for SEZ6 (average H-score 70, range 60–80).

Fig. 2

Immunohistochemical localization of DLL3 and SEZ6 in thymic neuroendocrine tumors. A grade 2 thymic NET has weak positivity for DLL3 (H-sore 20) and variable staining for SEZ6 (H-score 80)

Medullary Thyroid Carcinomas

Medullary thyroid carcinomas demonstrated strong expression of both markers. Ten of 11 cases (91%) were positive for both DLL3 and SEZ6 (Fig. 3), with staining intensities ranging from weak to strong in 20–100% of tumor cells for DLL3 (average H-score 199, range 50–300), and with moderate to strong staining intensities in 40–100% of cells for SEZ6 (average H-score 224, range 120–300). Staining was concordant in primary and metastatic foci. Seven tumors were low grade, and four were high grade; while the high-grade tumors tended to have high H-scores (DLL3 300, 300, 285, and 100; SEZ6 285, 200, 200, and 120), there was no correlation with staining intensity or H-score, since some of the low-grade tumors also had high H-scores; however, the only micromedullary thyroid carcinoma was negative for both DLL3 and SEZ6.

Fig. 3

Immunohistochemical localization of DLL3 and SEZ6 in medullary thyroid carcinomas. A high-grade medullary thyroid carcinoma expresses DLL3 strongly and diffusely (H-score 300) and SEZ6 strongly and in most tumor cells (H-score 285)

Parathyroid Neoplasms

We examined five parathyroid adenomas, including a lipoadenoma, a chief cell adenoma, an oncocytic adenoma, and two clear cell adenomas; all these tumors were negative for both DLL3 and SEZ6. Among the four parathyroid carcinomas examined, one (25%) had positivity for DLL3 with weak staining in 30% of cells (H-score 30) (Fig. 4). All cases were negative for SEZ6.

Fig. 4

Immunohistochemical localization of DLL3 in parathyroid carcinoma. The only parathyroid tumor to exhibit positivity for DLL3 was this parathyroid carcinoma (H-score 30)

Lung Neuroendocrine Neoplasms

Well-differentiated lung NETs (n = 11) frequently expressed DLL3, with nine tumors (81.8%) showing positivity ranging from 5 to 100% of tumor cells (average H-score 191; range 5–300) (Fig. 5). SEZ6 expression was less common, observed in only two cases (18.2%) (average H-score 45, actual scores 10 and 80); DLL3 was strongly expressed in these two tumors with H-scores of 240 and 285) (Fig. 5).

Fig. 5

Immunohistochemical localization of DLL3 and SEZ6 in lung neuroendocrine neoplasms. A well differentiated lung NET (top) expresses DLL3 with variable intensity (H-score 190) and SEZ6 focally (H-score 80). A large-cell lung NEC (bottom) has somewhat heterogeneous intense staining for DLL3 (H-score 270) and variable staining for SEZ6 (H-score 70)

Small-cell (n = 1) lung carcinoma expressed DLL3 moderately, and large-cell neuroendocrine carcinomas (n = 2) of the lung demonstrated strong DLL3 positivity (50–90% of cells; average H-score 180 combined, range 120 to 270) in all the tumors examined, including metastatic foci (Fig. 5). SEZ6 expression was seen in one large-cell NEC with weak positivity in 70% of cells (H-score 70) (Fig. 5) and not in small-cell lung carcinoma.

Gastrointestinal Neuroendocrine Neoplasms

Gastric NETs (n = 13) were mostly negative for DLL3; only one metastatic well-differentiated NET had moderate staining in 100% of the cells (H-score 200). SEZ6 positivity was observed in two cases, one type 1 ECL cell NET showing weak to moderate staining in 80% of the cells (H-score 120), and one metastatic well-differentiated grade 3 NET showing moderate staining in 100% of the cells (H-score 200). One gastric NEC was positive for both markers (H-score for DLL3 50 and for SEZ6 40).

Duodenal (n = 10) and ileal EC cell NETs (n = 10) were negative for DLL3. Eight of 10 (80%) duodenal NETs were weakly to strongly positive for SEZ6 in 50–100% of cells (average H-score 206; range 50–300) (Fig. 6). SEZ6 expression in ileal NETs was overall weaker, with and average H-score of 78 (range 30–160) and did not correlate with tumor grade. The two duodenal NECs were negative for DLL3, and one large-cell NEC was weakly to moderately positive for SEZ6 in 80% of the cells (H-score 110) (not shown).

Fig. 6

Immunohistochemical localization of SEZ6 in gastrointestinal neuroendocrine neoplasms. A duodenal gastrin-producing NET (left) has diffuse and strong positivity for SEZ6 (H-score 300). An appendiceal EC cell NET expresses SEZ6 weakly and diffusely (H-score 100 illustrated)

Appendiceal NETs (n = 10) included four L cell NETs and six EC cell NETs. Only two of 10 cases (one L cell NET and one EC cell NET) were weakly positive for DLL3 in 30 to 40% of the cells (average H-score 35, actual score 30 and 40). Eight of 10 cases were weakly to strongly positive for SEZ6 in 50–100% of the cases (average H-score 109; range 70–260) (Fig. 6). Neither stain was preferentially associated with tumor grade.

Colorectal NETs (n = 9) included six L cell and three EC cell tumors. One was metastatic to liver. All were DLL3-negative, with variable weak SEZ6 expression in four cases that included two L cell tumors (44.44%) (average H-score 45, range 10–100) (not shown). Of five colonic NECs, including adenocarcinoma with neuroendocrine differentiation, two were weakly to strongly positive for DLL3 (average H-score 50; actual scores 10 and 90). The only NEC positive for SEZ6 was a poorly differentiated adenocarcinoma with variable neuroendocrine differentiation; it was strongly positive for DLL3 and was also moderately positive for SEZ6 in 20% of the cells (H-score 40).

Pancreatic Neuroendocrine Neoplasms

Well-differentiated pancreatic NETs (n = 11) were largely DLL3-negative, with two G3 NETs showing focal weak positivity in 5% of the tumor cells (H-score 50 for both tumors) (Fig. 7). SEZ6 expression was more common, observed in seven cases (54.5%) with varying intensities, including strong staining in G3 tumors (H-scores ranging from 5 to 100; average: 45) (Fig. 8). While G3 tumors were more frequently positive and had stronger positivity, there was no strict correlation between tumor grade and SEZ6 reactivity.

Fig. 7

Immunohistochemical localization of DLL3 and SEZ6 in pancreatic neuroendocrine neoplasms. A grade 3 well-differentiated pancreatic NET (top) expresses DLL3 only very focally (H-score 5) and SEZ6 focally with some moderate and some strong intensity (H-score 100). A large-cell pancreatic NEC (bottom) has somewhat heterogeneous intense staining for DLL3 (H-score 270) and scant weak staining for SEZ6 (H-score 60)

Fig. 8

Immunohistochemical localization of SEZ6 in paragangliomas. An SDH-deficient extra-adrenal paraganglioma exhibits moderate positivity for SEZ6 in about 50% of tumor cells, yielding an H-score of 100

Pancreatic NECs (n = 2) showed strong DLL3 positivity (50–98% of cells) (average H-score 200) (Fig. 8), and one case had strong SEZ6 expression in 20% of the tumor cells (H-score 60) (Fig. 7).

Paragangliomas and Pheochromocytomas

Paragangliomas (n = 20, of which seven were adrenal pheochromocytomas) included three SDH-deficient tumors, one case of multifocal adrenal pheochromocytoma, one associated with VHL syndrome, one composite pheochromocytoma and ganglioneuroma in a patient with neurofibromatosis, and two metastatic tumors. Three extra-adrenal paragangliomas had weak to moderate DLL3 positivity in 10–100% of the tumor cells (average H-score 43; range 10–100); one was metastatic, one was SDH-deficient, and the other was non-metastatic. SEZ6 expression was variable in six cases, with average H-score of 73 (Fig. 8), range (5–160). Only one SDH-deficient paraganglioma expressed SEZ6.

Merkel Cell Carcinomas

All 13 cases of Merkel cell carcinoma including primary and metastatic disease showed weak to strong DLL3 positivity in 10–100% of cells (average H-score 178, range 10–300) (Fig. 6). SEZ6 expression was variable, with 10 of 13 cases (77%) showing positivity in 30–90% of cells (average H-score 128, actual scores 30 and 200) (Fig. 9).

Fig. 9

Immunohistochemical localization of DLL3 and SEZ6 in Merkel cell carcinomas. DLL3 expression is 60% moderate and 40% strong, providing an H-score of 240. SEZ6 expression is moderate in 90% of tumor cells yielding an H-score of 180

Non-neuroendocrine Neoplasms

We examined 53 non-neuroendocrine neoplasms including moderately and poorly differentiated colonic (n = 10) and pancreatic (n = 11) adenocarcinomas, hepatocellular carcinomas (n = 11), cholangiocarcinomas (n = 11), and high-grade differentiated, poorly differentiated and anaplastic thyroid carcinomas of follicular cell derivation (n = 10). Both DLL3 and SEZ6 were negative with only focal weak positivity for DLL3 in three tumors, a poorly differentiated colonic adenocarcinoma, a multifocal moderately to poorly differentiated hepatocellular carcinoma, and a moderately differentiated cholangiocarcinoma with weak to strong expression in 10 to 30% of the tumor cells (H-score ranging from 20 to 50; average 30). One steatohepatitic hepatocellular carcinoma was moderately positive for SEZ6 in 5% of the tumor cells (H-score 10). Among the 10 thyroid carcinomas, only two poorly differentiated oncocytic carcinomas were weakly positive in 10% of the cells for DLL3 (H-score 10), and only one of those tumors was moderately positive for SEZ6 in 5% of the cells (H-score 10).