Delta-Like Ligand 3 Expression and Functional Imaging in Gastroenteropancreatic Neuroendocrine Neoplasms

Abstract

Purpose: Delta-like ligand 3 (DLL3) is an emerging target across neuroendocrine cancers, but remains underexplored in gastroenteropancreatic neuroendocrine neoplasms (GEP NENs), including poorly differentiated gastroenteropancreatic neuroendocrine carcinomas (GEP NECs) and well-differentiated neuroendocrine tumors (NETs). We aimed to define the landscape of DLL3 expression and feasibility of DLL3-targeted imaging in this population.

Patients and methods: We completed DLL3 immunohistochemistry (IHC) on 379 tumor samples from patients with GEP NENs, analyzing associations between DLL3 IHC positivity, clinicopathologic features, and outcomes. [⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immuno-positron emission tomography-computed tomography (immunoPET-CT) imaging was performed in six patients with DLL3 IHC-positive advanced GEP NENs.

Results: Among GEP NECs, DLL3 expression was identified in 55/78 (71%) tumors, was enriched for small cell histology, and did not demonstrate prognostic significance. Among well-differentiated gastroenteropancreatic neuroendocrine tumors, DLL3 expression was identified in 5/235 (2%) of grade 1-2 and 25/66 (40%) grade 3 (G3) tumors, most commonly G3 pancreatic NETs (PanNETs; 22/52, 43%), with univariate analysis revealing increased mortality risk among patients with DLL3-positive advanced G3 PanNETs (hazard ratio 3.27 [95% CI, 1.09 to 9.78]). Between May 28, 2024, and February 10, 2025, six patients with DLL3 IHC-positive GEP NENs underwent [⁸⁹Zr]Zr-DFO-SC16.56 immunoPET-CT imaging, which delineated DLL3-avid tumor lesions in five of six patients (two of two GEP NECs, three of four G3 PanNETs). Tumor-specific uptake of [⁸⁹Zr]Zr-DFO-SC16.56 varied between patients, with maximum standard uptake values ranging from 7.4 to 36.7, with four of six cases demonstrating DLL3 avidity in ≥50% of tumor lesions.

Conclusion: DLL3 is expressed on a majority of GEP NECs and on a subset of high-grade PanNETs marked by poor outcomes. Functional imaging suggests DLL3 as a promising therapeutic target in both GEP NECs and high-grade PanNETs.

FIG 1.

Landscape of DLL3 IHC expression and clinicopathologic correlates of DLL3 expression in poorly differentiated GEP NECs. (A) DLL3 IHC positivity rates among GEP NECs overall and by primary site of disease. (B) Box and whisker plots describing median, IQR, and distribution of individual DLL3 IHC H-scores among tumor samples from poorly differentiated GEP NECs overall and by primary site of origin. (C) Box and whisker plots describing median, IQR, and distribution of individual DLL3 IHC H-scores among GEP NEC tumor samples with small cell histology (n = 26), large cell histology (n = 21), or histology NOS (n = 31). P value refers to comparison between small cell and large cell groups by Wilcoxon rank-sum test. (D) OncoPrint of genomic alterations by DLL3 status among GEP NEC tumors with available DLL3 IHC and MSK-IMPACT NGS. Only genes with at least 5% prevalence of oncogenic or likely oncogenic alterations in the cohort are displayed. (E) OS from diagnosis of advanced disease by DLL3 IHC status among patients with advanced GEP NECs. P value corresponds to log-rank test. DLL3, delta-like ligand 3; GEP NEC, gastroenteropancreatic neuroendocrine carcinoma; IHC, immunohistochemistry; MSK-IMPACT, Memorial Sloan Kettering-Integrated Molecular Profiling of Actionable Cancer Targets; NGS, next-generation sequencing; NOS, not otherwise specified; OS, overall survival.

FIG 2.

Landscape of DLL3 IHC expression in well-differentiated GEP NETs and clinicopathologic correlates of DLL3 expression in G3 well-differentiated PanNETs. (A) DLL3 IHC positivity rates among well-differentiated PanNETs and small bowel NETs by grade (G1, G2, G3). (B) Box and whisker plots describing median, IQR, and distribution of individual DLL3 IHC H-scores among tumor samples from PanNETs by grade. (C) Distribution of Ki-67 percentages (%) among G3 PanNETs by DLL3 (IHC) positive (n = 22) and negative (n = 30) status. P value refers to comparison between groups by Wilcoxon rank-sum test. (D) OncoPrint of genomic alterations by DLL3 status among G3 PanNET tumors with available DLL3 IHC and MSK-IMPACT NGS. Only genes with at least 5% prevalence of oncogenic or likely oncogenic alterations in the cohort are displayed. (E) OS from time of tumor evaluation by DLL3 IHC in patients with advanced G3 PanNETs. P value corresponds to log-rank test. DLL3, delta-like ligand 3; G1, grade 1; G2, grade 2; G3, grade 3; GEP NET, gastroenteropancreatic neuroendocrine tumor; IHC, immunohistochemistry; MSK-IMPACT, Memorial Sloan Kettering-Integrated Molecular Profiling of Actionable Cancer Targets; NGS, next-generation sequencing; OS, overall survival; PanNET, pancreatic NET.

FIG 3.

[⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging in patients with advanced poorly differentiated GEP NECs. (A-C) A patient with poorly differentiated pancreatic NEC metastatic to liver with previous progression of disease on carboplatin and etoposide and CAPOX underwent [⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging (patient 1). (A) H&E and DLL3 IHC images from a liver metastasis biopsy before start of systemic therapy, revealing DLL3 IHC H-score of 270. (B) MIP and (C) axial images from DLL3 immunoPET-CT, with corresponding CT completed 2 days after DLL3 immunoPET-CT. (D-I) A patient with gallbladder NEC with multiple previous lines of systemic therapy underwent [⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging after previous progression of disease on DLL3 TCE therapy (patient 2). (D) DLL3 IHC from a liver metastasis at biopsy confirmation of NEC histology before DLL3 TCE therapy revealed IHC H-score of 160. (E) Serial CT scans on DLL3 TCE therapy revealing partial response followed by progression of disease. At progression of disease on fourth-line carboplatin/etoposide, [⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT was completed, with (F) MIP and (H and I) axial images highlighting brain and liver metastases. (G) Biopsy of DLL3 PET-avid left supraclavicular nodal metastasis (corresponding to arrow in MIP image) revealed retained DLL3 IHC expression (H-score 120). For MIP and axial images, SUV scales are displayed. For MIP images, pixels in PET imaging with SUV 0 appear white and pixels with SUV ≥ upper thresholds appear black. For axial images, pixels with SUV 0 and SUV ≥ upper thresholds appear black and bright yellow, respectively. CAPOX, capecitabine and oxaliplatin; CT, computed tomography; DLL3, delta-like ligand 3; GEP NEC, gastroenteropancreatic neuroendocrine carcinoma; H&E, hematoxylin and eosin; IHC, immunohistochemistry; MIP, maximum-intensity projection; PET, positron emission tomography; SUV, standard uptake value; TCE, T-cell engager.

FIG 4.

[⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging in patients with advanced G3 well-differentiated PanNETs. (A and B) A patient with G3 PanNET metastatic to liver underwent [⁸⁹Zr]Zr-DFO-SC16.56 DLL3 immunoPET-CT imaging at progression of disease on third-line ¹⁷⁷Lu-DOTATATE (patient 5). (A) H&E and DLL3 IHC images from the patient's pancreatic primary tumor before start of systemic therapy. (B) MIP images from DLL3 immunoPET-CT and ⁶⁸Ga-DOTATATE PET-CT imaging completed 12 weeks before DLL3 immunoPET-CT, revealing 100% of tumor lesions positive for both DLL3 and SSTR avidity. (C and D) A patient with G3 PanNET metastatic to liver, lung, and mediastinum underwent DLL3 immunoPET-CT after progression of disease in the liver on second-line capecitabine and temozolomide. (C) H&E and DLL3 IHC images from progressive liver metastases, revealing DLL3 IHC 120. (D) MIP images from DLL3 immunoPET-CT and ⁶⁸Ga-DOTATATE PET-CT imaging completed 5 weeks before DLL3 immunoPET-CT, revealing DLL3 uptake in the progressive liver metastasis (SUV_max 14.4); the remainder of the disease was otherwise DLL3 PET-negative and SSTR PET-positive. The arrow notes progressive liver metastatic lesion, which was biopsied for DLL3 IHC evaluation. For all images, SUV scales are displayed, with pixels in PET imaging with SUV 0 appearing white and pixels with SUV ≥ upper thresholds appearing black. CT, computed tomography; DLL3, delta-like ligand 3; G3, grade 3; H&E, hematoxylin and eosin; IHC, immunohistochemistry; MIP, maximum-intensity projection; PanNET, pancreatic neuroendocrine tumor; PET, positron emission tomography; SSTR, somatostatin receptor; SUV, standard uptake value.