Delta-like ligand 3 (DLL3) landscape in pulmonary and extra-pulmonary neuroendocrine neoplasms

Abstract

Delta-like Ligand 3 (DLL3) targeting therapies are promising in small cell lung cancer (SCLC) treatment. However, DLL3 expression in SCLC and other neuroendocrine neoplasms (NEN) is heterogeneous and not well characterized. We describe the landscape of DLL3 at the mRNA and protein levels across SCLC, large cell neuroendocrine carcinoma (LCNEC), and non-small cell lung cancer. Additionally, we explore its expression in extra-pulmonary NEN (EP-NEN) using a standardized DLL3 IHC assay. DLL3 expression is enriched in SCLC, LCNEC along with combined histology lung cancers. Moreover, we find a wide range of DLL3 expression in high-grade EP-NEN. We describe heterogenous DLL3 expression not only in SCLC but also in different NEN types. This comprehensive characterization of DLL3 can help guide future clinical trial design targeting DLL3 in NEN including LCNEC and EP-NEN that are lacking standard of care treatment options.

Fig. 1. Gene and protein expression patterns of DLL3 in lung cancer cell lines.

Plots illustrating DLL3 gene (a) and protein (c) expression patterns of small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma (LCNEC), non-small cell lung carcinoma (NSCLC) and showing higher levels of DLL3 gene (b) and protein (d) expression in SCLC and LCNEC cell lines compared to NSCLC. Illustrate higher levels of intracellular/cytoplasmic DLL3 compared to cell surface/membrane location, evaluated by flow cytometry (e) and immunohistochemistry (f). Lung cancer cell lines with known DLL3 expression by IHC. g, h Microphotographs showing different levels of DLL3 IHC expression (negative in H460, moderate in H82 and high in SHP77) (g). Correlation graphs and Total-Scores, Membrane H-Score, and Cytoplasmic S-core. Panels contains Spearman’s correlation coefficient and P values (h).

Fig. 2. Plots showing the levels of DLL3 expression in neuroendocrine neoplasms.

Plots illustrating DLL3 total (a) and membrane (b) H-scores in all samples included in this study. Pulmonary neuroendocrine neoplasms (P-NEN) and Extrapulmonary neuroendocrine neoplasms (EP-NEN) (a, b). c Graph illustrating the levels of Total DLL3 in neuroendocrine carcinomas (Pulmonary and Extra pulmonary) by intensity (0, 1 + ,2 + ,3 + ) of each sample, DLL3 total percentage and P values. d Box plots displaying differences of Total DLL3 H-score between neuroendocrine carcinomas (NEC), epithelial neuroendocrine tumors (E-NET) and non-epithelial neuroendocrine tumors (NE-NET). e Box blots showing differences in Total DLL3 H-score between medullary thyroid carcinoma and other neuroendocrine carcinomas in this study.

Fig. 3. DLL3 IHC expression in patient samples with neuroendocrine carcinomas from different origin.

Representative images of DLL3 IHC staining intensities in four categories from left to right: negative (0), weak (1 + ), moderate (2 + ), and strong (3 + ) lung: small cell lung carcinoma (b) and large cell neuroendocrine carcinoma (a, c, d); uterine cervix: small cell carcinoma (e, f, h), and large cell neuroendocrine carcinoma (g); bladder: small cell carcinoma (i, j, k, l), prostate: AVPC-NEC (m, n, o, p); Merkel cell carcinoma (MCC) of the skin (q, r, s, and t); Head & Neck (H&N): parotid gland neuroendocrine carcinoma (u, x), NEC metastatic from unknowing origin (v), laryngeal neuroendocrine carcinoma (w); and medullary thyroid carcinoma (MTC) (y, z, za, zb) (×40).

Fig. 4. Microphotographs of two combined tumors (large cell neuroendocrine carcinoma with adenocarcinoma).

Left side: low magnification microphotograph of combined tumor stained with H&E (×4). a Miniatures showing higher magnification of the large cell neuroendocrine carcinoma pattern (b) and adenocarcinoma acinar pattern (×40). c Low magnification of the same tumor stained with DLL3 (4X). d Miniatures of the same areas (b, c) showing large cell neuroendocrine carcinoma expressing DLL3 (e), and adenocarcinoma without DLL3 expression (×40) (f). Right side: Microphotograph at medium magnification (×10) of another combined tumor stained with H&E (g). Miniatures showing higher magnification of the large cell neuroendocrine carcinoma pattern (h) and adenocarcinoma solid pattern (i) (×40). Same area stained with DLL3 (j) (10X). Miniatures at higher magnification showing large cell neuroendocrine carcinoma expressing DLL3 (l) and adenocarcinoma without DLL3 expression (k) (×40).