Immunohistochemistry (IHC)

IHC staining was performed according to the manufacturer's instructions, using the Ventana Discovery (Ventana Medical System) automated staining system. In brief, slides were incubated with the primary antibody DLL3 (clone SP347, Ventana, SN 678, RTU). Representative tumor blocks from FFPE tissue were cut in 4-μm thick sections.

DLL3 staining was considered positive if staining was membranous in at least 1% of tumor cells. Protein expression of DLL3 was assessed in two different ways: on the one hand by estimating the percentage of positive tumor cells from all tumor cells (tumor proportion score, TPS, range 0–100%) as previously described (5) and, on the other hand, semi-quantitatively using the H-score method according to Yan et al. by converting the staining intensity (SI) (range 0–3) and the TPS (range 0–100) to a H-score (range, 0–300) (10). SI was indicated as strongly positive (SI 3), moderately positive (SI 2), weakly positive (SI 1) and negative (SI 0). To dichotomize samples into positive and negative staining, TPS < 50% and H-score ≤ 150 were defined as DLL3 low expression (DLL3-low), and TPS ≥ 50% and H-score > 150 were defined as DLL3 high expression (DLL3-high) (5, 10).