Comparison of four DLL3 antibodies performance in high grade neuroendocrine lung tumor samples and cell cultures

Abstract

Background: Small cell lung cancer (SCLC) is usually diagnosed in the advanced stage. It has a very poor prognosis, with no advancements in therapy in the last few decades. A recent phase 1 clinical study, using an antibody-drug conjugate directed against DLL3, showed promising results. A prerequisite for this therapy is an immunohistochemical test for DLL3 expression. The antibody used in the clinical trial was bound to a specific platform, which is not available in all pathology laboratories. In this study, the expression of DLL3 was analyzed using different DLL3 antibodies in high-grade neuroendocrine tumors of the lung and cell cultures. Additionally, correlation of DLL3 expression with Rb1 loss and TP53 mutation was evaluated.

Methods: The study cohort consisted of surgically resected cases, 24 SCLC and 29 large cell neuroendocrine carcinoma (LCNEC), from which tissue microarrays (TMAs) were constructed. The validation cohort included 46 SCLC samples, mostly small biopsies. Additionally, well-characterized SCLC cell lines were used. Immunohistochemical analysis was performed using four different DLL3 antibodies, as well as TP53 and Rb1 antibodies. Expression was evaluated microscopically and manually scored.

Results: The comparison of all DLL3 antibodies showed poor results for the overall agreement, as well as positive and negative agreement. Differences were observed regardless of the applied cut-off values and the tumor type. The antibody used in the clinical trial was the only which always positively stained the tumor cells obtained from cell cultures with known DLL3 expression and was negative on cells that did not express DLL3. There was no correlation between p53 and DLL3 expression in SCLC and LCNEC. RB1 loss in SCLC showed statistical significant correlation with the DLL3 positivity (p = 0.037), while no correlation was found in LCNEC.

Conclusion: The DLL3 antibody used in the clinical trial demonstrated superiority in the detection of DLL3 expression. Cell cultures, which can be used for DLL3 antibodies as positive and negative probes, were established. Evidence of DLL3 expression in high proportions of patients with LCNEC might provide basis for studies of new therapy options in this group of patients.

Keywords: Cell culture; DLL3; Large cell neuroendocrine carcinoma; RB1; Small cell carcinoma; TP53.

Fig. 1

Presentation of correlation of VenA tumor cell positivity in SCLC and LCNEC, with AbcA (a), NovA (b) and TherA (c), none of which was significant

Fig. 2

DLL3 staining in one sample in validation cohort, demonstrating difference between VenA (D) with strong staining intensity in all tumor cells, while all other antibodies (TherA-A, AbcA-B, NovA-C) are showing weaker intensity and positivity is not present in all tumor cells. Bar 50 μm

Fig. 3

Immunocytochemical stainings of NCI-H69s with VenA, AbcA, NovA, and TherA (left to right). VenA shows an intense membranous but also a cytoplasmic staining, the latter concentrated as dots, most likely representing accumulation within endoplasmic reticulum. AbcA stained less than half of the cells, whereas NovA and TherA stained approximately 50% of the cells, but in variable intensity. Bar 50 μm

Fig. 4

Immunocytochemical stainings of NCI-H69a with VenA (a), AbcA (b), NovA (c), and TherA (d). VenA (a) is completely negative in the carcinoma cells, whereas AbcA (b) and TherA (d) stained many cells, and NovA (c) stained almost all cells intensely. Bar 20 μm