Cancer Res Treat. 2019;51(3):1231-1240.

Alterations in PD-L1 Expression Associated with Acquisition of Resistance to ALK Inhibitors in ALK-Rearranged Lung Cancer.

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제1저자 김수정, 김소연 · 교신저자 김동완

PMID 30653748 PMC6639241 DOI 10.4143/crt.2018.486 IF 4.2 혈액종양내과

소속 (5)

Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.
Department of Internal Medicine, Seoul National University, Seoul, Korea.
Department of Pathology, Seoul National University Hospital, Seoul, Korea.
Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.

Abstract

PURPOSE The purpose of this study was to evaluate the relationships between the resistance of anaplastic lymphoma kinase (ALK)‒positive non-small cell lung cancer (NSCLC) to ALK inhibitors and the programmed cell death-1/programmed cell death-ligand 1 (PD-L1) pathway, we evaluated alterations in PD-L1 following acquisition of resistance to ALK inhibitors in ALK-positive lung cancer.

MATERIALS AND METHODS We established ALK inhibitor-resistant cell lines (H3122CR1, LR1, and CH1) by exposing the parental H3122 ALK-translocated NSCLC cell line to ALK inhibitors. Then, the double-resistant cell lines H3122CR1LR1 and CR1CH1 were developed by exposing the H3122CR1 to other ALK inhibitors. We compared the alterations in PD-L1 expression levels using western blotting, flow cytometry, and quantitative polymerase chain reaction. We also investigated gene expression using RNA sequencing. The expression of PD-L1 in the tumors from 26 ALK-positive metastatic NSCLC patients (11 ALK inhibitor-naïve and 15 ALK inhibitor-resistant patients) was assessed by immunohistochemistry and analyzed.

RESULTS PD-L1 was expressed at higher levels in ALK inhibitor-resistant cell lines than in the ALK inhibitor-naïve parental cell line at the total protein, surface protein, and mRNA levels. Furthermore, PD-L1 expression in the double-resistant cell lines was much higher than that in the single resistant cell lines. RNA sequencing demonstrated that expression of immune-related genes were largely involved in ALK inhibitor resistance. The mean value of the PD-L1 H-score was 6.5 pre-treatment and 35.0 post-treatment, and the fold difference was 5.42 (p=0.163).

CONCLUSION PD-L1 expression increased following acquisition of ALK inhibitor resistance in ALK-positive NSCLC cell lines and tumors.

Keywords

Anaplastic lymphoma kinaseB7-H1 antigenDrug resistanceLung neoplasms

Figures

**Fig. 1.** Programmed cell death–ligand 1 (PD-L1) protein levels in the parental H3122 and its subclonal cell lines with single- and double-resistant cell lines by Western blot (band intensities were measured by densitometry using Image J and normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serving as a loading control) (A) and fluorescence-activated cell sorting analysis. Histograms depict PD-L1 expression (B). PD-L1 mRNA levels. The error bars indicate the standard deviation (C).

**Fig. 2.** The heatmap of log2 fold changes of gene expression in anaplastic lymphoma kinase (ALK) inhibitor-resistant cell lines compared to parental H3122 cell lines. The image represents the top 20 absolute log2 fold changes in each comparison (H3122 vs. R1 and H3122 vs. R2; total: 32 genes). Eight genes among the top 20 absolute log2 fold changes in both the R1 and R2 groups are shown in bold texts.

**Fig. 3.** Representative images of programmed cell death–ligand 1 (PD-L1) immunohistochemistry in non-small cell lung cancer patients (×400). PD-L1 score was graded as absent (score 0) (A), weak (score 1) (B), moderate (score 2) (C), or strong (score 3) (D). A score of 2 or 3 was deemed positive for PD-L1 expression.

**Fig. 4.** Changes in programmed cell death–ligand 1 (PD-L1) before and after crizotinib treatment in anaplastic lymphoma kinase–positive non-small cell lung cancer tumor tissues based on H-score (A), PD-L1 score (0, 1, 2, 3) (B), and positivity (C).

그림 출처: PubMed Central 공개 논문 (PMC6639241)

Conflict of interest statement

Conflict of interest relevant to this article was not reported.

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