Cancer Res Treat.  2019 Jul;51(3):1231-1240. 10.4143/crt.2018.486.

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

Affiliations
  • 1Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea. kimdw@snu.ac.kr
  • 2Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.
  • 3Department of Internal Medicine, Seoul National University, Seoul, Korea.
  • 4Department of Pathology, Seoul National University Hospital, Seoul, Korea.
  • 5Department 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.

Keyword

Anaplastic lymphoma kinase; Lung neoplasms; Drug resistance; B7-H1 antigen

MeSH Terms

Antigens, CD274
Blotting, Western
Carcinoma, Non-Small-Cell Lung
Cell Line
Drug Resistance
Flow Cytometry
Gene Expression
Humans
Immunohistochemistry
Lung Neoplasms*
Lung*
Lymphoma
Parents
Phosphotransferases
Polymerase Chain Reaction
RNA, Messenger
Sequence Analysis, RNA
Antigens, CD274
Phosphotransferases
RNA, Messenger

Figure

  • 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).


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