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

• Kim SJ
• Kim S
• Kim DW
• Kim M
• Keam B
• Kim TM
• Lee Y
• Koh J
• Jeon YK
• Heo DS

• KMID: 2454314
• DOI: http://doi.org/10.4143/crt.2018.486

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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