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. 2021 Sep 11;13(18):4562.
doi: 10.3390/cancers13184562.

PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis

Jutta Kirfel  1 , Christiane Charlotte Kümpers  1 , Anke Fähnrich  2   3 , Carsten Heidel  1 , Mladen Jokic  1 , Ignacija Vlasic  1   4 , Sebastian Marwitz  5   6 , Torsten Goldmann  5   6 , Helen Pasternack  1 , Sabine Bohnet  7 , Danny Jonigk  8   9 , Mark P Kühnel  8   9 , Anne Offermann  1 , Hauke Busch  2   3 , Sven Perner  1   5
Affiliations

PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis

Jutta Kirfel et al. Cancers (Basel). .

Abstract

Background: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade-responsive biology is warranted.

Methods: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration ("hot" vs. "cold" phenotype). The study comprises LUAD cases (n = 138) with "hot" (≥150 lymphocytes/HPF) and "cold" (<150 lymphocytes/HPF) tumor immune phenotype and positive (>50%) and negative (<1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis.

Results: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup.

Conclusion: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification.

Keywords: cold; hot; immune phenotype; lung adenocarcinoma (LUAD); programmed cell death-ligand 1 (PD-L1); protein; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Flowchart showing working steps of the study.
Figure 2
Figure 2
Characterization of the cohort. (A) PD-L1 expression in lung adenocarcinoma correlates with total immune infiltration. Representative images of PH (lane 1), PC (lane 2), NH (lane 3), and NC (lane 4) LUADs (B) Bar graph showing the distribution of the cohort in subgroups PH, PC, NH, and NC. Scale bar: 50 µm.
Figure 3
Figure 3
Relation between PD-L1 expression and immune infiltration. (A) Representative images of CD8 and CD4 expression in PH LUAD (lane 1,2) and NH LUAD (lane 3,4) (objective magnification ×10; scale bar = 50 µm). (B) CD8/CD4 quotient of the four groups showing that increased cytotoxic T-cell presence depends on PD-L1 expression and high total immune infiltration. Scale bar: 50 µm.
Figure 4
Figure 4
Immune profiling using the nCounter Immune Profiling panel. (A) Volcano plot showing differentially expressed genes between PH and NH LUADs (p-value > 0.01 und fold change > 0.25.). Analyzed targets CD274, KIT, and STAT1 marked. (B) Cluster analysis using Euclidean distance of normalized NanoString mRNA expression between PH (orange) and NH (in brown) LUADs assembled into a heatmap. Analyzed targets CD274, KIT, and STAT1 marked in the list on the right side. (C) Barplot depicting the p-values of the most upregulated molecular pathways according to a GSEA analysis in PH LUADs.
Figure 5
Figure 5
STAT1 is upregulated in PH LUADs. (A) Boxplot of transcriptomic analysis showing STAT upregulated in PH LUADs (black) when compared to NH LUADs (green). (B) STAT1 validation on the protein level. Boxplot of immunohistochemistry analysis showing STAT1 is more expressed in the PH group than in the NH group. STAT1 expression was quantified as the percentage of STAT1-positive tumor cells of all tumor cells. (C) Representative images of a PH LUAD (1) with STAT1 expression (2). (D) Representative images of a NH LUAD (3) without STAT1 expression (4) (objective magnification ×40; scale bar = 20 µm).
Figure 6
Figure 6
KIT is downregulated in PH LUADs. (A) Boxplot of transcriptomic analysis showing KIT downregulated in PH LUADs (black) when compared to NH LUADs (green). (B) KIT validation on the protein level. Boxplot of immunohistochemistry analysis showing KIT more expressed in the NH group than in the PH group. KIT expression was quantified as the percentage of KIT-positive tumor cells of all tumor cells. (C) Representative images of a PH LUAD (1) without KIT expression (2) (D) Representative images of an NH LUAD (3) with KIT expression (4) (objective magnification ×40; scale bar = 20 µm).
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