Profiling non-small cell lung cancer reveals that PD-L1 is associated with wild type EGFR and vascular invasion, and immunohistochemistry quantification of PD-L1 correlates weakly with RT-qPCR

Abstract

Most lung cancer patients are diagnosed at an advanced stage, limiting their treatment options with very low response rate. Lung cancer is the most common cause of cancer death worldwide. Therapies that target driver gene mutations (e.g. EGFR, ALK, ROS1) and checkpoint inhibitors such anti-PD-1 and PD-L1 immunotherapies are being used to treat lung cancer patients. Identification of correlations between driver mutations and PD-L1 expression will allow for the best management of patient treatment. 851 cases of non-small cell lung cancer cases were profiled for the presence of biomarkers EGFR, KRAS, BRAF, and PIK3CA mutations by SNaPshot/sizing genotyping. Immunohistochemistry was used to identify the protein expression of ALK and PD-L1. Total PD-L1 mRNA expression (from unsorted tumor samples) was quantified by RT-qPCR in a sub-group of the cohort to assess its correlation with PD-L1 protein level in tumor cells. Statistical analysis revealed correlations between the presence of the mutations, PD-L1 expression, and the pathological data. Specifically, increased PD-L1 expression was associated with wildtype EGFR and vascular invasion, and total PD-L1 mRNA levels correlated weakly with protein expression on tumor cells. These data provide insights into driver gene mutations and immune checkpoint status in relation to lung cancer subtypes and suggest that RT-qPCR is useful for assessing PD-L1 levels.

Fig 1. EGFR but not KRAS was negatively correlated with PD-L1 membranous protein expression in the lung cancer patient’s cohort.

A total of 232 lung tumors were evaluated for PD-L1 expression on tumor cells. All patients were screened previously for molecular alterations. EGFR positive patients were shown to negatively correlated to PD-L1 (p = 0.02; Fisher exact test) 26% of EGFR+ patients had PD-L1 expression versus 74% had PD-L1 expression in the same population.

Fig 2. PD-L1 expression by IHC correlates with PD-L1 mRNA expression by qPCR.

A total of 49 fresh lung tumors were evaluated for PD-L1 expression by IHC and quantified for PD-L1 mRNA by RT-qPCR. PD-L1 expression was evaluated on tumor cells only (TC), and on both tumor and immune cells (TC+IC). (A) PD-L1 expression on tumor cells (IHC) is significantly correlated with PD-L1 mRNA expression (qPCR). (B) Also, PD-L1 expression tumor and immune cells (IHC) is significantly correlated with PD-L1 mRNA expression (RT-qPCR).

Fig 3. CD8 expression by qPCR correlates with PD-L1 expression by IHC for 50% cut-off.

A total of 49 fresh lung tumors were evaluated for PD-L1 expression by IHC and quantified for CD8, CD3 and CD45 mRNA by RT-qPCR. (A) CD45 (RT-qPCR) did not correlate with PD-L1 (IHC, 1% and 50% cut-off). (B) Also, CD3 marker (qPCR) was not significantly correlated with PD-L1 (IHC, 1% and 50% cut-off). (C) CD8 marker (RT-qPCR) was significantly correlated with PD-L1 (IHC) for 50% cut-off but not for 1% cut-off.