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. 2016 Feb;18(2):195-205.
doi: 10.1093/neuonc/nov172. Epub 2015 Aug 30.

PD-L1 expression and prognostic impact in glioblastoma

Edjah K Nduom  1 , Jun Wei  1 , Nasser K Yaghi  1 , Neal Huang  1 , Ling-Yuan Kong  1 , Konrad Gabrusiewicz  1 , Xiaoyang Ling  1 , Shouhao Zhou  1 , Cristina Ivan  1 , Jie Qing Chen  1 , Jared K Burks  1 , Greg N Fuller  1 , George A Calin  1 , Charles A Conrad  1 , Caitlin Creasy  1 , Krit Ritthipichai  1 , Laszlo Radvanyi  1 , Amy B Heimberger  1
Affiliations

PD-L1 expression and prognostic impact in glioblastoma

Edjah K Nduom et al. Neuro Oncol. 2016 Feb.

Abstract

Background: Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact.

Methods: Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas.

Results: The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%-86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome.

Conclusions: The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.

Keywords: PD-1; PD-L1; cancer stem cells; glioblastoma; immune suppression.

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Figures

Fig. 1.
Fig. 1.
(A) Representative microphotograph of immunohistochemical staining of PD-L1 using the EPR1161(2) antibody in a positive placenta control. (B) PD-L1 expression staining with the EPR1161(2) antibody on representative HEK293 cells stabily transfected with PD-L1 (right panel) versus untransfected cells (left panel). Transfection efficiency by flow cytometry was 33%–35%. (C) Representative examples of the glioblastoma (GBM) tumor stained with the EPR1161(2) antibody demonstrating distinct cell-surface staining of positive cells (100x magnification). Arrows demonstrate clusters of round cells with a morphology consistent with lymphocytes. (D) High magnification (200x) microphotograph demonstrating cell surface staining of small round cells. (E) Representative microphotograph demonstrating cell surface staining of GBM cells (left panel) and high magnification (200x) microphotograph of the same region. (F) Representative histogram demonstrating PD-L1 expression relative to isotype controls as assayed by flow cytometry in a fresh GBM specimen. Only live cells were included in the gated analysis. PD-L1 expression is represented by the solid black line, and the isotype control is the dotted grey line.
Fig. 2.
Fig. 2.
Glioblastoma (GBM) tumor infiltrating CD4+ and CD8+ T cells express both PD-1 and PD-L1. The top FACS plot shows the percentages of tumor infiltrating CD4+ and CD8+ T cells in an enriched mononuclear cell preparation by Percoll gradient isolation from a fresh GBM specimen. The histogram plots presenting positive PD-1 and PD-L1 expression on both CD4+ and CD8+ T cell subsets (red line: isotype; blue line: PD-1 or PD-L1). Replicated in n = 5 specimens with similar results.
Fig. 3.
Fig. 3.
PD-L1 expression on glioma cells. (A) Histogram plots showing expression levels of PD-L1 on glioma stem cells. (B) Histogram plots showing expression levels of PD-L1 on representative glioma cell lines. The dotted line represents the isotype control, and the black line denotes PD-L1 expression.
Fig. 4.
Fig. 4.
PD-L1 expression and outcome in glioblastoma (GBM). (A) Kaplan-Meier survival estimates of patients with GBM in relation to expression levels of PD-1 and PD-L1 in the tumors based on the The Cancer Genome Atlas (TCGA) datasets. Median survival for the PD-L1 high-expressing group was was 11.42 months versus 14.9 months for the low. Median survival for the PD-1 high-expressing group was 10.63 months versus 14.68 for the low. (B) Kaplan-Meier survival estimates of patients from the GBM tissue microarray comparing high versus low PD-L1 expression by the immunohistochemistry. (C) Kaplan-Meier survival estimates of patients with GBM when accounting for both PD-1 and PD-L1 expression levels in the tumors based on the TCGA datasets. Median survival for the high group was 9.67 months versus 15.88 month for the low.
Fig. 5.
Fig. 5.
Expression of markers of cytotoxicity and outcome in glioblastoma (GBM). (A) Kaplan-Meier survival estimates of patients with a GBM in relation to expression levels of Granzyme A (GZMA). Median survival for the high-expressing group was 10.54 months versus 14 months for the low. (B) Kaplan-Meier survival estimates of patients with GBM in relation to expression levels of PRF1. Median survival for the high-expressing was 10.63 months versus 15.08 for the low. (C) Kaplan-Meier survival estimates of patients from the TCGA dataset when expression of both GZMA and PRF1 are high (median survival: 5.38 mo) compared with low expression (median survival: 14.59 mo). (D) Kaplan-Meier survival estimates show a difference in survival when there is co-associated high expression of GZMA, PRF1 and PD-L1 (median survival: 5.54 mo) relative to low expression (median survival:16.73 mo) or other combinations of expression.
Fig. 6.
Fig. 6.
Correlation of markers of cytotoxicity and immunosuppression in glioblastoma. (A) High expression of PRF1 is correlated positively with PD-L1. (B) High expression of PD-1 is positively correlated with PRF1 and (C) GZMA. (D) GZMA and PRF1 expression are positively correlated with each other.
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