Immune checkpoint blockade as a potential therapeutic target: surveying CNS malignancies

Abstract

Background: Expression of programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) across glioma grades is undocumented, and their interactions with commonly expressed genetic and epigenetic alterations are undefined but nonetheless highly relevant to combinatorial treatments.

Methods: Patients with CNS malignancies were profiled by Caris Life Sciences from 2009 to 2016. Immunohistochemistry findings for PD-1 on tumor-infiltrating lymphocytes (TIL) and PD-L1 on tumor cells were available for 347 cases. Next-generation sequencing, pyrosequencing, immunohistochemistry, fragment analysis, and fluorescence in situ hybridization were used to determine isocitrate dehydrogenase 1 (IDH1), phosphatase and tensin homolog (PTEN), and tumor protein 53 mutational status, O(6)-DNA methylguanine-methyltransferase promoter methylation (MGMT-Me) status, PTEN expression, plus epidermal growth factor receptor variant III and 1p/19q codeletion status.

Results: PD-1+ TIL expression and grade IV gliomas were significantly positively correlated (odds ratio [OR]: 6.363; 95% CI: 1.263, 96.236)-especially in gliosarcomas compared with glioblastoma multiforme (P = .014). PD-L1 expression was significantly correlated with tumor grade with all PD-L1+ cases (n = 21) being associated with grade IV gliomas. PD-1+ TIL expression and PD-L1 expression were significantly correlated (OR: 5.209; 95% CI: 1.555, 20.144). Mutations of PTEN, tumor protein 53, BRAF, IDH1, and epidermal growth factor receptor or MGMT-Me did not associate with increased intratumoral expression of either PD-1+ TIL or PD-L1 in glioblastoma multiforme even before false discovery rate correction for multiple comparison.

Conclusions: Targeting immune checkpoints in combination with other therapeutics based on positive biomarker selection will require screening of large patient cohorts.

Keywords: PD-1; PD-L1; glioblastoma; immune checkpoint; low-grade glioma.

Fig. 1.

Representative IHC staining of PD-1+ TIL and PD-L1 in glioblastoma. (A) Hematoxylin and eosin staining of a glioblastoma (WHO grade IV), showing typical hypercellularity and multiple areas of vascular proliferation. Magnification is at 20x. (B) Image of PD-1-positive staining (2–5 per high-power fields) on TIL in a glioblastoma (using antibody MRQ-22). High-power field = 40x. (C) Image of PD-L1–positive staining (2+, 10%) on tumor cells of a glioblastoma (WHO grade IV) (using antibody SP142). (D) Image of PTEN-positive staining (3+) and over 50% expression on tumor cells. (E) Image of a PTEN-negative staining in a glioblastoma. Bar = 100 μm.

Fig. 2.

PD-1+ TIL and PD-L1 expression based on WHO tumor grade and pathology. (A) The expressions of PD-1 increases with tumor grade and is significantly associated with grade IV gliomas; PD-L1 is only associated with grade IV gliomas. (B) PD-1+ TIL expression was more commonly associated with astrocytic tumors than with oligodendroglioma. O, oligodendroglioma; AO, anaplastic oligodendroglioma; MOA, mixed oligoastrocytoma; AMOA, anaplastic mixed oligoastrocytoma; LGA, low-grade astrocytoma; AA, anaplastic astrocytoma; GS, gliosarcoma. Number of positive and total cases are shown.

Fig. 3.

Simultaneous PD-1/PD-L1 expression relative to WHO tumor grade and pathology. PD-L1 expression was analyzed relative to that of PD-1+ TIL. Positive staining for both PD-1 on TIL and PD-L1 was found in 4.3% of all cranial tumors studied. PD-1+ TIL and PD-L1 were coexpressed in grade IV gliomas. LGA, low-grade astrocytoma; AA, anaplastic astrocytoma; GS, gliosarcoma; O, oligodendroglioma; MOA, mixed oligoastrocytoma; E, ependymoma; AO, anaplastic oligodendroglioma; AMOA, anaplastic mixed oligoastrocytoma.

Fig. 4.

Association of PD-1+ TIL and PD-L1 with molecular determinants in glioblastomas. (A) PTEN, PD-1+ TIL, and PD-L1 were assessed using IHC analysis. (B) PTEN mutational status was determined by next-generation sequencing (NGS). (C) TP53 mutation status was determined by NGS. (D) EGFRvIII was detected using fragment analysis, using as the cutpoint the presence of a signal that was 5-fold higher than was seen in the background. (E) BRAF mutations as determined by NGS. (F) MGMT methylation status was defined according to pyrosequencer-based analysis of 5 CpG sites. None of those mutations showed a significant correlation with either PD-1+ TIL or PD-L1 expression (P-values ranged from .79 to 1 after correction for multiple testing).

Fig. 5.

Lack of association of PD-1 and PD-L1 with IDH1 mutational status in gliomas. The presence of IDH1 mutations was determined by next-generation sequencing, and expression of PD-1 and PD-L1 was assessed by IHC analysis. Gliomas were categorized according to WHO criteria. Regardless of glioma grade or pathology, IDH1 mutations were not commonly associated with either PD-1+ TIL or with glioma PD-L1 expression (P-values are all equal to 1 after correction for multiple testing). O, oligodendroglioma; AO, anaplastic oligodendroglioma; LGA, low-grade astrocytoma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme.