Programmed Death Ligand-1 Expression in Gliomas: A Study of Histopathological and Molecular Associations
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.325352
Source of Support: None, Conflict of Interest: None
Keywords: Gliomas, immunotherapy targets, programmed death ligand-1Key Message: Immunotherapy drugs may serve as novel treatment options for gliomas that are aggressive tumours with poor prognosis. Evaluation of PD-L1 expression across spectrum of gliomas may have prognostic implications.
Gliomas constitute 51.4% of all brain tumors. According to the World Health Organization (WHO) classification, gliomas are divided into grades I–IV, with each higher grade corresponding to an augmented level of malignancy and poorer survival. The treatment options include surgery followed by chemo-radiotherapy., The 2016 WHO classification for central nervous system (CNS) tumors mandates integrated phenotypic and genotypic categorizations of CNS tumors. The genotypic classification is achieved with the aid of immunohistochemistry (IHC) for isocitrate dehydrogenase-1(IDH-1), alpha thalassemia, and mental retardation syndrome (ATRX) and p53.
Programmed death 1(PD1) and its ligand (PDL1) regulates T cell activation and proliferation. Blockage of immune check points with monoclonal antibodies has emerged as a promising new approach for the treatment of glial neoplasms. The consequences of PD1/PDL1 binding are loss of proliferation, apoptosis, and decreased cytokine production. Immunotherapy is currently under trial for high-grade gliomas (HGG).
The current study was undertaken with the objectives to analyze the expression of PD-L1 in adult diffuse gliomas (ADG) in WHO grade II, III, and IV and to corelate its expression with demographic features, IDH-1, ATRX, and p-53 mutation status.
The current study was a tertiary hospital-based retrospective case series that included 30 cases of ADG including 10 grade II, 10 grade III, and 10 cases of glioblastoma (GBM). Cases diagnosed and rendered a composite diagnosis including the histologic type, WHO grade, and defining of recommended molecular alterations. Waiver of consent was obtained from the Institutional ethical committee for the use of formalin fixed paraffin embedded tissue blocks.
The hematoxylin and eosin (H and E) stained sections were used to evaluate the histomorphology. IHC was performed for IDH-1, ATRX, and p-53 for molecular categorization [Table 1]. IHC was performed as per the standard recommended protocol. Blocking was done using 3% hydrogen peroxide, followed by antigen retrieval and incubation with the primary antibody. The envision secondary antibody was used (Dakopatts, Denmark). Visualization was done using diaminobenzidine (DAB). All the sections were adequately counter stained with hematoxylin and visualized. All the slides were run in batches including positive and negative controls.
Prediluted Ventana PDL1(SP-263) rabbit monoclonal antibody, Optiview DAB IHC detection kit, and Optiview Amplification kit (Ventana Medical Systems, Tuscon, AZ) were used on the benchmark XT system as per the manufacturer's instructions. A positive control (placental tissue) and a negative control (by omitting the primary antibody) were run with every batch. PDL1 staining was assessed as membranous and/or cytoplasmic staining. The immunostaining for PDL1 was assessed in the tumor cells and the immune cells. The cut of criteria used was ≥1% staining in the tumor or immune cells.
The statistical analysis was done using the Statistical Package for the Social Sciences software version 16. The frequency of PDL1 expression in both the tumor cells and immune cells was assessed. Chi-square test was used to assess associations between two categorical variables. A P value of <0.05 was considered as statistically significant.
The current study included 10 cases each of ADG grade II and III and GBM. Grade II tumors included 26.67% (n = 8) cases of diffuse astrocytoma and 6.67% (n = 2) cases of oligodendroglioma (ODG). Grade III tumors included 20% (n = 6) cases of anaplastic astrocytoma, and 13.33% (n = 4) cases of anaplastic ODG. GBM constituted 33.33% (n = 10) cases in the current study. The age range of the patients varied from 13 to 66 years with a mean age of 38.33 years. M:F ratio was 0.87:1. IDH-1 was mutated in 43.33% (n = 13) cases, p-53 in 50% (n = 15) cases, and ATRX loss was observed in 3.33% (n = 1) cases.
PDL1 expression was identified in 33.3% (n = 10/30) cases. The pattern of staining was both diffuse and heterogeneous [Figure 1]a and [Figure 1]b. The diffuse pattern was identified in 70% (n = 7/10) cases, while 30% (n = 3/10) cases had a heterogeneous pattern of staining. The percentage of tumor cells staining for PDL1 varied from 5 to 70%. PDL1 staining was prominent around zones of necrosis in cases of GBM [Figure 1]c. PDL1 expression in immune cells was observed in 6.67% (n = 2/30) cases. Coexpression of PDL1 in both tumor cells and immune cells was observed in one case [Figure 1]d, [Figure 1]e, [Figure 1]f.
PDL1 expression was identified in 10 cases that included three males and seven females. PDL1 expression was identified in 20% (n = 2) grade II tumors, 30% (n = 3) grade III tumors, and 50% (n = 5) GBMs. All neoplasms with PDL1 expression were phenotypically astrocytic tumors. Tumors with oligodendroglial phenotype were all negative for PDL1 [Figure 2]. Tumors with PDL1 expression were IDH-1 wild type, which was statistically significant when compared with the PDL1 negative group (P = 0.013). p-53 mutation was associated with PDL1 expression in 60% cases; however, this was not statistically significant. ATRX loss was observed in one case with PDL1 expression [Table 2].
The term “innate immune resistance” is used for PDL1 expression effects on the tumor oncogenic signaling pathways. Expression of PDL1 has been assessed in cell lines and glioma cells. PDL1 expression in glioma cell lines was detected in 2003 by Wintterle et al. Reported expression of PDL1 in gliomas varies from 6.1 to 100%. In the current study, PDL1 was observed in 30% using the cutoff of ≥1%.
Common patterns of PDL1 expression include the membranous and diffuse/fibrillary pattern., In the current study, all the cases had an admixture of both the staining patterns.
In glioma tissues, PDL1 may have heterogeneous expression. In the current study, 30% cases harbored heterogeneous expression. In the study conducted by Yao et al., PDL1 expression in tumor center was significantly lower than that present at edges of the tumor. This result is associated with the invasion of gliomas., The upregulation of PDL1 at edge of the tumor constitutes the “molecular shield.” The outcome of this phenomenon is immune surveillance escape during invasion into adjacent brain parenchyma and greater malignant potential. In the current study, PDL1 expression was prominent around the zones of geographic necrosis in cases of GBM. This finding may be attributed to the role of the vascular mechanisms in PDL1 expression.
PDL1 testing has been done using various available clones of PDL1 that include CAL 10, 5H1, SP263, SP142, E1L3N, and 28-8. We have used SP263 clone that yields strong and robust signal intensity. The interpretation of results is simpler with the use of either SP263 or SP142 clones.
We have observed a significant association of PDL1 expression in HGG. The study conducted by Wintterle et al. included 10 cases, wherein PDL1 expression was identified in all nine GBMs and one case of glioma WHO grade III. In a large study conducted by Garber et al., PDL1 expression was identified in 6.1% cases and was limited to GBMs. The greater levels of PDL1 expression particularly in HGG indicate that development of worst forms of tumor is possibly ascertained by the selection of tumor cells with greater PDL1 expression. The development of a novel therapeutic target in HGG may be based on this result. There is marked variation of PDL1 expression among the various WHO grades in the limited studies that have been published in literature; hence, conclusive results for the relation of PDL1 expression with grade still need to be derived.,,,
In the current study, ODGs were all negative for PDL1. This finding is in concordance with the results obtained from the study conducted by Garber et al., wherein ODG cases lacked PDL1 staining. This finding may indicate that tumor cells or microenvironment in ODG inhibit PDL1 expression. Additional studies are recommended for decisive results.
IDH mutation is present in ODGs both grade II and anaplastic, diffuse or anaplastic astrocytomas, and GBMs with decreasing frequency., The association between PDL1 expression and IDH mutation in glial tumors has been evaluated in limited studies published in literature. We have observed a significant association of PDL1 positivity with wild-type IDH expression. Pratt et al. have also observed PDL1 expression was significantly associated with IDH wild-type GBM. IDH wild-type cases are associated with a worse prognosis and aggressive behavior. Addition of PDL1 therapy to these poor prognosis neoplasms can be evaluated in clinical trials.
p53 is a tumor suppressor gene and is associated with bad prognosis. In the current study, p53 mutation was associated with PDL1 in 60% cases. This finding possibly implies that PDL1 expression is frequent in HGGs with poor prognosis.
ATRX plays a major role in DNA repair and transcription regulation., Association between PDL1 expression and ATRX has not been well documented in literature. In the current study, ATRX loss was identified in one case. ATRX loss is associated with longer progression free survival.
Preliminary results indicate expression of PDL1 is associated with poor prognosis and reduced survival. Immunotherapy may be investigated as a treatment option in HGGs as an adjunct to conventional chemo-radiation.
PDL1 is a novel therapeutic target in gliomas. PDL1 expression was identified in 33.3% cases, all cases that harbored PDL1 expression were phenotypically astrocytic tumors and its expression was higher IDH-1 immunonegative gliomas. The expression of PDL1 among the various grades was also assessed and 50% GBMs expressed PDL1. The current study is an attempt to evaluate the expression of PDL1 over the varied spectrum of gliomas. However, owing to the limited sample size results indicate the preliminary data.
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Conflicts of interest
There are no conflicts of interest.
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