Immunohistochemical Assessment of Programmed Death ligand1 and LC3B in GBM

Gliomas account for almost 30% of all primary brain tumours, and are responsible for the majority of deaths from primary brain tumours .

Glioblastoma is the most common primary malignant brain tumor in adults, accounting for 60-70% of gliomas and 15% of primary brain tumors . Glioblastoma is the most aggressive and undifferentiated type of glioma and designated WHO Grade IV .

Programmed cell death 1 (PD-1) is a protein receptor that functions as an immune checkpoint. It conveys an inhibitory signal to T cells. In cancer, It protects tumors from cytotoxic T lymphocytes(CTL) . The inter¬action of PD1 with its ligand PD-L1 on surface of T-cells suppresses their function and hindering the recognition of tumor cells .PD-L1 expression is inversely correlated with the density of intratumoral CD8+(Cluster of Differentiation 8) T cells .

Autophagy is a catabolic process that leads to cellular degradation and recycling by lysosomal digestion. Thus allowing cells to adapt to stress). Autophagy promotes tumor development, progression and therapy resistance due to its pro-survival role under stress as radiation and chemotherapy. LC3B(microtubule associated protein light chain 3 B) is a commonly used marker to monitor autophagy .PD-L1 has been related to autophagy. Recent findings from experiments with murine melanoma cells and human ovarian cancer cells indicated cells expressing high levels of PD-L1 are more sensitive to autophagy inhibitors than cells that weakly express PD-L1. This finding provides potential opportunities for using autophagy inhibitors in PD-L1-overexpressing cells as a new avenue in cancer management . Thus not studied in GBM before ,our study is pure novel study.

The authors hypothesize that both PD-L1 and LC3B are highly expressed in GBM cells and high PDL1 expression is associated with low CTL count . It is possible that elevated levels of PD-L1 and LC3B markers indicate immune inhibition ,increased autophagy and poor prognosis thus their inhibition will have therapeutic benefit.

To date and to the best of our knowledge, this hypothesis has not been fully tested especially on our locality. To explore our hypothesis, this work is organized into three specific aims:

1. To evaluate relationship between PD-L1 expression and CTL count in GBM.
2. To evaluate the expression of PDL-1 and LC3B in GBM and to assess their relationship.
3. To explore the prognostic relevance of PDL-1 and LC3B in GBM by relating the level of their expression to some established prognostic factors (patient age, multifocal tumor, total or subtotal excision, tumor regrowth , karnofsky performance score and histopathological feature as: necrosis).

Formalin-fixed-paraffin-embedded tissue specimens of about sixty one cases of GBM and nearby apparently normal brain tissue will be obtained from private laboratory.

Histological diagnosis of H\&E stained sections will be reviewed and the histological prognostic criteria will be assessed.

Immunohistochemical staining for PDL-1,CD8 and LC3B using mouse monoclonal antibodies with immunoperoxidase staining will be used as a visualization method. Antibodies dilutions, antigen retrieval methods, and incubation time will all be conducted according to the manufacturer's instructions.

Sample size calculation was carried out using G*Power 3 software . A calculated sample of 61 glioblastoma patients will be needed to detect an effect size of 0.1 (HR ranged between 1.54 and 1.74 in the multivariate Cox Hazard Proportion Regression for the PD-L1 expression level), with an error probability of 0.05 and 80% power on a one-tailed test.

Correlation between PD-L1 ,CTL count and LC3B expression and prognostic criteria of GBM will be performed using a computer software SPSS(Statistical Package for Social Sciences).