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Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Published on: February 8, 2018
Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative
Rui Ferreira Marques1,2, Daniel Antunes Moreno3, Luciane da Silva3
1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.
Introduction:
Medulloblastoma is the most common and lethal pediatric malignant brain tumor. It comprises four main molecular subgroups: WNT-activated, SHH-activated, Group 3, and Group 4. Medulloblastoma treatment is surgical resection, craniospinal radiation, and chemotherapy. However, many patients do not respond to therapy, and most suffer severe side effects. Cancer immunotherapy targeting immune checkpoints (IC) (PD-1, PD-L1, and CTLA4) has been getting disappointing outcomes in brain tumors. Nevertheless, other less explored immune checkpoints may be promising candidates for medulloblastoma therapy.
Objectives:
In the present study, we aimed to characterize the expression profile of 19 immune checkpoints in medulloblastoma.
Methods:
We analyzed 88 formalin-fixed paraffin-embedded medulloblastomas previously classified for each molecular subgroup and three non-tumoral brain tissue. mRNA levels of 19 immune checkpoint-related genes were quantified using the nCounter (PanCancer Immune Profiling Panel) assay. Further in silico analysis was performed in two larger public microarray datasets, one of which enabled comparisons between tumoral and non-tumoral tissues. Immunohistochemistry of PD-L1 was performed in a subset of cases. Microsatellite instability was also molecularly analyzed.
Results:
We observed an absence of expression of the canonic ICs, namely PDCD1 (PD-1), CD274 (PD-L1), and CTLA4, as well as CD80, CD86, BTLA, IDO1, CD48, TNFSF14, CD160, CEACAM1, and CD244. PD-L1 protein expression was also practically absent. We found higher mRNA levels of CD24, CD47, CD276 (B7-H3), and PVR, and lower mRNA levels of HAVCR2, LAG3, and TIGIT genes, with significant differences across the four molecular subgroups. Compared to the non-tumor tissues, the expression levels of CD276 in all subgroups and CD24 in SHH, Group 3, and Group 4 subgroups are significantly higher. The in silico analysis confirmed the expression profile found in the Brazilian cohort, including the lower/absent expression of the canonic ICs. Moreover, it confirmed the overexpression of CD24 and CD276 in medulloblastomas compared with the non-tumor tissue. Additionally, CD276 and CD24 high levels were associated with worse survival.
Conclusion:
These results highlight the low or absence of mRNA levels of the canonic targetable ICs in medulloblastomas. Importantly, the analysis revealed overexpression of CD24 and CD276, which can constitute prognostic biomarkers and attractive immunotherapy targets for medulloblastomas.
Insights
This study found that common immune checkpoints (ICs) are absent in medulloblastoma. However, CD24 and CD276 (B7-H3) are overexpressed, suggesting they are potential biomarkers and immunotherapy targets for this pediatric brain tumor.
Area of Science:
- Pediatric Oncology
- Neuro-oncology
- Cancer Immunology
Background:
- Medulloblastoma is a lethal pediatric brain tumor with limited treatment options.
- Current immunotherapies targeting PD-1, PD-L1, and CTLA-4 have shown poor outcomes in brain tumors.
- Exploring novel immune checkpoints is crucial for developing effective medulloblastoma therapies.
Purpose of the Study:
- To comprehensively characterize the expression profile of 19 immune checkpoints in medulloblastoma.
- To identify potential novel immunotherapy targets and prognostic biomarkers for medulloblastoma.
Main Methods:
- Analyzed mRNA levels of 19 immune checkpoint genes in 88 medulloblastoma samples using the nCounter assay.
- Performed in silico analysis on public microarray datasets for cross-validation and comparison with non-tumoral tissues.
- Conducted immunohistochemistry for PD-L1 and analyzed microsatellite instability.
Main Results:
- Canonical immune checkpoints (PD-1, PD-L1, CTLA-4) showed low or absent mRNA and protein expression.
- Significant overexpression of CD24 and CD276 (B7-H3) was observed in medulloblastoma compared to normal brain tissue.
- Higher levels of CD24 and CD276 correlated with worse patient survival and varied across molecular subgroups.
Conclusions:
- Canonical immune checkpoints are not promising therapeutic targets in medulloblastoma.
- CD24 and CD276 (B7-H3) represent potential prognostic biomarkers and novel immunotherapy targets for medulloblastoma.
- Further research into CD24 and CD276-targeted therapies is warranted for pediatric brain tumors.
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