Digital expression profile of immune checkpoint genes in medulloblastomas identifies CD24 and CD276 as putative immunotherapy targets

Medulloblastoma is a common and aggressive type of brain tumor in children, consisting of four molecular subgroups. Traditional methods of treatment with surgery, radiotherapy and chemotherapy result in an incomplete response with serious side effects. Immunotherapy using the blockade of immune checkpoints such as PD-1, PD-L1 and CTLA4 has increased its prominence in solid tumor therapies. However, its application has been disappointing in brain tumors. Thus, alternative and poorly described immunological checkpoints could be revealed as interesting targets for medulloblastoma therapy. In this study, we analyzed the expression of 19 immune checkpoints in 88 medulloblastomas and three non-tumor brain tissues by Nanostring microarray analysis technology and performed in silico analysis in more than 1300 medulloblastomas. Our analysis revealed a low/absent expression of the majority of targets analyzed and, in particular, of the traditional immune checkpoints. On the other hand, it was observed a higher expression of CD24 and CD276 in comparison with non-tumor tissues. These two immunological checkpoints were also associated with patient survival and were bioinformatically validated in the mentioned databases.  Hence, our results show that CD24 and CD276 can serve as prognostic biomarkers and potential immunotherapy targets for medulloblastomas.

Authors and Affiliations:

Rui Ferreira Marques^1,2, Daniel Antunes Moreno³, Luciane da Silva³, Leticia Ferro Leal^3,4, Fla´ via Escremim de Paula⁵, Iara Santana⁶, Gustavo Teixeira⁶, Fabiano Saggioro⁷, Luciano Neder⁷, Carlos Almeida Junior⁸, Bruna Mançano⁸ and Rui Manuel Reis^1,2,3,5*

¹Instituto de Investigação em Ciências da Vida e da Saúde (ICVS), Escola de Ciências da Saúde da Universidade do Minho, Braga, Portugal,

²ICVS/3B's –PT Laboratório Associado do Governo, Braga, Guimarães, Portugal,

³Centro de Investigação em Oncologia Molecular, Hospital de Cancro de Barretos, Barretos, Brasil,

⁴Faculdade de Ciências da Saúde de Barretos Dr. Paulo Prata (FACISB), Escola de Medicina, Barretos, Brasil,

⁵Laboratório de Diagnóstico Molecular, Hospital do Cancro de Barretos, Barretos, Brasil,

⁶Departamento de Patologia, Hospital de Oncologia de Barretos, Barretos, Brasil,

⁷Departamento de Patologia e Medicina Forense, Escola Médica de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brasil

⁸Hospital De Cancro Infantil De Barretos, Barretos, Brasil

Abstract:

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.

Journal: Frontiers in Immunology, Section: Cancer Immunity and Immunotherapy. | https://doi.org/10.3389/fimmu.2023.1062856

Link: https://www.frontiersin.org/articles/10.3389/fimmu.2023.1062856/full