Immune Landscape in PTEN-Related Glioma Microenvironment: A Bioinformatic Analysis

Introduction: PTEN gene mutations are frequently found in the genetic landscape of high-grade gliomas since they influence cell proliferation, proangiogenetic pathways, and antitumoral immune response. The present bioinformatics analysis explores the PTEN gene expression profile in HGGs as a prognostic factor for survival, especially focusing on the related immune microenvironment. The effects of PTEN mutation on the susceptibility to conventional chemotherapy were also investigated. Methods: Clinical and genetic data of GBMs and normal tissue samples were acquired from The Cancer Genome Atlas (TCGA)-GBM and Genotype-Tissue Expression (GTEx) online databases, respectively. The genetic differential expressions were analyzed in both groups via the one-way ANOVA test. Kaplan–Meier survival curves were applied to estimate the overall survival (OS) and disease-free survival (DFS). The Genomics of Drug Sensitivity in Cancer platform was chosen to assess the response of PTEN-mutated GBMs to temozolomide (TMZ). p < 0.05 was fixed as statistically significant. On Tumor Immune Estimation Resource and Gene Expression Profiling Interactive Analysis databases, the linkage between immune cell recruitment and PTEN status was assessed through Spearman’s correlation analysis. Results: PTEN was found mutated in 22.2% of the 617 TCGA-GBMs patients, with a higher log2-transcriptome per million reads compared to the GTEx group (255 samples). Survival curves revealed a worse OS and DFS, albeit not significant, for the high-PTEN profile GBMs. Spearman’s analysis of immune cells demonstrated a strong positive correlation between the PTEN status and infiltration of Treg (ρ = 0.179) and M2 macrophages (ρ = 0.303). The half-maximal inhibitor concentration of TMZ was proven to be lower for PTEN-mutated GBMs compared with PTEN wild-types. Conclusions: PTEN gene mutations prevail in GBMs and are strongly related to poor prognosis and least survival. The infiltrating immune lymphocytes Treg and M2 macrophages populate the glioma microenvironment and control the mechanisms of tumor progression, immune escape, and sensitivity to standard chemotherapy. Broader studies are required to confirm these findings and turn them into new therapeutic perspectives.


Introduction
High-grade gliomas (HGGs) are common neoplasms of the central nervous system accounting for 70% of brain tumors [1][2][3][4][5]. Glioblastoma (GBM) is the most lethal and represents 60% of newly diagnosed gliomas in the adult population [6,7]. The current standard of care in the management of HGGs is maximum surgical resection, adjuvant chemoradiation, and six cycles of temozolomide [8].
Despite advances in surgical techniques, diagnostics, and target therapeutic strategies, the 5-year survival rate persists under 10% and the median overall survival (OS) still ranges between 14 and 16 months [9]. The poor prognosis and high mortality rate of GBM are attributable to aberrant angiogenesis, extreme mitotic activity, immune escape mechanisms, and intrinsic genome-wide heterogeneity [10][11][12][13][14][15][16][17]. In 2021, Louis and colleagues published the fifth edition of the WHO classification of brain cancers, which reflects the advances in translational medicine, taxonomy, and genetics in neuro-oncology.
They reported a novel tumors nomenclature aimed at integrating histological features, key diagnostic genes, and molecular characteristics underlying oncogenesis [18].
Moreover, the PTEN pathway has a close interaction with the Wnt/β-catenin signals involved in embryogenesis and the determination of neural patterning [22].
Lack/mutation of the PTEN gene, found in 40% of GBMs, influences neurogenesis, and gliogenesis and heightens the DNA damage repairing and the malignant progression of brain tumors [20,25,27].
The prognostic significance of the PTEN gene is also related to the maintenance of the immune microenvironment [28]. Recent transcriptomic pieces of evidence support the correlation between PTEN mutation and the amendment of immune infiltrating cells expression [29,30]. The immune suppression mechanisms intrude on the host antitumor responses and are liable for the failure of conventional therapeutic approaches [31][32][33][34][35]. The number of reported studies on the immunogenomics and immunosuppressive microenvironment of HGGS opened the way for tailored treatments against glioma resilience [36][37][38][39]. Based on these assumptions, we conducted a bioinformatics analysis to examine the PTEN gene expression profile in HGGs as a prognostic factor for survival. We investigated the cluster of the immune infiltrates within the PTEN-related microenvironment intending to identify the contribution of each subpopulation to the immune escape mechanisms. The effects of PTEN mutation on the sensitivity to standard chemotherapy were also explored.

Statistical Bioinformatics Analysis
R (https://www.r-project.org) (accessed on 7 March 2022) and Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) software were used for statistical analysis. Continuous and categorical variables were reported as mean and percentages, respectively.
Top mutation trends and gene patterns were estimated in the TCGA-GBM cohort, focusing on nucleotide variations of the PTEN gene. The differential expressions of PTEN mRNA levels were determined in TCGA-GBM and GTEx groups with the aim to assess the significance of the PTEN gene in the glioma genome compared to healthy brain tissue. A one-way ANOVA test was used for the analysis.
Kaplan-Meier survival curves were used to estimate the prognostic value of the PTEN gene mutations. The overall survival (OS), disease-free survival (DFS), and comparisons between the high-or low-PTEN mutation profile in TCGA-GBM patients were assessed using the log-rank test. Hazard ratios (HDs) were calculated with the Cox proportional risk regression model. The Genomics of Drug Sensitivity in Cancer (GDSC) database (https://www.cancerrxgene.org/) (accessed on 15 November 2021) was the source used for the appraisal of the chemotherapeutic response of PTEN-mutated HGGs [41]. The R software package "Prophetic" was used and the half-maximal inhibitor concentration (IC50) of temozolomide (TMZ) was assessed by one-way ANOVA analysis [41]. p-value was set at <0.05 for all the tests.

Estimation of Immune Infiltrating Cells
On behalf of the glioma immune microenvironment, the T cells CD4+, CD8+, T reg , NK cells, monocytes/macrophages, and tumor-infiltrating endothelial cells were considered in the TCGA-GBM cohort. Tumor Immune Estimation Resource 2.0 (TIMER2.0) was used to identify the correlation between mRNA PTEN expression and the transcriptional profile of each tumor immune cell in the TCGA-GBM project.
Assuming the purity adjustment, Spearman's correlation method was employed, where rho (ρ) > 0 and ρ < 0 denoted a positive and negative correlation between the variables, respectively. Based on the Gene Expression Profiling Interactive Analysis (GEPIA), the differential subexpression of mRNA PTEN in the immune subtypes, for both TCGA-GBM and GTEx samples, was clustered with the one-way ANOVA method, and p < 0.05 was assumed as statistical. The results were reported as boxplots.

Prediction of Chemotherapeutic Response
Based on the GDSC pharmacogenomic database, lower differential targeted responses to TMZ were found in PTEN-mutated and PTEN wild-type samples, with a median IC50 of 531.13 and 701.55 µM, respectively (Figure 9).

Discussion
The present study analyzes mutation profiles and immune signatures of PTENassociated microenvironment estimating GBM patients' prognosis, survival, and chemotherapy response. The PTEN gene regulates the cell cycle and DNA repair mechanisms. Its expression modulates cell proliferation, neural development, and gliogenesis [21,27,42].
PTEN mutations are hallmarks of glioma malignancy and influence the patients' survival [43][44][45][46][47][48][49]. As recently reported by Erira and his group, alterations of PTEN genes are related to glioma proliferation. PTEN mutation may induce post-translational changes in low-grade gliomas, leading to malignant progression [50].  [51]. In 2016, Han and colleagues explored the genetic linkage between PTEN expression and patients' outcomes in a meta-analysis where a worse prognosis was revealed for PTEN-mutated gliomas [49]. Zhang and his group, 2021, conducted an online bioinformatics analysis about PTEN mutation as a prognostic signature for HGGs. They designed a tailored risk score based on the individual PTEN status, aiming to simplify HGGs diagnosis, prognosis, and treatment planning. They identified 14 independent prognostic genes in PTENwild-type tumors and 3 for the PTEN-mutated ones. These last proved to be related to the worse survival [52].
Amid the PTEN-related genetic mechanisms underlying the tumorigenesis, the maintenance of the glioma immune microenvironment is critical. Based on this rationale, our analysis also aimed at typifying the subpopulations in the glioma immune niche.
In accordance with the evidence in the literature, these data denoted that brain tumor growth and progression are sustained by genetic mechanisms of immune tolerance and exhaustion. The suppression of T, B, and NK cells activity within the PTEN tumor microenvironment suggested immune-mediated biological processes are involved in pathways for glioma immune evasion and resistance to chemotherapies [53][54][55][56].
Our results also verified that the immunosuppressive anticancer microenvironment is sustained by the recruitment of monocytes, which in the glioma context are converted into macrophages, with an explicit M2 polarization. M2 macrophages are known to hold an immunosuppressive role [67,68]. M2 phenotypes induce the differential expression of receptors, cytokines, and chemokines. They produce IL-10, IL-1, and IL-6, stimulating tumorigenesis and negatively affecting the prognosis [69,70]. The M2 macrophages, detected in perivascular areas, enhance the VEGF and COX2 production resulting in increased and aberrant angiogenesis [71][72][73].
Opposing our results, the latest study by Zhou and colleagues published in 2022, found the high expression of PTEN related to a better prognosis for HGGs patients [78]. They discovered via the transwell and flow cytometry that the PTEN gene may inhibit the M1/M2 polarization and M2 macrophages recruitment. These data suggest a potential positive role of the PTEN as an antitumoral immunoregulatory gene [78].
The discrepancy in our study can be explained by the distinct patients cohorts involved, such as the Chinese Glioma Genome Atlas (CGGA) database, and the different techniques applied for data analysis. However, above all, they explored the effects of different PTEN statuses, split into PTEN deletion, PTEN mutated, and PTEN wild-type. This distinction allowed us to assess the specific impact of each PTEN gene expression on glioma immunity. The study demonstrates that, despite advances in genomics, further research is needed to shed light on PTEN activity and its immunological role in tumor progression.
The composition of immune infiltrates explains the prognosis concomitant to the PTEN status and, above all, the ineffective response to standard therapies. TMZ is currently the first line of treatment for HGGs in combination with surgery and radiotherapy [79,80]. It is still debated whether the PTEN mutation may influence sensitivity to radiochemotherapy [81][82][83]. PTEN controls the Wnt/β-catenin and PI3K/Akt/mTOR signaling pathways and arrests the cell cycle at the G2/M phase. TMZ alkylates DNA at this stage, hence PTEN overexpression affects the complex biochemical mechanism of drug alkylation encouraging TMZ activity [84,85].
In 2012 Carico and colleagues conducted a clinical study involving newly diagnosed GBM treated with TMZ. They reported greater effectiveness of TMZ in GBMs with PTEN loss. Inaba et al. investigated the effects of TMZ related to PTEN status founding an increased efficacy in cases of PTEN mutation [83]. Similarly, our analysis of the GDSC database revealed a lower IC50 for PTEN-mutated GBMs (531.13 µM) in comparison with wild-types (701.55 µM).
Apart from conventional chemotherapy, the identification of immune phenotypes and molecular interactions within the tumor microenvironment has been recognized as crucial to widening the spectrum of tailored strategies against the immune escape mechanisms [86][87][88].

Limitations of the Study
The present study has several undeniable limitations, among which are the relatively limited number of patients and short follow-up (average 14.7 months). Other potential biases were the different patients' ethnicity, limited data about the histological classification, and heterogeneity of radiochemotherapy regimens.

Conclusions
PTEN mutations frequently occur in malignant brain tumors, contributing to their progression, reduced OS, and DFS. Within the glioblastoma microenvironment, the PTENrelated immune landscape mainly consists of T reg and M2 macrophages. They repress the antitumor immune activation and are responsible for the triggering of the glioma cell growth, invasion, and aberrant vasculogenesis.
PTEN expression and related glioma microenvironment also influence the sensitivity to conventional radiochemotherapy.
Prospective and randomized trials are necessary to validate these data and to develop novel target treatments.  Institutional Review Board Statement: All procedures performed in the study were in accordance with the ethical standards of the institution and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Data Availability Statement: All data are included in the main text.

Conflicts of Interest:
The authors declare no conflict of interest.