Systematic Review of Molecular Targeted Therapies for Adult-Type Diffuse Glioma: An Analysis of Clinical and Laboratory Studies

Gliomas are the most common brain tumor in adults, and molecularly targeted therapies to treat gliomas are becoming a frequent topic of investigation. The current state of molecular targeted therapy research for adult-type diffuse gliomas has yet to be characterized, particularly following the 2021 WHO guideline changes for classifying gliomas using molecular subtypes. This systematic review sought to characterize the current state of molecular target therapy research for adult-type diffuse glioma to better inform scientific progress and guide next steps in this field of study. A systematic review was conducted in accordance with PRISMA guidelines. Studies meeting inclusion criteria were queried for study design, subject (patients, human cell lines, mice, etc.), type of tumor studied, molecular target, respective molecular pathway, and details pertaining to the molecular targeted therapy—namely the modality, dose, and duration of treatment. A total of 350 studies met the inclusion criteria. A total of 52 of these were clinical studies, 190 were laboratory studies investigating existing molecular therapies, and 108 were laboratory studies investigating new molecular targets. Further, a total of 119 ongoing clinical trials are also underway, per a detailed query on clinicaltrials.gov. GBM was the predominant tumor studied in both ongoing and published clinical studies as well as in laboratory analyses. A few studies mentioned IDH-mutant astrocytomas or oligodendrogliomas. The most common molecular targets in published clinical studies and clinical trials were protein kinase pathways, followed by microenvironmental targets, immunotherapy, and cell cycle/apoptosis pathways. The most common molecular targets in laboratory studies were also protein kinase pathways; however, cell cycle/apoptosis pathways were the next most frequent target, followed by microenvironmental targets, then immunotherapy pathways, with the wnt/β-catenin pathway arising in the cohort of novel targets. In this systematic review, we examined the current evidence on molecular targeted therapy for adult-type diffuse glioma and discussed its implications for clinical practice and future research. Ultimately, published research falls broadly into three categories—clinical studies, laboratory testing of existing therapies, and laboratory identification of novel targets—and heavily centers on GBM rather than IDH-mutant astrocytoma or oligodendroglioma. Ongoing clinical trials are numerous in this area of research as well and follow a similar pattern in tumor type and targeted pathways as published clinical studies. The most common molecular targets in all study types were protein kinase pathways. Microenvironmental targets were more numerous in clinical studies, whereas cell cycle/apoptosis were more numerous in laboratory studies. Immunotherapy pathways are on the rise in all study types, and the wnt/β-catenin pathway is increasingly identified as a novel target.


Introduction
As the most common brain tumor in adults, gliomas have sustained the focus of scientific research for the past several decades. Recently, more attention has been drawn to the diagnostic criteria of gliomas with the restructured 2021 WHO Classification of Tumors of the Central Nervous System, specifically focusing more on molecular biomarkers as a means of categorization [1]. Within this classification adult-type diffuse gliomas are the most prevalent tumor types, defined on the basis of molecular expression of isocitrate dehydrogenase (IDH) and the 1p/19q codeletion. These glioma subtypes include astrocytoma (IDH-mutant astrocytoma), oligodendroglioma (IDH-mutant and 1p19q-codeleted), and glioblastoma (GBM) (IDH-wildtype) [1]. The typical management of adult-type diffuse glioma begins with a resection or biopsy, followed by possible radiotherapy and/or chemotherapy with the alkylating agent, temozolomide, or the combination procarbazine, lomustine, and vincristine (PCV) [2]. Even with this regimen, recurrence is prevalent, and the prognosis is dismal, particularly in GBM, which has an average survival of 14-16 months [3].
As gliomas are becoming more molecularly defined, so too is their treatment progressing more towards the targeting of molecular pathways [4]. Compared with traditional chemotherapeutic drugs, molecularly targeted antitumor therapy has the advantage of strong specificity with minimal damage to normal tissues. Molecular-targeted glioma therapies have gained traction in the scientific literature, with many analyses centered on identifying mechanisms pertinent to glioma growth [5]. The Raf/MEK/Erk pathway has been of particular interest as a targetable pathway due to its preponderance among gliomas [5]. Additionally, a systematic review by Da Silva et al. highlighted the molecular targeted therapies in clinical trials for GBM, identifying four categories of targets: targeting the potential for unlimited replication, growth autonomy and migration, cell cycle and apoptosis, and angiogenesis [6].
To date, there has yet to be a systematic review of the literature characterizing molecular targeted therapy in adult-type diffuse gliomas. A comprehensive understanding of the progress in this field-both in terms of existing therapies and novel targets-is integral to guiding advancement in treatment development, integration into clinical trials, and more adequate treatment options for diffuse glioma patients.

Methods
A systematic review was performed, characterizing the current state of molecular targeted therapies for gliomas, to inform scientific progress and guide advancement in this area of study. The protocol was conducted in accordance with PRISMA (preferred reporting items for systematic reviews and meta-analyses) guidelines.

Search Strategy
A literature search of English-text articles was conducted through January 2023 using PubMed and Web of Science. Categories of concepts related to both molecular targeted therapy and glioma, both adhering to the 2021 WHO classification as well as prior classifications (including language such as low-or high-grade glioma), were searched; results were combined via Boolean operators (Appendix A).
Additionally, a search with the same search terms was conducted on clinicaltrials.gov to assess clinical trials relating to molecular targeted therapy for adult-type diffuse glioma.

Selection Criteria
Article titles and abstracts were screened for relevance by two authors (L.M. and N.K.G.), and duplicates were removed. The remaining articles were then screened in full text by three authors (L.M., N.K.G., and N.C.). Inclusion criteria used were: any clinical or laboratory studies testing molecular targeted therapies for glioma or laboratory studies identifying novel molecular targets for glioma with a target of adult-type diffuse glioma or its subtypes. Exclusion criteria used were: brain tumors other than adult-type diffuse glioma-such as medulloblastoma; any study of pediatric tumor focus, etc.; papers centering on the delivery technology rather than the molecular target; systemic therapies or adjuvants to molecular targeted therapies; studies with no molecular target identified; or papers that were correspondences, reviews, or commentaries. Conflict resolution at all stages of article selection was via discussion between authors.

Data Extraction
The data extraction for the systematic review included the following: first author, year of publication, design (multi-institutional retrospective analysis, in vivo, in vitro, and in vivo, etc.), study subject (patients, human cell lines, mice, etc.), ages of subjects, type of tumor studied, molecular target, respective molecular pathway, and the modality and results of the molecular targeted therapy investigated.
The data extraction for clinicaltrials.gov was limited to ongoing trials-defined as those with a status of completed, recruiting, or active, non-recruiting. The extracted variables included the following: title of the study, year started and year of most recent update, tumor type, NCT number, sponsoring or collaborating organization, molecular target of interest, intervention utilized, as well as the phase of the study (Phase 1, 2, or 3), status (active or recruiting), funding sources (NIH, industry, or other), and results, if available.

Data Categorization
Published studies were then divided into three main categories: clinical studies testing existing molecular targeted therapies; laboratory studies testing existing molecular targeted therapies; and laboratory studies identifying a novel molecular target.
Unless specifically stated otherwise in the study, tumor types were classified by molecular associations with respective cell lines in the literature. For instance, those classified under GBM included cell lines known to harbor wild-type IDH (U87, U251, T98G, and A172) and human tumors classified specifically as GBM [7][8][9][10][11]. In instances where molecular mutation information was not readily available or numeric glioma grading was utilized (grades I-IV), these were classified as simply "Glioma".
Ongoing clinical trials were also queried in this manner and organized by tumor type.

Statistical Analysis
A meta-analysis was not conducted; therefore, descriptive data is reported for most variables in this study. To compare means by group, ANOVA testing was utilized. The chosen type 1 error rate was set to p < 0.05. All statistical analyses were performed via IBM SPSS Statistics for Macintosh (version 28.0.1.1) (Armonk, NY, USA).

Quality Assessment
The quality of evidence was determined by study design and graded using a level of evidence scheme adapted from Ackley et al. (Table 1) [12]. Table 1. Level of Evidence and Quality Assessment.

Level of Evidence (LoE) Description
Level I Evidence from a systematic review or meta-analysis of randomized control trials (RCTs) or evidence-based clinical practice guidelines based on RCTs.

Level II
Evidence obtained from at least one well-designed RCT (e.g., a large multi-site RCT).
Level III Evidence obtained from well-designed controlled trials without randomization (i.e., quasi-experimental). Level IV Evidence from well-designed case-control or cohort studies.

Level of Evidence (LoE) Description
Level V Evidence from systematic reviews of descriptive and qualitative studies. Level VI Evidence from a single descriptive or qualitative study.
Level VII Evidence from the opinions of authorities and/or reports of expert committees.
Level of effectiveness rating scheme adapted from Ackley et al. 2007 [12].
The level of evidence for the clinical studies varied. Eight studies had Level II evidence (15%), as they were multi-institutional clinical trials. Most published clinical studies had Level IV evidence (34/52; 65%), consisting of single institutional phase II or prospective trials. The rest of the clinical studies (10/52, 19%) were case reports or series, classifying them as Level VI studies (Table 1 and Table S1).
Of the laboratory studies testing existing molecular targeted therapies, all were queried for whether or not they utilized spheroid or 3-dimensional (3D) technologies for cell culture as part of their methodology. Fifty-nine (31%) of studies adopted tumor sphere or 3D technology (Table S2).

Laboratory Studies Identifying Novel Molecular Targets
There were 108 laboratory studies identifying novel molecular targets for treating glioma ( Table 4).
The most common funding source was industry-related funding (54/119, 45%), followed by the National Institute of Health (NIH) (45/119, 38%) (Table S3). All ongoing clinical trials were in phase I or II.

Discussion
This systematic review examined the current evidence on molecular targeted therapy for adult-type diffuse glioma. The majority of clinical and laboratory studies focused on GBM, with few studies examining IDH-mutant astrocytomas, oligodendrogliomas, or unspecified gliomas. In both clinical and laboratory settings, protein kinase pathwaysparticularly PI3K/Akt/mTOR and Ras/BRAF/Mek/Erk-were the most commonly targeted molecular pathways. The next most common molecular targets in published clinical studies and clinical trials were microenvironmental targets-including angiogenesis, cellcell adhesion, or ion/cation regulation-followed by cell cycle/apoptosis pathways and immunotherapy. The second most common molecular targets in laboratory studies were cell cycle/apoptosis pathways, followed by microenvironmental targets, and then immunotherapy pathways. The wnt/β-catenin pathway was also prevalent in the studies identifying novel targets. The level of evidence for published clinical studies varied, with the majority being Level IV-consistent with early-phase, single-institution clinical trials; all laboratory studies were quasi-experimental designs. Published clinical studies testing molecular targeted therapies, in general, were published more recently than laboratory studies. Lastly, clinical trials on protein kinase pathways began earlier than other clinical trial types, particularly trials testing cell cycle/apoptosis targets or immunotherapy.

Adult-Type Diffuse Glioma Subtypes
Though the overwhelming majority of studies centered on GBM, the literature shows that adult gliomas found more frequently in practice tend to harbor IDH mutations [7,344]. The reason for the overrepresentation of GBM-focused studies and the underrepresentation of IDH-mutant astrocytoma or oligodendroglioma is multifactorial. First off, the updated WHO classification is a recent development as of 2021; because the majority of the works in this study occurred prior to the molecular subtype differentiation, there were likely studies that self-identified as GBM studies that may have included tumors with an IDH mutation or 1p19q co-deletion. To the best of our ability, we retroactively identified studies that specifically identified these molecular statuses, but those were few in number. Additionally, it is likely that GBM has received more research funding and scientific attention than other brain tumors, perhaps due to its more aggressive nature and mortality rates. Therefore, the funding for studies investigating IDH-mutant astrocytoma or oligodendroglioma may be less robust. Of note, the ongoing clinical trials for glioma vastly favor GBM as well, receiving the majority of funding from industry sources. Further studies to quantify the distribution of research funding between glioma subsets would be necessary to confirm this association. Lastly, the standard cell lines for all glioma research tend to be glioblastoma models, particularly U87, U373, and U251, as also reflected in our study [345] (Table S2).

Protein Kinase Pathways
In terms of molecular targets, protein kinase pathways-especially PI3K/Akt/mTOR and Ras/BRAF/Mek/Erk-were the most prevalent in the clinical and laboratory studies analyzing existing therapies and novel targets to treat adult-type diffuse glioma. (Tables 2-4) These results are consistent with previous studies that have demonstrated a predominance in the PI3K/Akt/mTOR and Ras/BRAF/Mek/Erk protein kinase pathways in molecularly targeted glioma treatment [5,6]. The importance of these pathways in glioma has been welldescribed in the literature; ultimately, these tumors harbor mutations that continuously activate these protein kinase signaling pathways, leading to increased tumorigenesis and progression [346][347][348].
Both the PI3K/Akt/mTOR and Ras/BRAF/Mek/Erk protein kinase pathways are also downstream of receptors such as EGFR, one of the most significant signaling pathways clinically implicated in glioma [349]. A systematic review of molecular targeted therapy clinical trials for GBM identified EGFR as the most prevalent molecular target [6]. Nonetheless, studies have demonstrated limited clinical benefit of anti-EGFR therapies, theorized to be secondary to PTEN-mediated resistance of GBM to this therapy type [350].
Similar to the published clinical studies on this topic, protein kinase pathways were by far the most predominant molecular targets tested in ongoing clinical trials. Interestingly, these therapeutics were also investigated much earlier on average. This finding is likely due to the fact that protein kinase inhibitors are some of the earliest molecular target therapies in the field of targeted oncologic interventions, thus being able to start clinical trials for the treatment of glioma as early as 2001 [351]. Perhaps, in the coming years, as the analysis of existing molecularly targeted therapies progresses from earlier stage clinical testing or laboratory testing, there will be a shift favoring more of the scientifically novel approaches-such as immunotherapeutics, cell cycle inhibitors, or more specifically localized targeting-in clinical trials.
Additional protein kinase pathways targeted in laboratory studies included HER2 receptors, epithelial membrane protein-2 (EMP2), and STAT3, to name a few [108,117,140]. HER2 expression tends to be low in GBM, and though one clinical trial examining a HER2 inhibitor has yet to show therapeutic gain, laboratory studies have promising evidence for efficacy [140,352]. EMP2 has been implicated in bevacizumab resistance and thus shows promise as a molecular target for preventing resistance in conjunction with this common therapeutic [117,353]. STAT3 plays a role in astrocyte development and has tumor suppressive roles in glial malignancies; this target shows promise in laboratory research using tetrandrine as an inhibitor [108]. Despite varying clinical evidence of efficacy, protein kinase-targeted therapies remain a prevalent area of study for both individual inhibitors and combined therapies.

Cell Cycle/Apoptosis Pathways
Interestingly, a prevalent molecular target in laboratory studies-both testing existing therapies and identifying novel targets-were cell cycle/apoptosis pathways. This difference may be attributed to the fact that clinical studies tend to focus on targets with existing FDA-approved therapies or targets that are more well-established in the literature, while studies with the goal of establishing new targets or testing newly developed therapies can explore a wider range of targets with less established evidence.
The use of cell cycle or apoptosis pathways as targets stems from the use of these pathways in the treatment of other tumors, in particular. In the present study, only four clinical studies included cell cycle/apoptosis pathway inhibitors, namely the cyclin-dependent kinase (CDK) 4/6 inhibitor palbociclib, the mouse double minute 2 (MDM2) inhibitor idasanutlin, the ribonucleotide reductase inhibitor Motexafin Gadolinium, and the 26S proteasome inhibitor bortezomib [36,[57][58][59]. CDK and MDM2 inhibitors were also prevalent in laboratory studies testing existing therapies [150,156,160,161,165,176]. Two CDKs were identified as novel molecular targets-namely CDK 5 and 10-and other novel targets include other apoptosis regulators such as E2F1, trichothiodystrophy group A protein (TTDA), and protease activated receptor 2 (PAR2) [267,269,274,279,280].

Immunotherapy Pathways
The immune checkpoint blockade adopted in the glioma therapeutics model follows treatment paradigms for melanoma, lung cancer, colon cancer, and hepatocellular carcinoma; the therapies used to treat these tumors tend to block programmed cell death protein 1 (PD1), a protein known for attenuating the host immune response to tumor cells, or cytotoxic T lymphocyte antigen-4 (CTLA-4), a molecule that inhibits T-cell activation [355][356][357][358]. The clinical studies identified in the present study investigating immunotherapeutic pathways targeted PD1 using the inhibitor nivolumab [54,359]. Other immunotherapy targets that were found to be effective in vivo included the inhibition of CD73 with antibodies, extracellular matrix metalloproteinase (EMMPRIN) with icaritin, and NFκB with BAY117082 [207,210,211]. Novel immunotherapeutics for GBM and oligodendroglioma include cluster of differentiation 204 (CD204), S100A, and the CE7 epitope of the L1-CAM adhesion molecule [92,306,308].

Wnt/β-Catenin Pathway
The wnt/β-catenin pathway was much more prevalent in earlier stages of laboratory research identifying new targets, likely because the role of wnt/β-catenin in glioma progression is a more recent scientific advancement [310][311][312][313][314][315][316]349]. It is likely that in the upcoming years, the distribution of molecular targets may shift from protein kinase pathway-targeted therapies towards the wnt/β-catenin pathway or a combinatory approach of the two. Ongoing clinical trials have yet to target these pathways, but it is likely that this will soon change.

Study Design
The majority of laboratory studies utilized GBM cell lines or GBM patient samples. The frequent use of the U87 cell line in laboratory studies may be attributed to its widely accepted use as a model for GBM [360]. The use of technology such as spheroid or 3D cell culture is highly relevant in the context of therapies for gliomas. These technologies more accurately represent the tumor microenvironment and allow for better design of patient-specific treatments. Nearly one-third of laboratory studies testing existing therapies utilized this technology, implying that these studies are likely closer to translation to human studies.
The use of patient-derived GBM and glioma samples also highlights the importance of personalized medicine approaches in glioma treatment; nonetheless, this use also limits the generalizability of the conclusions, as most of these studies did not investigate molecular subtypes.

Implications
Molecular targeted therapy is predicted to revolutionize glioma therapy [361][362][363]. Particularly looking at the NCT Neuro Master Match (N2M2) trial, which uses molecular signatures of GBM to inform treatment, future studies will likely use the molecular identities of tumors to designate treatment [36]. These findings portend a shift in molecular targeted therapy research as well, wherein laboratory studies testing existing treatments will enter Phase I/II clinical trials and studies identifying novel targets will advance into the development and testing of therapies in a laboratory setting. Specifically, we will likely see a broadening of the current clinical studies and ongoing clinical trials-including more immunotherapeutics and microenvironmental pathway testing-in addition to testing of wnt/β-catenin pathway inhibitors in vitro and in vivo in the coming years.

Limitations
There are several limitations to our analysis that should be considered. First, there was significant heterogeneity in the patient populations, interventions, and outcomes reported across clinical studies. The quality of the studies included in our analysis also varied, with the majority having low levels of evidence due to being case reports or series. Notably, only 52 clinical studies were identified, which may be an underrepresentation of the true number of current clinical research studies investigating molecular targeted therapies for glioma. For instance, for GBM alone, a study analyzing the clinical trials related to molecular targeted therapy totaled 257 [6]. In contrast, the sum of published literature and ongoing clinical trials identified in this study totaled 171. This discrepancy is likely due to the fact that clinical trial titles may utilize specific drug names rather than the term "molecular targeted therapy" or broad names of categories within molecular targeted therapies.
Additional limitations include the fact that the studies had varying methodological quality and targeted different molecular pathways, making it difficult to draw definitive conclusions. The categorization of molecular targets is an imperfect model as well, for pathways such as STAT3 can simultaneously qualify as involving protein kinase inhibitors and angiogenesis, for instance [108]. The categorization of tumor types has also changed drastically since the WHO 2021 guideline change. This study retroactively reflects the updated tumor classification for these studies, using the literature to classify the mutation status of known cell lines. This may create a discrepancy between GBM literature released prior to 2021 and current models, but it more accurately reflects what these studies can add to future glioma literature. Our study, while comprehensive and broad in scope, is restricted by the vast variation, particularly in histological methodology and molecular marker identification capabilities.
Other limitations inherent to a systematic review are that of the search terms-for there may be studies about molecular targeted therapies that do not self-identify as such; publication bias from only including published studies; limiting the studies to only those available in English for full-text screen; and the lack of meta-analysis to quantify the data.

Future Directions
Future studies should aim to address these limitations by conducting larger multiinstitutional clinical trials with standardized protocols and consistent reporting of outcomes. Studies should also consider investigating the effectiveness of combination therapies that target multiple molecular pathways simultaneously.

Conclusions
Here, we identify the current state of molecular target therapy research for adult-type diffuse gliomas, broadly found to be among one of three stages: validating molecular targeted therapies through published human clinical studies, testing existing therapies in a laboratory setting, and identifying novel molecular targets in a laboratory setting. We also queried clinicaltrials.gov for ongoing clinical trials on this topic. All studies predominantly investigated GBM, with few mentioning IDH-mutant astrocytomas or oligodendrogliomas. The most common molecular targets in all study types were protein kinase pathways such as PI3K/AKT/mTOR and Ras/BRAF/Mek/Erk. Microenvironmental targets were more numerous in clinical studies, whereas cell cycle/apoptosis were more numerous in laboratory studies. Immunotherapy pathways are few in number but on the rise in all study types, and the wnt/β-catenin pathway has been increasingly identified as a novel target.
Ultimately, these findings provide insight into the current state of molecular targeted therapy for glioma, highlighting the need for further investigation and the potential for this approach to improve patient outcomes.