Table 10.

Summary of the advantages and disadvantages of each diagnostic method, including MRI, tissue biopsy, and various liquid biopsy-based methods.

Diagnostic Method	Advantages	Disadvantages
MRI	Noninvasive procedure with no known risk Provides initial diagnosis and anatomic characterization of GBM through a non-invasive procedure	Challenges in distinguishing GBM from other brain diseases and concurrent pathological processes Challenges in correlating MRI features with molecular characteristics Challenges in differentiating true tumor recurrence from PsP
Tissue biopsy	Clinically validated Provides histological evaluation Enables histologic and molecular characterization of the tumor Reveals spatial and temporal tumor heterogeneity	Highly invasive procedure with associated risks Localized sampling of tissue Longer time to complete the procedure Low sensitivity Limited or no repeated sampling Fails to accurately reveal intra-tumoral heterogeneity Unable to assess tumor activity in real time Organ penetration required Cannot reveal tumor evolution Lacks real-time monitoring of treatment response. High cost of sample collection
ctDNA	Higher levels than those of CTCs High specificity ctDNA quantity correlates with tumor burden and disease stage Easier to collect and established detection techniques available Ability to detect CNAs and rearrangements with NGS Relatively inexpensive	ctDNA level varies depending on the tissue type and cancer stage Gliomas have the lowest detectable levels of ctDNA ctDNA concentration in cancer is very low (180 ng/mL) and potentially even lower in GBM ctDNA has a short half-life (<2.5 h) Released mainly by apoptotic or necrotic cells and therefore represents only a subpopulation of tumor cells Sensitivity of detection limited
miRNAs	Relative high stability in biofluids (half-life ~16.4 h) Fast High sensitivity Ubiquitous appearance in biofluids Tissue-specific expression patterns of certain miRNAs Can be measured using high-throughput platforms High sequence homology between animal models and humans facilitates translation of miRNA biomarkers Novel miRNA quantification methods such as dynamic chemical labeling for point-of-care clinical detection Knowledge of a wide range of expression levels of miRNAs Relatively inexpensive	No standardized methods for RNA extraction and sequencingLess specific than ctDNA Low sample yield Low specificity Measurement subject to sample quality Lack of consensus regarding controls and standardization of assays Biological variability can be high, possibly influenced by smoking, diet, and other environmental factors Low levels of expression of many individual miRNAs
CTCs	Highly specific Offer insights into protein, DNA, and RNA levels Immunoaffinity enrichment: highly specific Size and density separation: heterogeneous sample of tumor cells, faster and less expensive than immunoaffinity enrichment, label-free CTCs obtained	High blood volume required Low sample yield Lack of standardized methods for isolating and characterizing CTCsLow presence in blood CTCs have a short half-life (1–2.4 h)May not represent the whole tumor
EVs/Exosomes	Fast and relatively inexpensive Carry RNAs, proteins, and lipids, all of which are protected from enzyme degradation Able to cross an intact BBB Released by both normal and cancer cells	Lack of standardized methods for isolating EVs EVs are highly heterogeneous EVs have a short half-life (<30 min) Exosomes have a short half-life (<30 min) Released by non-neoplastic cells, resulting in a background of non-tumoral EVs in the blood Requires a high volume of blood Produces a low sample yield
Metabolites	Metabolomics enables endogenous metabolite profiling Reveals information about the current pathophysiological status of patients Offers insights into the tumor’s unique biochemical landscape Reveals alterations in the cellular phenotype due to its specific focus on biochemical changes Reveals information about PK and PD drug processes Enables the monitoring of disease progression and treatment response through changes in metabolic and lipidomic profiles	Complex data analysis. The vast amount of data generated requires sophisticated analysis techniques, which can be challenging and time-consuming. Variability in sample preparation, instrument calibration, and data interpretation can affect the reproducibility and accuracy of results Metabolites have a short half-life (<100 min) Can be costly, requiring specialized equipment and expertise Translating findings from metabolic and lipidomic profiling into clinical practice may take time and further validation The heterogeneity of GBM may lead to challenges in interpreting metabolic and lipidomic profiles, as different regions of the tumor may exhibit distinct profiles
Circulating nucleosome-associated histonemodifications	Circulating nucleosome-associated histone modifications are highly stable Can be detected by ELISA and ChLIA Epigenetics is an emerging and intensive field of research	Low specificity
Circulating proteins as potential biomarkers	Circulating proteins can be detected in blood (i.e., serum or plasma), CSF, and urine Circulating proteins are more accessible and can be repeated multiple times, facilitating the regular monitoring of the disease Circulating proteins can potentially detect glioblastoma at an early stage and monitor tumor progression or response to therapy in real time They have shown promise in guiding diagnosis, assessing treatment responses, and understanding mechanisms of treatment resistance Protein biomarkers can reflect the dynamic changes in the tumor microenvironment, providing specific information about the state of the disease Potential for Personalized Medicine: Analysis of circulating proteins can help tailor personalized treatment strategies based on the specific protein expression profile of an individual’s tumor Circulating proteins can provide insights into the heterogeneous nature of glioblastomas, potentially revealing multiple aspects of tumor biology	Variability in sample preparation, instrument calibration, and data interpretation can affect the reproducibility and accuracy of the results Variability and sensitivity. Circulating protein levels can be influenced by various factors unrelated to glioblastoma, such as inflammation, infection, or other comorbidities, leading to potential false positives or variability in the results Limited sensitivity and specificity. Some circulating proteins may not be specific to glioblastoma and could be elevated in other types of cancers or diseases, complicating the interpretation of the results Circulating proteins have a short half-life (<1 h) Some GBM-related proteins might be present at very low levels in the circulation (blood or CSF), making their detection challenging and requiring highly sensitive analytical techniques Advanced and sensitive technologies are required to accurately measure circulating proteins, which can be costly and technically demanding Many potential protein biomarkers for glioblastoma are still in the research phase and require extensive clinical validation before they can be reliably used in a clinical setting Further investigation is needed to determine their translational significance in clinical settings.

Abbreviations: BBB, blood–brain barrier; ChLIA, chemiluminescence immunoassay; CTCs, circulating tumor cells; ctDNA, circulating tumor DNA; ELISA, enzyme-linked immunosorbent assay; EVs, extracellular vesicles; GBM, glioblastoma; PD, pharmacodynamics; PK, pharmacokinetics; miRNAs, microRNAs; MRI, magnetic resonance imaging; PsP, pseudoprogression.