Chromosomal Alterations and Genomic Instability in Glioblastoma: Implications for Diagnosis and Therapeutic Strategies

Frequent loss of function in various tumor suppressor genes, including Cyclin Dependent Kinase Inhibitor 2A/2B (CDKN2A/2B) and Protein Tyrosine Phosphatase Receptor Type D (PTPRD), expressing cell cycle regulatory proteins associates with loss of chromosome 9P in glioblastoma [6]. In addition to numerical Chromosomal Instability (CIN), double-stranded DNA breaks with possible rearrangement increase the rate of intrachromosomal aberrations in structural CIN, leading to chromosome segment gains or losses, chromosomal fusion, and chromothripsis [7, 8], a catastrophic process where the lagging chromosome undergoes massive rearrangement [9]. Whole-genome sequencing analysis has linked chromothripsis to the amplification of key oncogenes such as EGFR, Mouse Double Minute 2/4 (MDM2/4), and Cyclin-Dependent Kinase 4 (CDK4), which promote tumorigenesis by enhancing cell proliferation, inhibiting tumor suppressor pathways, and enabling uncontrolled cell cycle progression [10, 11]. RNA sequencing studies of Isocitrate Dehydrogenase (IDH)-wild-type Glioblastoma Multiforme (GBM) further identified chromothripsis-driven novel gene fusions, including the fusion of Septin-14 (SEPT14) and Vesicular overexpressed in cancer, prosurvival protein 1 (VOPP1) with EGFR forms EGFR-SEPT14 and EGFR-VOPP1, respectively, which are implicated in aberrant signaling and tumor progression. These findings underscore the dual impact of chromothripsis in amplifying oncogenes and creating fusion proteins with potential oncogenic properties, highlighting its central role in the aggressive biology of GBM [12]. Thus, the resulting intra-tumor heterogeneity driven by chromosomal alteration features sub-clones with different growth rates, malignant potential, resistance to therapy, propensity to invade and metastasize, and other phenotypes [5]. Such evidence attracts attention to focus on discussing the impact of CIN-based chromosomal aberration on GBM progression, as CIN could serve as a platform to develop novel therapeutics for difficult-to-treat cancers [4]. Compounds like Hesperadin , an Aurora B kinase inhibitor, represent promising avenues in this context of targeting cell cycle abnormalities.

Schematic Representation of Chromosomal Variations

Chromosomal variations can be divided into two major groups: structural and numerical. These variations collectively contribute to genomic instability, an important hallmark of cancer and developmental disorders.

Glioblastoma: Characteristics, Incidence, and Evolving Classification

Glioblastoma, alternatively known as GBM, is the most common and lethal primary malignancy of the central nervous system. As per the 2016 World Health Organization (WHO) classification [13], GBM is categorized into either IDH wild-type or IDH mutant depending upon the mutational status in the metabolic enzyme, IDH. GBM primarily affects the cerebral hemispheres of adult brains and specifically the brainstem region in children [14]. The Central Brain Tumor Registry of the United States (CBTRUS) statistical report demonstrates an incidence rate of 3.22 per 100,000 in the U.S., with a median age of 64 years. It accounts for 38.7% of Central Nervous System (CNS) tumors, with 23,000 GBM cases reported in India in 2016 [15]. Males are reported to have a higher incidence rate of GBM compared to females, and the increasing number of GBM cases in subsequent years indicates a concerning upward trend in GBM incidence globally. This trend is characterized by poor survival, usually less than 18 months in treated patients, and a 5-year survival under 5% [16, 17, 18]. Recent comprehensive analyses further highlight that glioblastoma is characterized by extensive intratumoral heterogeneity, genomic instability, and diffuse infiltrative growth, which collectively contribute to poor clinical outcomes and limited therapeutic success [19]. Hence, accurate identification of cancer subtypes has become a major priority for improving treatment strategies.

According to the revised 2021 World Health Organization classification of tumors of the central nervous system (WHO CNS5), glioblastoma is categorized under adult-type diffuse gliomas, characterized by their infiltrative growth into the surrounding CNS parenchyma, distinguishing them from circumscribed (non-diffuse) gliomas [20]. Under the new schema, only IDH-wildtype diffuse astrocytic tumors in adults are considered glioblastoma. Tumors that are IDH-mutant, even with high-grade histologic features, are now classified as astrocytoma, IDH-mutant, WHO grade 4, rather than "glioblastoma, IDH-mutant" (WHO CNS5) [20, 21]. Additionally, molecular criteria have gained importance: an IDH-wildtype diffuse astrocytoma lacking classic histologic grade 4 features (necrosis, microvascular proliferation) may nevertheless be classified as glioblastoma if one or more of the following are present: Telomerase Reverse Transcriptase (TERT) promoter mutation, EGFR amplification, or combined +7/-10 chromosomal copy number alterations [21, 22]. Therefore, the 'AMEN' score-based histological characterization, including nuclear Atypia, Mitosis index, Endothelial cell proliferation, and Necrosis, proposed within the WHO glioblastoma classification framework may be insufficient to clearly distinguish between primary and secondary GBM. In addition, inter-observer variability among pathologists can lead to inconsistent diagnoses [23]. Hence, integrating the knowledge of histology with molecular profiles, including IDH mutant variance and chromosomal aberrations of CNS tumors, facilitates recognition of distinct molecular subtypes, tumor heterogeneity, and spatial variability [20]. Therefore, the introduction of the 2021 WHO classification system compensates for the limitations of the 2016 WHO classification, which was based on histopathological grading, IDH mutation, and 1p/19q co-deletion status as key molecular markers, failing to clearly distinguish GBM subtypes [24, 25]. Thus, in parallel with considering mutational status in IDH and histological features, chromosomal alteration supports diagnostic precision of GBM in patients.

Classification of Glioma and Glioblastoma Development Pathways

Glioma and glioblastoma development pathways are characterized by several key features:

• A. Glioma Classification: Gliomas are physically classified based on the type of cells they originate from and their ability to invade neighboring brain parenchyma. Isocitrate Dehydrogenase (IDH) serves as a primary molecular marker for differentiating primary and secondary glioblastoma emerging from diffuse gliomas.
• B. and C. Development Pathways: Primary and secondary glioblastoma development pathways involve specific genetic alterations, including mutations in cell cycle regulatory proteins (p53 and RB1), alterations in the expression of growth factor receptors, CDKN2A/2B deletion, and chromosomal aberrations in Chromosome 7 and 10.

Astrocytoma, IDH-mutant, CNS WHO Grade 4

Astrocytoma, IDH-mutant, CNS WHO grade 4 comprises the subset of IDH-mutant diffuse astrocytic neoplasms that either display classic grade 4 histology (necrosis and/or microvascular proliferation) or harbor molecular high-risk alterations. Most notably, homozygous deletion of CDKN2A/2B alone is sufficient to designate an IDH-mutant tumor as WHO grade 4 under the 2021 WHO criteria [20, 26]. Clinically and molecularly, this category sits at the upper end of the IDH-mutant astrocytoma spectrum (grades 2–4) and typically includes tumors with the canonical co-occurrence of IDH1/IDH2 mutation, Tumor Protein 53 (TP53) mutation, and Alpha-Thalassemia/Mental Retardation Syndrome X-linked (ATRX) loss. Within grade 4 IDH-mutant astrocytomas, one also observes subgroups defined by additional focal copy-number alterations such as CDKN2A/2B homozygous deletion, CDK4/Cyclin D2 (CCND2) amplification, MDM2/MDM4 alterations, and focal gains or losses that are acquired during malignant progression [27, 28, 29].