Metastatic extraneural glioblastoma diagnosed with molecular testing

Abstract Glioblastoma, the most common malignant brain tumor in adults, is associated with a median overall survival duration of less than 2 years. Extraneural metastases occur in less than 1% of all patients with glioblastoma. The mechanism of extraneural metastasis is unclear. We present a case of extensive extraneural, extraosseous, epidural, and soft-tissue metastasis of glioblastoma. The diagnosis of metastatic glioblastoma was made only after next-generation sequencing (NGS) of the metastatic paraspinal lesions was completed. The CDK4, pTERT, PTEN, and TP53 molecular alterations seen in the initial intracranial glioblastoma were found in the paraspinal tumor, along with the addition of MYC, which is implicated in angiogenesis and epidermal-to-mesenchymal transition. Immunohistochemical stains showed that neoplastic cells were negative for GFAP. In conclusion, this case raises awareness about the role of NGS in the diagnosis of extraneural glioblastoma. This diagnosis was not possible with histology alone and only became evident after molecular profiling of the metastatic lesions and its comparison to the original tumor.


Introduction
Glioblastoma, the most common malignant brain tumor in adults, is associated with a median overall survival duration of less than 2 years. 1 Among patients with glioblastoma, extraneural metastases are very rare and comprise less than 1% of all cases. 2 It is postulated that the incidence of extraneural metastasis is low because the microenvironment outside of the central nervous system (CNS) is inadequate for glioma cell growth and/or because the survival of patients with glioblastoma is dismal.The most common extraneural metastatic sites are the lungs, lymph nodes, and bones. 3,4The pathogenesis of extraneural metastasis is unclear, but potential mechanisms include direct invasion through dura and bone, venous invasion via the leptomeningeal sinuses or intracerebral veins, and lymphatic drainage of cerebrospinal fluid into the extraneural tissue. 5Even though molecular markers of poor prognosis of intracranial glioblastoma such as isocitrate dehydrogenase (IDH) wild-type and methyl-guanine methyl transferase (MGMT) promoter unmethylated status have been identified, data about the acquired molecular changes that allow the tumor to grow outside of the nervous system are limited.Metastatic lesions are rarely biopsied and profiled molecularly; however, molecular analysis is necessary to confirm the diagnosis of metastatic glioblastoma by ruling out other primary neoplastic processes.
Here, we present a case of extensive extraneural, extraosseous, epidural, and soft-tissue metastasis of glioblastoma, which posed a diagnostic challenge because of its atypical imaging and histological features.The diagnosis of metastatic glioblastoma was made only after next-generation sequencing

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The Oncologist, 2024, Vol. 29, No. 9 (NGS) of the metastatic paraspinal lesions showed that they shared molecular alterations with the original intracranial tumor, indicating clonal evolution in the extraneural metastases.This unique case of molecularly defined metastatic glioblastoma highlights the challenges involved in establishing a histologic diagnosis and demonstrates the evolution of molecular aberrations noted in the metastatic foci.

Intracranial tumor
The MD Anderson Solid Tumor Genomic Assay (STGA) was used to analyze the newly diagnosed intracranial tumor: NGS-based analysis included gene panel mutations in the coding sequence of 134 genes and selected amplifications in 47 genes (overlap: 146 genes total) in our CLIA-certified molecular diagnostics laboratory.
In addition, unstained sections were obtained to perform mutation analysis precision panel (MAPP) assays with a custom high-throughput next-generation sequencing-based CLIA assay that targeted hybridization-based capture technology for detection of sequence variants/mutations in 610 genes, copy number variants in 583 genes, selected gene rearrangement in 34 genes, and selected genomic immune-oncology signatures including microsatellite instability and tumor mutational burden.The MAPP assay uses the NovaSeq 6000 next-generation sequencing platform and bidirectional paired-end sequencing to identify nucleic acid variants for all coding regions from most genes in the panel.Reported somatic mutations are identified by comparison to the human genome reference sequence GRCh37/hg19 and reviewed in OncoSeek against a process-matched normal control.

Results
An overview of the patient's clinical history is depicted in Figure 1.A right-handed man in his late 40s presented with new-onset progressive headaches.Magnetic resonance imaging (MRI) of the brain demonstrated a large, heterogeneously enhancing cystic lesion in the right parietal lobe (Figure 2a).He underwent gross total resection of the lesion, and the pathology findings were consistent with glioblastoma.NGS sequencing using STGA identified CDK4 amplification and pTERT, PTEN, and TP53 mutations (Figure 1).The tumor was IDH wild-type and the MGMT promoter region was unmethylated.The patient's postoperative neurological examination was unremarkable, except for left homonymous hemianopia.His Karnofsky performance scale score was 90.He underwent standard-of-care radiation therapy (60 Gy, 30 fractions) and concurrent temozolomide, which he tolerated well.
The patient's post-chemoradiation therapy MRI showed subtle enhancement surrounding the surgical cavity.After completion of one cycle of adjuvant temozolomide, MRI of the brain demonstrated a new enhancing lesion in the right temporal lobe outside of the initial radiation field, and the right parietal resection cavity was stable (Figure 2b).The patient received radiation therapy (52 Gy, 15 fractions) and concurrent temozolomide for the right temporal lesion.
Three weeks after completion of radiation therapy, the patient presented with back pain.Computed tomography (CT) of the chest, abdomen, and pelvis with contrast and CT of the thoracic and lumbar spine without contrast were unremarkable, except for mild degenerative disc disease of the thoracic and lumbar spine.Two weeks later, an MRI of the spine showed diffuse skeletal lesions with paraspinal and epidural tumor extension throughout the cervical, thoracic, and lumbar spine and pelvis and enhancement of the nerve roots of the cauda equina.MRI of the brain showed multifocal areas of dural/ extra-axial disease in the right frontal and temporal lobes.The patient was started on a clinical trial with a CDK4 inhibitor.On cycle 1, day 21, he was admitted to the hospital with worsening back pain.A neurological examination showed no significant changes since the last assessment, 3 weeks earlier.MRI of the spine showed extensive extraosseous epidural and soft-tissue lesions (Figure 2c, d).Positron emission tomography (PET)/ CT imaging showed numerous sites of abnormal fluorodeoxyglucose (FDG) avidity within the skeleton, extending into the adjacent soft-tissues and the spinal canal (Figure 2e, f).
To further characterize the neoplasm, NGS of tumor tissue obtained from the second paraspinal biopsy was initiated using The MD Anderson Mutation Analysis Precision Panel (MDA MAPP).The CDK4 amplification and pTERT, PTEN, and P53 mutations seen in the initial intracranial glioblastoma were found in the paraspinal tumor, along with MYC amplification.Additional molecular aberrations found on the metastatic foci obtained on MDA MAPP were HSP90AB1 and vascular endothelial growth factor (VEGF)-A amplifications and GRIN2A mutation (Figure 1).It is notable that MYC was included in the analysis of both intracranial and metastatic lesions, but HSP90AB1, VEGFA, and GRIN2A were not included in the NGS panel used for the analysis of the intracranial tumor.While awaiting the final results of molecular testing (10 months after the initial diagnosis of glioblastoma), the patient developed progressive neurological decline and paraparesis that quickly progressed to paraplegia despite high-dose steroids.Palliative radiation therapy was attempted but aborted owing to poor tolerance.The patient was transitioned to hospice care and passed away 1 month later.

Discussion
Here, we present a rare case of glioblastoma with rapid clinical deterioration that required NGS analysis to make the diagnosis of extraneural metastases.Histologic and extensive immunohistochemical testing of the extraneural tumor, which was biopsied twice, and the bone marrow were insufficient to classify the metastatic tumor.Identification of the molecular alterations seen in the original tumor that included CDK4 amplification and pTERT, PTEN, and P53 mutations conthe diagnosis of extraneural glioblastoma.In addition to these molecular alterations, amplification of MYC, HSP90AB1, and VEGF-A and mutation of GRIN2A were present in the metastatic tumors.While MYC amplification was an acquired molecular event (included in the NGS panel used for the initial and metastatic tumor), it is not clear whether HSP90AB1, VEGFA, or GRIN2A were present at the time of initial diagnosis.Nevertheless, the 3 gene amplifications in the metastatic tumors are implicated in angiogenesis and epidermal-mesenchymal transition.The MYC oncogene encodes a transcription factor that regulates several  genes related to cell cycle progression, differentiation, and cellular transformation. 6MYC amplification is noted in 0.88% of patients with glioblastoma, 7 and it is the most frequently amplified gene across all cancer types, occurring in 21% of cases based on recent cancer genome atlas analysis. 8MYC enhances the self-renewal capacity of glioblastoma stem-like cells and maintains their tumorigenic potential. 9,10In addition, overexpression of MYC has been shown to induce epithelialmesenchymal transition, angiogenesis, and tumorigenesis in mammary epithelial cells. 11,12HSP90AB1 (heat shock protein 90 kDA, alpha, class B, member 1) is a member of the large family of HSP proteins, which are molecular chaperones that enable proper folding, refolding, and stabilization of vital proteins within the cell.HSP90AB1 alterations are found in approximately 1% of all cancers, with the highest prevalence in glioblastoma, followed by colorectal adenocarcinoma and lung adenocarcinoma. 7HSP90AB1 stabilizes VEGF-A-induced pro-angiogenic protein BAZF (BCL-6 associated zinc finger protein) and may positively regulate angiogenesis. 13The role of HSP90AB1 in glioblastoma remains unclear.VEGF-A-the prototype member of the VEGF family-predominantly regulates the process of angiogenesis in the CNS and directly interacts with VEGF receptors expressed on cancer cells, stimulating disease progression. 14EGF overexpression is an adverse prognostic factor in many cancers, including glioblastoma. 15Lastly, mutations in GRIN2A, which encodes the NMDA receptor subunit NR2A, have been associated with poor survival in melanoma, 16 but its role in glioblastoma remains unclear.Although the role of these alterations in the aggressive nature of patient's tumor is unclear, it is plausible that they are contributing factors.We have previously reported a partial response that lasted for 10 years in a patient with glioblastoma with CDK4 amplification and FGFR3-TACC3 chromosomal fusion, treated with bevacizumab and valproic acid, that targeted multiple pathways associated with tumor progression and angiogenesis. 17ther investigators have reviewed 28 published cases of glioblastoma metastatic to the vertebra. 18They reported that the mean age at presentation was 38.4 years and the average overall survival was 26 months.Interestingly, the clinical presentation ranged from asymptomatic disease to varying degrees of pain, extremity weakness, or other neurologic deficits.In 8 (28.6%) patients, the diagnosis was made via autopsy.The median survival duration after diagnosis of vertebral metastasis was 10 months. 18n another series of 10 patients seen at the Memorial Sloan Kettering Cancer Center with extraneural metastases of glioblastoma (9 patients) and gliosarcoma (1 patient), the median age was 39 years; 7 patients were men and 3 women. 19All patients had surgical resection and radiation therapy; and 9 patients received temozolomide, with subsequent individualized chemotherapy.The median overall survival from initial diagnosis was 19.6 months (range 11.2-57.5 months) and from extraneural metastasis, 5 months (range 1-16.1 months).The most common genomic alterations identified in 8 patients were P53 (n = 5), RB1 (n = 5), PTEN (n = 4), TERT (n = 4), ATRX (n = 4), NF1 (n = 3), IDH1 (n = 1), EGFR amplification (n = 2), and EGFR mutation (n = 1).In 3 of these 5 cases, the TP53 mutation was found in both the primary and metastatic sites.ATRX, PTEN, RB1, TERT, IDH1, and NF1 were found in both primary and metastatic sites; TP53 and RB1 were both mutated in 4 out of 7 (57%) sequenced primary tumors.The authors concluded that several risk factors emerged for extraneural metastasis of glioblastoma and gliosarcoma, including sarcomatous dedifferentiation, disruption of normal anatomic barriers during surgical resection, and tumor suppressor gene alterations. 19

Conclusions
Our case raises awareness regarding the diagnosis of extraneural glioblastoma and highlights the important role of molecular analysis in the diagnosis of extraosseous, epidural, or soft-tissue metastases of glioblastoma.The mechanism of metastasis for diffuse gliomas is not well-understood, in part due to the rarity of these cases.In our case, next-generation sequencing provided insights into understanding the pathogenesis and evolution of intracranial glioblastoma from the initial tumor to the metastatic site.The widespread use of next-generation sequencing as a diagnostic tool has the potential to shed light on the acquired molecular aberrations that drive extraneural metastasis in glioblastoma.

Figure 2 .
Figure 2. a. MRI T1 post-contrast axial image demonstrates a large, heterogeneously enhancing cystic lesion in the right parietal lobe; b.MRI T1 post-contrast axial image demonstrates an enhancing lesion in the right temporal lobe outside of the initial radiation field (white arrow); c-d.MRI of the cervical and thoracic spine and lumbosacral image demonstrates extensive extraosseous epidural and soft-tissue metastasis.Examples are shown with an arrow at the level of C6, T2, T5-T6, and L2; e-f.PET/CT demonstrates numerous sites of abnormal FDG avidity within the skeleton, extending into the adjacent soft-tissues and the spinal canal.g-h.H&E staining of initial intracranial glioblastoma: g.Neoplastic cellular proliferation replacing normal brain.Note the presence of relatively normal brain matter on the left side (H&E 10×).h.Higher magnification shows cells with round to oval nuclei, moderate amounts of eosinophilic cytoplasm, and several mitotic figures-all features of grade 4 glioblastoma (H&E 40×).i-j.H&E staining of extraneural paraspinal metastatic glioblastoma: i. Poorly differentiated malignant neoplasm with focal areas of necrosis (H&E 20X); j.Tumor is composed of large cells with indistinct cell borders, round to oval nuclei, and nucleoli.Mitotic figures are present (H&E 60×).