Neuro-oncology is a complex field undergoing rapid changes with the advancement and evolution of sophisticated genetic testing. Evidence continues to grow in support of broad molecular and cytogenetic analysis for patients with brain tumors. The Genomics Laboratory within Mayo Clinic's Department of Laboratory Medicine and Pathology has developed two unique assays to support comprehensive patient care for these patients. The first is a next-generation sequencing assay that detects both gene mutations and rearrangements — all specific to brain tumors. The panel detects mutations within 118 genes and rearrangements within 81 genes. The second assay is a chromosomal microarray that assesses copy number variants across the genome. Clinical studies at Mayo Clinic have shown that this broad, but tumor-specific, genetic analysis has significant — and sometimes unexpected — clinical impact. The following case studies highlight the importance and clinical utility of these assays in diagnosis, prognosis, and therapy selection.

Case #1

Background

A 3-month-old boy presented with progressive increase of head circumference and decreased oral intake. Imaging studies revealed a large postcontrast enhancing right cerebellar tumor. His family history was significant for childhood medulloblastoma. The patient underwent tumor excision at an outside institution, and then the case was sent to Mayo Clinic for neuropathology consultation.

Histopathological findings

• The resection specimen showed an embryonal “small blue cell” tumor with multinodular reticulin-free areas displaying variable neurocytic maturation within a neuropil-like fibrillary matrix surrounded by densely packed, hypercellular reticulin-rich areas. By immunohistochemistry, the tumor cells were positive for synaptophysin, GAB1 (cytoplasmic), YAP (nuclear and cytoplasmic) and beta-catenin (cytoplasmic). The expression of p53 was low. These findings were consistent with the diagnosis of desmoplastic/nodular medulloblastoma (historically defined) and medulloblastoma, SHH-activated and TP53-wild-type (genetically defined), WHO grade IV.
• Given the reported positive family history for medulloblastoma, Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel was performed.

Figure 1. Histopathological findings

Genetic testing

• The NGS panel showed a SUFU mutation and no TP53 mutation, further supporting the diagnosis of medulloblastoma, SHH-activated and TP53-wild-type, WHO grade IV.
• The SUFU mutation was identified at 100% allelic frequency, suggesting a germline (i.e., constitutional) rather than somatic (i.e., tumor-specific) origin, in keeping with the family history for medulloblastoma.
• FISH testing was also performed and showed no evidence of monosomy 6 (associated with WNT Group) nor MYCN or MYC gene amplification (associated with Groups 3 and 4).

Figure 2. SUFU mutation at 100% variant allelic frequency

Teaching points

• Genetically defined medulloblastoma is currently divided into the following main subtypes: WNT-activated, SHH-activated, and non-WNT/non-SHH Group 3 and Group 4.¹
• Histologically defined desmoplastic/nodular medulloblastoma is almost exclusively SHH-activated. SHH-activated medulloblastoma is further divided in TP53-mutant and TP53-wild-type.
• SUFU mutations have been described primarily in the “medulloblastoma, SHH-activated and TP53-wild-type” subgroup, which encompasses the majority of very young children.²
• Preclinical studies have shown that SHH-activated medulloblastoma with a SUFU mutation is primarily resistant to small-molecule inhibitors of SHH component smoothened (SMO) protein (e.g., vismodegib and sonidegib).³ 
• Hereditary cancer syndromes associated with SUFU germline mutations, which may or may not fulfill the diagnosis of nevoid basal cell carcinoma syndrome (Gorlin syndrome), have been frequently described in very young children (<3 years) with an SHH-activated medulloblastoma.⁴ 
• Tumor genetic testing may suggest a somatic (i.e., tumor-specific) or germline (i.e., hereditary) origin for an identified mutation. If likely germline in origin, as seen in this case, the specific genomic coordinates for the identified mutation can guide follow-up, targeted germline testing.

Case #2

Background

A 14-year-old girl with a history of two prior subtotal resections of a left cerebellar pilocytic astrocytoma presents with progressive enlargement of the residual tumor. The neurologic exam was essentially normal except for mild left appendicular ataxia. The patient was referred to Mayo Clinic to attempt a gross total tumor resection.

Histopathological findings

• The resection specimen showed a pilocytic astrocytoma (WHO grade I) with a biphasic densely fibrillated and loosely arranged microcystic pattern, and a relatively solid pattern of growth. Rosenthal fibers and eosinophilic granular bodies were present. Features of anaplasia, such as brisk mitotic activity or necrosis, were not observed.
• Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel and chromosomal microarray were performed to further characterize this tumor, given the progressive residual tumor growth. 

Figure 4. Histopathological findings

Genetic testing

• The NGS panel revealed a fusion between BRAF and KIAA1549, which is the most frequent genetic abnormality observed in pilocytic astrocytoma, supporting this diagnosis.^5-7
• The chromosomal microarray showed a segmental copy gain of 7q34 (including BRAF and KIAA1549) consistent with similar molecularly defined tandem duplications that result in the aberrant KIAA1549-BRAF fusion product, also supporting the diagnosis of a pilocytic astrocytoma.

Figure 5. KIAA1549 (exon 16)-BRAF (exon 11) fusion

Figure 6. Chromosomal microarray pattern with segmental copy gain of 7q34

Teaching points

• Pilocytic astrocytoma is a single pathway disease driven by activating alterations involving the MAPK pathway. KIAA1549-BRAF fusion is the most frequent MAPK pathway genetic abnormality observed in pilocytic astrocytoma (>70%) and a potentially therapeutic targetable event.^8,9 
• KIAA1549-BRAF fusion is the result of a 1.9 megabase tandem duplication at 7q34, which disrupts the regulatory domain of BRAF leading to constitutive activation of this oncogene.⁵ This fusion event can be detected by RNA-based fusion analysis, and its footprint (focal duplication) can be identified by chromosomal microarray.
• The prevalence of KIAA1549-BRAF fusion varies with tumor location and is more frequent in cerebellar than in extra-cerebellar tumors.⁸ 

Case Study #3

Background

A 10-year-old boy involved in a car accident had an incidentally discovered left brainstem/IV ventricle exophytic predominantly non-postcontrast enhancing tumor. The radiological differential diagnoses included ependymoma/subependymoma. The patient was referred to Mayo Clinic for tumor resection.

Figure 7. Axial MRI with contrast: Well-circumscribed tumor involving left medulla/IV ventricle with very focal postcontrast enhancement

Histopathological findings

• The resection specimen revealed a low-grade astrocytoma with a somewhat biphasic appearance, consisting of densely fibrillated and loosely arranged areas. Scattered Rosenthal fibers were present, especially in the densely fibrillated areas. Proliferative activity was very low with no mitoses and a very low Ki67 labeling index. Neither microvascular proliferation nor necrosis were present.
• Olig2 immunostain supported an astrocytic (rather than ependymal) differentiation, and neurofilament immunostain disclosed an infiltrating pattern of growth with numerous axons present throughout the tumor.
• Taken together, the main differential diagnosis included a pilocytic astrocytoma versus a low-grade diffuse astrocytoma.
• Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel was performed to further characterize this tumor given the infiltrative pattern or growth.

Figure 8. Histopathological findings

Figure 9. OLIG2, neurofilament, KI67

Genetic testing

• The NGS panel revealed a KCTD16-NTRK2 fusion. Although not functionally characterized or previously reported, this fusion is predicted to maintain an intact NTRK2 C-terminal kinase domain similar to other oncogenic NTRK2 fusions, indicating that the KCTD16-NTRK2 fusion is also likely oncogenic.
• As NTRK2 fusions have been recurrently reported in pilocytic astrocytoma, the finding of a KCTD16-NTRK2 fusion supported the integrated diagnosis of pilocytic astrocytoma, WHO grade I with KCTD16-NTRK2 fusion.⁸

Figure 10. KCTD16 (exon 3)-NTRK2 (exon 16) fusion

Teaching points

• Pilocytic astrocytoma is a single pathway disease driven by activating alterations involving the MAPK pathway. NTRK2 fusions also activate the MAPK pathway and have been recurrently, albeit infrequently, observed in pilocytic astrocytoma, especially in extra-cerebellar tumor.⁸
• There is clinically approved histology-agnostic multi-kinase inhibitor for adult and pediatric patients with advanced solid tumors, including CNS tumors, harboring NTRK1/2/3 fusions without a known acquired resistance mutation.^10,11
• Targeted therapies represent an alternative adjuvant treatment option for patients with tumors not amenable to complete surgical resection and tumors that recur/progress.

Case #4

Background

A 15-year-old boy recently presented with new-onset seizures. Imaging studies showed a left parieto-occipital tumor with postcontrast enhancement. The patient underwent tumor excision at an outside institution, and the case was sent for neuropathology consultation at Mayo Clinic.

Histopathological findings

• The resection specimen disclosed a high-grade poorly differentiated neuroepithelial tumor. Necrosis was present but microvascular proliferation was not observed.
• Immunohistochemical stains showed absence of IDH1-R132H, GFAP, H3K27M, and Olig2 staining, variable ATRX loss, and strong p53 overexpression.
• Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel was performed to further characterize this tumor.

Figure 11. Histological findings

Figure 12. H3K27M, ATRX, p53

Genetic testing

• The NGS panel confirmed the lack of an IDH1-IDH2 mutation and revealed an H3F3A G34R* mutation in addition to an ATRX and two TP53 mutations.
• This mutation profile is characteristic of a distinctive high-grade neuroepithelial tumor with H3 G34-mutation, an emergent new entity among primary CNS tumors in adolescents and young adults.

*Recommended genetic nomenclature (HGVS): G35R

Figure 13. H3F3A, ATRX, and TP53 mutations

Teaching points

• High-grade neuroepithelial tumors with H3F3A G34 mutations are typically non-midline high-grade neoplasms with microscopic characteristics of either glioblastoma or CNS embryonal tumor occurring in both pediatric and young adult patients.^12-15
• H3F3A G34 mutations are associated with global hypomethylation and with a distinct gene expression profile, including downregulation of OLIG1/2 genes, resulting in Olig2 loss of protein expression by immunohistochemistry, as seen in this case.¹³
• Recent integrative clinical, histopathological, and molecular study of H3F3A G34-mutant CNS tumors suggested that, despite the histopathological heterogeneity, these tumors had a unifying epigenetic signature, and therefore could be outlined as a separate entity of malignant supratentorial CNS tumors.^13,15 
• Patients with H3F3A G34-mutant tumors have a better prognosis than patients with tumors harboring H3 K27M mutations.¹⁵ Additionally, MGMT promoter methylation was frequently observed and associated with better prognosis, whereas the presence of oncogene amplification seemed to be a negative prognostic marker in these tumors.¹⁶

Case #5

A 13-year-old boy presented with a two-year history of seizures. Imaging studies revealed a left mesial temporal non-postcontrast enhancing tumor. The patient underwent tumor resection at Mayo Clinic.

Figure 14. T1-weighted axial MRI with contrast and T2-weighted axial MRI without contrast: left mesial temporal non-postcontrast enhancing tumor

Histopathological findings

• The biopsy showed a low-grade glioma with low cellularity, mild cytological atypia, and an apparent infiltrative growth pattern. Mitotic activity, microvascular proliferation, and necrosis were absent. Proliferative index by Ki67 was low.
• Immunohistochemical stains disclosed atypical cells to be immunoreactive for Olig2 and negative for CD68 and KP1 immunostain, supporting glial/astrocytic differentiation.
• The histopathological features were consistent with a low-grade diffuse glioma.
• Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel was performed to further characterize this tumor.

Figure 15. Histopathological findings

Figure 16. Olig2, KP1, Ki67

Genetic testing

• The neuro-oncology NGS panel identified an MYB-PCDHGA1 fusion.
• MYB-PCDHGA1 fusions are predicted to result in loss of the MYB negative regulatory region and have been shown to be associated with MYB overexpression.¹⁷
• Similar fusions have been reported in “pediatric-type” diffuse gliomas, supporting the diagnosis of a “diffuse glioma, MYB-altered” in this case.¹⁸

Figure 17. MYB (exon 9)-PCDHGA1 (exon 2) fusion

Teaching points

• “Pediatric-type” diffuse gliomas are distinct from “adult-type” diffuse gliomas, as they are typically WHO grade II tumors that rarely progress.¹ 
• While “adult-type” diffuse low-grade gliomas are typically IDH mutant, the “pediatric-type” are IDH-wild-type.
• “Pediatric-type” diffuse astrocytic tumors are characterized by MYB/MYBL1 fusions, and “pediatric-type” tumors with oligodendroglial morphology frequently show FGFR1 alterations rather than IDH/TERT promoter mutations and 1p/19q co-deletion typical of adult oligodendroglioma.^17,19

Case #6

A 17-year-old girl presented with a one-month history of intermittent, brief sensory changes on her left arm. The physical exam revealed intertriginous freckling, café-au-lait macules, and multiple subcutaneous nodules. Her past family medical history was unremarkable. Imaging studies revealed a right parietal enhancing solid-cystic tumor. The patient underwent tumor resection at Mayo Clinic.

Histopathological findings

• The resection specimen revealed an astrocytic glioma with large pleomorphic, xhantic, and spindle cells. Intensely eosinophilic granular bodies were also present and the tumor had a predominantly solid growth pattern. Mitotic activity and necrosis were absent. The morphological findings were consistent with a low-grade pleomorphic xanthoastrocytoma (PXA).
• Immunohistochemical stains confirmed the glial nature of this tumor with reactivity to Olig2 and S100 stains. Tumor cells were negative for BRAF V600E immunostain, indicating the absence of a BRAF V600E mutation in the tumor.
• Mayo Clinic Laboratories’ Neuro-Oncology Expanded NGS Panel and chromosomal microarray were performed to further characterize this tumor.

Figure 19. Histopathological findings

Figure 20. S100, Olig2, BRAFV600E

Genetic testing

• The NGS panel identified an NF1 mutation. The identified NF1 mutation had not been previously reported as a somatic (i.e., tumor-specific) mutation but had been recurrently observed as a germline mutation in association with neurofibromatosis type 1 syndrome (NF1). This patient was subsequently shown to meet clinical criteria for NF1, indicating that the identified NF1 mutation was most likely germline (i.e., constitutional) in origin. 
• Chromosomal microarray revealed focal chromosomal losses with homozygous deletion of CDKN2A/B genes, a finding frequently seen in PXA.²¹
• The molecular profile supports the histopathological diagnosis of PXA (WHO grade II).

Figure 21. Chromosomal pattern with 9p loss including CDKN2A/B homozygous loss in addition to partial chromosomal losses

Teaching points

• PXA is a rare primary CNS tumor genetically characterized by frequent MAPK pathway alterations, largely represented by the BRAF V600E mutation (50%-80%) and recurrent copy number changes including CDKN2A/B homozygous deletion (90%).^20-21
• Neither BRAF V600E mutations nor CDKN2A/B homozygous deletion is associated with the presence of histological anaplastic features.^20-21 
• While there is no distinct association with hereditary syndromes, PXA has been reported in the context of neurofibromatosis type 1 syndrome.¹⁹ Given the high frequency of MAPK pathway alterations in sporadic PXA, such association is not unexpected and these NF1-associated tumors are thought to be driven by NF1 rather than BRAF alterations, as seen in this case.

Case #7

Background

A 4-year-old boy presented with headaches and vomiting. Imaging studies revealed a large left frontal tumor. The patient underwent tumor resection at an outside institution, and the case was sent for genetic testing at Mayo Clinic.

Histopathological findings

• The representative hematoxylin-eosin section of the paraffin block sent for testing showed a tumor with a biphasic appearance. Highly cellular areas with poorly differentiated embryonal tumor cells of relatively large size and often radially arranged around blood vessels were intermixed with paucicellular neuropil-rich areas with ganglion cells. Although not obvious in the available representative section sent for testing, true multilayered rosettes were reportedly observed in other sections of the tumor. Mitotic activity was present and necrosis was also reportedly present.
• Immunohistochemical studies performed at the referring institution were reported to show synaptophysin reactivity in the tumor, including the neuropil areas, and intact nuclear protein expression by INI1 immunostain.
• Overall, the findings were thought to be consistent with an embryonal tumor with multilayered rosettes, and Mayo Clinic Laboratories’ Neuro-Oncology NGS Expanded Panel and chromosomal microarray were performed for further tumor characterization.

Figure 22. Histopathological findings

Genetic testing

• The NGS panel was noninformative in this case and revealed only a single variant of uncertain significance.
• Chromosomal microarray disclosed amplification of 19q13.42 (including the C19MC region), multiple gains within 11q, and gain of chromosome 2, which have been recurrently reported in embryonal tumors with multilayered rosettes (ETMR), C19MC-altered. Additional copy number changes also were present. 
• Taken together, the molecular profile supports the integrated diagnosis of an embryonal tumor with multilayered rosettes, C19MC-altered, WHO grade IV (2016 WHO classification of CNS tumors).

Figure 23. Chromosomal microarray pattern with amplification of 19q14.42 in addition to other copy number changes

Figure 24. Amplified 19q14.42 includes the C19MC cluster

References

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