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 46-year-old man presented with seizures and was found to have a right temporal lobe ring-enhancing tumor. The patient underwent tumor resection at Mayo Clinic.
Histopathological findings
The resected specimen showed a high-grade infiltrating glioma composed by monomorphous tumor cells with round nuclei and high nuclear-to-cytoplasmic ratio. Mitotic activity was brisk and microcalcifications were focally present. Microvascular proliferation and necrosis were observed. Immunohistochemical studies excluded the most common IDH1 mutation (p.R132H), while ATRX protein expression was retained.
The diagnoses considered included: 1) IDH-wild-type small-cell glioblastoma; 2) IDH-mutant glioblastoma; and 3) IDH-mutant and 1p/19q co-deleted anaplastic oligodendroglioma.
T1-weighted axial MRI: right temporal enhancing lesion
Genetic testing
Neuro-oncology next-generation sequencing (NGS) panel was consistent with the absence of an IDH mutation and revealed a TERT promoter mutation.
Chromosomal microarray showed intact 1p/19q status, EGFR amplification associated with gain of chromosome 7, loss of chromosome 10, CDKN2A/B homozygous loss, and gain of chromosomes 19 and 20.
The overall genetic findings, in conjunction with the morphology, led to the final integrated diagnosis of IDH-wild-type small-cell glioblastoma (WHO grade IV).
Teaching points
Small-cell glioblastoma is a subtype of IDH-wild-type glioblastoma that morphologically overlaps with anaplastic oligodendroglioma due to the nuclear regularity, high nuclear-to-cytoplasmic ratio, microcalcifications, and lack of cellular pleomorphism.
TERT promoter mutations are characteristic of both IDH-wild-type glioblastoma and IDH-mutant oligodendroglioma, which is molecularly defined by the concurrent presence of an IDH mutation and 1p/19q co-deletion. The finding of a TERT promoter mutation in the absence of an IDH mutation, as seen in this case, is consistent with an integrated diagnosis of IDH-wild-type glioblastoma.
Frequent molecular features of small-cell glioblastoma include EGFR amplification (~70%) and losses of chromosome 10 (>95%), as observed in this case.1
A typical chromosomal microarray pattern for glioblastoma, IDH-wild-type and TERT promoter-mutant
Case #2
Background
A 65-year-old man presented with visual changes and was found to have a large right occipital lobe heterogeneously enhancing tumor. The patient underwent tumor excision at an outside institution, and the case was sent to Mayo Clinic for neuropathology consultation.
Histopathological findings
The resection specimen showed a well-demarcated relatively solid glioma with perivascular arrangement consistent with pseudorosettes. Mitotic activity was low (up to 4/10 HPF), and neither necrosis nor microvascular proliferation was observed. Tumor cells were immunoreactive for GFAP, which highlighted radially arranged perivascular processes, showed a “dot-like” cytoplasmic staining with EMA, and absent Olig2 expression.
These findings were consistent with the histopathological diagnosis of ependymoma (WHO grade II).
T1-weighted axial MRI: right occipital enhancing lesion
Genetic testing
Neuro-oncology NGS panel revealed a fusion between C11orf95 and RELA, supporting the integrated diagnosis of RELA fusion-positive ependymoma (WHO grade II).
Chromosomal microarray showed chromothripsis of chromosome 11 (including RELA), which is the mechanism that mediates the C11orf95-RELA fusion event. Additionally, gain of 1q and whole chromosome 7 were present. These findings were also consistent with a RELA fusion-positive ependymoma.
Teaching points
RELA fusion-positive ependymoma is a newly introduced subtype of supratentorial ependymomas (2016 WHO Classification of CNS Tumors), genetically defined by the presence of gene fusions involving RELA.
RELA fusions, of which C11orf95-RELA is the most frequent, are mediated by chromothripsis of chromosome 11 and occur exclusively in supratentorial ependymomas.2,3
RELA fusion-positive ependymomas account for approximately 85% and 60% of pediatric and adult supratentorial ependymomas, respectively, and available data suggest that they are associated with adverse outcomes.3
Chromosome 11 chromothripsis in RELA fusion-positive ependymoma
Integrated genomics view (IGV) of the chimeric C11orf95 (exon 3) and RELA (exon 2) fusion gene
Case #3
Background
A 37-year-old man presented with a recurrent right cerebellar contrast-enhancing tumor. The patient has a history of a right cerebellar anaplastic astrocytoma, resected 7 years ago and treated with radiotherapy. Imaging studies revealed a recurrent right cerebellar contrast-enhancing tumor. The patient underwent surgical re-excision at Mayo Clinic.
Histopathological findings
The resected specimen showed a high-grade neoplasm with an infiltrative growth pattern and areas showing either an astrocytic or embryonal morphology. Slides from the prior surgery were not available for comparison. Immunohistochemical studies excluded the most common IDH1 mutation (p.R132H), while demonstrating retained ATRX protein expression and focal synaptophysin immunoreactivity.
The diagnoses considered included: 1) a recurrent high-grade astrocytoma with a primitive neuronal component, or 2) a second embryonal tumor.
T1-weighted axial MRI: right occipital enhancing lesion
Genetic testing
Neuro-oncology NGS panel revealed a TERT promoter and a PTCH1 mutation. While this result could be seen in the context of a high-grade IDH-wild-type astrocytoma, this mutation profile has been recurrently observed in adult sonic hedgehog (SHH)-activated medulloblastoma. Indeed, clinico-pathological correlation favored this tumor to be a medulloblastoma, SHH-activated, and TP53-wild-type (WHO grade IV).
Chromosomal microarray showed gain of 3q, loss of 7q, and copy-neutral loss of heterozygosity (cnLOH) of 20p. While unusual for a diffuse glioma, this copy-number pattern has been described in SHH-activated medulloblastoma, further supporting this diagnosis.
Teaching points
Medulloblastoma can now be classified in four main subtypes based on molecular features, according to the 2016 WHO Classification: 1) WNT-activated, 2) SHH-activated and TP53-mutant, 3) SHH-activated and TP53-wild-type, and 4) non-WNT/non-SHH.
TERT promoter mutations have been primarily observed in adult SHH- and WNT-activated tumors.4,5
PTCH1 mutations have been described almost exclusively in medulloblastoma, primarily in the medulloblastoma, SHH-activated and TP53-wild-type subgroup, which encompasses the majority of adult medulloblastoma.6
Therapies targeting the canonical SHH pathway, such as small-molecule inhibitors of SHH-component smoothened (SMO) protein (e.g., vismodegib and sonidegib), are available. Preclinical and early clinical studies suggest potential clinical benefit in treating patients with SHH-activated medulloblastoma harboring a PTCH1 mutation using SMO inhibitors.5
Chromosome 11 chromothripsis in RELA fusion-positive ependymoma
Case #4
Background
A 3-month-old girl was found to have a large, heterogeneous tumor involving the left cerebral hemisphere. She underwent a tumor resection at an outside institution, and the case was sent for a neuropathology consultation at Mayo Clinic.
Histopathological findings
The resection specimen showed a highly cellular and mitotically active embryonal tumor. Scattered tumor cells had rhabdoid features, characterized by eccentrically located nuclei with prominent nucleoli and dense eosinophilic globular cytoplasm. Immunohistochemical studies showed that tumor cells had diffuse Olig2 expression with focal GFAP, EMA, and smooth muscle actin staining, along with loss of INI1 nuclear expression. These findings are diagnostic of an atypical teratoid/rhabdoid tumor (AT/RT) (WHO grade IV).
Genetic testing
Neuro-oncology NGS panel revealed a SMARCB1 mutation with high allelic frequency/read counts (approximately 87%) in the context of high tumor content (80%–90%), consistent with biallelic inactivation of SMARCB1 in the tumor and suggesting a somatic (rather than germline) origin for this mutation.
Chromosomal microarray revealed a 22q (including SMARCB1 gene) cnLOH as the sole copy-number abnormality. The 22q cnLOH resulted in duplication of the acquired SMARCB1 mutation and biallelic inactivation of this gene.
Teaching points
AT/RT is a high-grade CNS embryonal tumor predominantly occurring in young children, defined by inactivation of either SMARCB1 (a.k.a., INI1) or, extremely rarely, SMARCA4 (a.k.a., BRG1).
Approximately 35% to 40% of AT/RTs may occur in the context of the rhabdoid tumor predisposition syndrome (RTPS).7
RTPS is an autosomal dominant disorder that arises in the vast majority of cases from a de novo germline SMARCB1/SMARCA4 pathogenic alteration. Therefore, most individuals with RTPS report a negative family history. Additionally, families may show incomplete penetrance and gonadal mosaicism, resulting in an apparent negative family history.
Given the high frequency of RTPS in patients with an AT/RT, genetic testing may identify the SMARCB1/SMARCA4 pathogenic alteration and suggest a somatic (i.e., tumor-specific) or germline (i.e., constitutional) origin. If likely somatic in origin, as seen in this case, follow-up germline testing is likely not necessary.
Copy-neutral loss of heterozygozity of chromosome 22q (including SMARCB1) in an AT/RT
Case #5
Background
A 38-year-old woman was diagnosed with a right frontal glioblastoma at an outside institution. Studies performed at the outside institution showed mutant IDH1 (p.R132H) expression and 1p/19q co-deletion by FISH analysis, a profile more in keeping with oligodendroglioma. Given the discordance between the histopathological diagnosis and the genetic profile, the case was sent to Mayo Clinic for a neuropathology consultation.
Histopathological findings
The infiltrating glioma cells showed astrocytic morphology with high mitotic activity, necrosis, and microvascular proliferation. IDH1 R132H immunostain was positive while ATRX protein expression was lost.
The possibility was considered that this might represent an IDH-mutant glioblastoma and the reported 1p/19q co-deletion result by FISH might represent a false positive.
T1-weighted axial MRI: right occipital enhancing lesion
Genetic testing
Neuro-oncology NGS panel revealed an IDH1 R132H mutation, an ATRX mutation, and a TP53 mutation (with twice the allelic frequency of the IDH1 and ATRX mutations). No TERT promoter mutation was observed.
Chromosomal microarray was near tetraploid and extremely complex. Abnormalities included partial loss of 1p and 19q, multiple subterminal deletions, chromothripis of 5q and 17, and cnLOH of 17p (including TP53). The cnLOH resulted in duplication of the TP53 mutation accounting for the double allelic frequency observed by NGS.
These genetic findings, including the overall complexity of the array, are characteristic of IDH-mutant high-grade infiltrating astrocytomas, and in conjunction with the morphology, led to the final integrated diagnosis of an IDH-mutant glioblastoma (WHO grade IV).
Teaching points
Approximately 5% of TERT-mutant IDH-wild-type glioblastomas and 10% of IDH-mutant astrocytomas will have partial deletions of both 1p and 19q. Chromosomal microarray can differentiate between whole and partial arm deletions, while FISH analysis cannot.
IDH-mutant astrocytomas usually acquire TP53 and ATRX mutations. This combination of mutations generates defects in homologous recombination and, thus, chromosomal instability. This pattern is easily evaluated by chromosomal microarray.
IDH-mutant astrocytomas usually have cnLOH of 17p resulting in duplication of the acquired TP53 mutation, as seen in this case. When such tumors do not have 17p cnLOH, they often acquire two TP53 mutations.
The median survival of IDH-mutant glioblastomas is approximately three years when treated with concurrent temozolomide and radiotherapy.8 The median survival of WHO grade III IDH-mutant astrocytomas and IDH-mutant codeleted oligodendrogliomas is approximately 5.5 years and 14.7 years, respectively, when treated with PCV and radiotherapy.9
A very complex chromosomal microarray pattern
Mayo Clinic Laboratories
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