Whole tumor analysis reveals early origin of the TERT promoter mutation and intercellular heterogeneity in TERT expression.

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.

Is Next-Generation Sequencing Alone Sufficient to Reliably Diagnose Gliomas?

The power and widespread use of next-generation sequencing (NGS) in surgical neuropathology has raised questions as to whether NGS might someday fully supplant histologic-based examination. We therefore sought to determine the feasibility of relying on NGS alone for diagnosing infiltrating gliomas. A total of 171 brain lesions in adults, all of which had been analyzed by GlioSeq NGS, comprised the study cohort. Each case was separately diagnosed by 6 reviewers, based solely on age, sex, tumor location, and NGS results. Results were compared with the final integrated diagnoses and scored on the following scale: 0 = either wrong tumor type or correct tumor type but off by 2+ grades; 1 = off by 1 grade; 2 = exactly correct. Histology alone was treated as a seventh reviewer. Overall reviewer accuracy ranged from 81.6% to 94.2%, while histology alone scored 87.1%. For glioblastomas, NGS was more accurate than histology alone (93.8%-97.9% vs 87.5%). The NGS accuracy for grade II and III astrocytoma and oligodendroglioma was only 54.3%-84.8% and 34.4%-87.5%, respectively. Most uncommon gliomas, including BRAF-driven tumors, could not be accurately classified just by NGS. These data indicate that, even in this era of advanced molecular diagnostics, histologic evaluation is still an essential part of optimal patient care.

Pleomorphic xanthoastrocytoma: a brief review.

Pleomorphic xanthoastrocytoma (PXA) is a rare primary CNS tumor. Recent advances in the molecular characterization are helping to define subtypes of tumor. The discovery of BRAF mutations within a substantial percentage of PXA fosters a clearer understanding of the pathophysiology of these tumors with clear prognostic and therapeutic implications. These findings are expected to provide insight into the spectrum of clinical behavior observed in PXA, ranging from cure with surgery to diffuse dissemination throughout the neuraxis. This review details the clinical presentation including radiographic appearance of PXA. Pathology, including molecular pathology is discussed. Therapeutic management including surgical resection, radiotherapy and systemic therapies are reviewed.

Myeloid-Derived Suppressive Cells Promote B cell-Mediated Immunosuppression via Transfer of PD-L1 in Glioblastoma.

The potent immunosuppression induced by glioblastoma (GBM) is one of the primary obstacles to finding effective immunotherapies. One hallmark of the GBM-associated immunosuppressive landscape is the massive infiltration of myeloid-derived suppressor cells (MDSC) and, to a lesser extent, regulatory T cells (Treg) within the tumor microenvironment. Here, we showed that regulatory B cells (Breg) are a prominent feature of the GBM microenvironment in both preclinical models and clinical samples. Forty percent of GBM patients (n = 60) scored positive for B-cell tumor infiltration. Human and mouse GBM-associated Bregs were characterized by immunosuppressive activity toward activated CD8+ T cells, the overexpression of inhibitory molecules PD-L1 and CD155, and production of immunosuppressive cytokines TGFß and IL10. Local delivery of B cell-depleting anti-CD20 immunotherapy improved overall survival of animals (IgG vs. anti-CD20 mean survival: 18.5 vs. 33 days, P = 0.0001), suggesting a potential role of Bregs in GBM progression. We unveiled that GBM-associated MDSCs promoted regulatory B-cell function by delivering microvesicles transporting membrane-bound PD-L1, able to be up-taken by tumoral B cells. The transfer of functional PD-L1 via microvesicles conferred Bregs the potential to suppress CD8+ T-cell activation and acquisition of an effector phenotype. This work uncovered the role of B cells in GBM physiopathology and provides a mechanism by which the GBM microenvironment controls B cell-mediated immunosuppression.See related Spotlight on p. 1902.

Drak/STK17A Drives Neoplastic Glial Proliferation through Modulation of MRLC Signaling.

Glioblastoma (GBM) and lower grade gliomas (LGG) are the most common primary malignant brain tumors and are resistant to current therapies. Genomic analyses reveal that signature genetic lesions in GBM and LGG include copy gain and amplification of chromosome 7, amplification, mutation, and overexpression of receptor tyrosine kinases (RTK) such as EGFR, and activating mutations in components of the PI3K pathway. In Drosophila melanogaster, constitutive co-activation of RTK and PI3K signaling in glial progenitor cells recapitulates key features of human gliomas. Here we use this Drosophila glioma model to identify death-associated protein kinase (Drak), a cytoplasmic serine/threonine kinase orthologous to the human kinase STK17A, as a downstream effector of EGFR and PI3K signaling pathways. Drak was necessary for glial neoplasia, but not for normal glial proliferation and development, and Drak cooperated with EGFR to promote glial cell transformation. Drak phosphorylated Sqh, the Drosophila ortholog of nonmuscle myosin regulatory light chain (MRLC), which was necessary for transformation. Moreover, Anillin, which is a binding partner of phosphorylated Sqh, was upregulated in a Drak-dependent manner in mitotic cells and colocalized with phosphorylated Sqh in neoplastic cells undergoing mitosis and cytokinesis, consistent with their known roles in nonmuscle myosin-dependent cytokinesis. These functional relationships were conserved in human GBM. Our results indicate that Drak/STK17A, its substrate Sqh/MRLC, and the effector Anillin/ANLN regulate mitosis and cytokinesis in gliomas. This pathway may provide a new therapeutic target for gliomas.Significance: These findings reveal new insights into differential regulation of cell proliferation in malignant brain tumors, which will have a broader impact on research regarding mechanisms of oncogene cooperation and dependencies in cancer.See related commentary by Lathia, p. 1036.

Farewell to GBM-O: Genomic and transcriptomic profiling of glioblastoma with oligodendroglioma component reveals distinct molecular subgroups.

INTRODUCTION: Glioblastoma with oligodendroglioma component (GBM-O) was recognized as a histologic pattern of glioblastoma (GBM) by the World Health Organization (WHO) in 2007 and is distinguished by the presence of oligodendroglioma-like differentiation. To better understand the genetic underpinnings of this morphologic entity, we performed a genome-wide, integrated copy number, mutational and transcriptomic analysis of eight (seven primary, primary secondary) cases. RESULTS: Three GBM-O samples had IDH1 (p.R132H) mutations; two of these also demonstrated 1p/19q co-deletion and had a proneural transcriptional profile, a molecular signature characteristic of oligodendroglioma. The additional IDH1 mutant tumor lacked 1p/19q co-deletion, harbored a TP53 mutation, and overall, demonstrated features most consistent with IDH mutant (secondary) GBM. Finally, five tumors were IDH wild-type (IDHwt) and had chromosome seven gains, chromosome 10 losses, and homozygous 9p deletions (CDKN2A), alterations typical of IDHwt (primary) GBM. IDHwt GBM-Os also demonstrated EGFR and PDGFRA amplifications, which correlated with classical and proneural expression subtypes, respectively. CONCLUSIONS: Our findings demonstrate that GBM-O is composed of three discrete molecular subgroups with characteristic mutations, copy number alterations and gene expression patterns. Despite displaying areas that morphologically resemble oligodendroglioma, the current results indicate that morphologically defined GBM-O does not correspond to a particular genetic signature, but rather represents a collection of genetically dissimilar entities. Ancillary testing, especially for IDH and 1p/19q, should be used for determining these molecular subtypes.

Comprehensive evaluation of thyrotropinomas: single-center 20-year experience.

PURPOSE: To present a single-center 20-year experience with operated thyrotropinomas, including prevalence, clinical, biochemical and histological characteristics, and postoperative outcomes. METHODS: Retrospective series of histopathologically-proven thyrotropinomas (1993-2013), divided in two groups: A (active, central hyperthyroidism) and B (silent, no hyperthyroidism). RESULTS: Of 1628 operated pituitary adenomas, 20 were ß-TSH-positive (1.2%). In increments of 5 years, proportion of thyrotropinomas was 1, 1, 0.04 and 1.77% respectively. Median follow-up was 10.4 months (1.2-150). Group A: 6 patients (5 men), age 41 ± 12 years presented with hyperthyroidism (3), pituitary incidentaloma (2) and acromegaly (1). Tumor diameter was 2.1 ± 1.2 cm, FT4 2.68 ± 2.73 ng/dL; TSH 6.50 ± 3.68 µIU/mL. Glycoprotein alpha subunit (GSU) was uniformly elevated. Two patients had biochemical evidence of acromegaly. Tumors were plurihormonal (5 GH-positive); none atypical. Postoperative euthyroidism was achieved in 4 of 6 patients (66%). Group B: 14 patients (7 men), age 47 ± 14 years presented with acromegaly (6), mass effect (4), incidentaloma (3) and galactorrhea (1). Tumor diameter was 2.0 ± 1.0 cm. Free T4 (1.00 ± 0.24 ng/dL) and TSH (2.02 ± 1.65 mIU/L) were lower than in group A (p < 0.01). GSU was elevated in all tested cases. Nine patients had biochemical evidence of acromegaly. Tumors were plurihormonal (12 GH-positive); none atypical. Gross total resection was achieved in 12 of 14 (86%), and 1 (7%) recurred. CONCLUSION: In our series, more thyrotropinomas were operated in recent years. These tumors were often plurihormonal with heterogenous clinical presentation and frequent GH co-secretion. Surgical outcomes were good but long-term follow up is necessary.

Biomarker-driven diagnosis of diffuse gliomas.

The diffuse gliomas are primary central nervous system tumors that arise most frequently in the cerebral hemispheres of adults. They are currently classified as astrocytomas, oligodendrogliomas or oligoastrocytomas and range in grade from II to IV. Glioblastoma (GBM), grade IV, is the highest grade and most common form. The diagnosis of diffuse gliomas has historically been based primarily on histopathologic features, yet these tumors have a wide range of biological behaviors that are only partially explained by morphology. Biomarkers have now become an established component of the neuropathologic diagnosis of gliomas, since molecular alterations aid in classification, prognostication and prediction of therapeutic response. Isocitrate dehydrogenase (IDH) mutations are frequent in grades II and III infiltrating gliomas of adults, as well as secondary GBMs, and are a major discriminate of biologic class. IDH mutant infiltrating astrocytomas (grades II and III), as well as secondary GBMs, are characterized by TP53 and ATRX mutations. Oligodendrogliomas are also IDH mutant, but instead are characterized by 1p/19q co-deletion and mutations of CIC, FUBP1, Notch1 and the TERT promoter. Primary GBMs typically lack IDH mutations and demonstrate EGFR, PTEN, TP53, PDGFRA, NF1 and CDKN2A/B alterations and TERT promoter mutations. Pediatric gliomas differ in their spectrum of disease from those in adults; high grade gliomas occurring in children frequently have mutations in H3F3A, ATRX and DAXX, but not IDH. Circumscribed, low grade gliomas, such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma and ganglioglioma, need to be distinguished from diffuse gliomas in the pediatric population. These gliomas often harbor mutations or activating gene rearrangements in BRAF.

Molecular pathways in gliomagenesis and their relevance to neuropathologic diagnosis.

Gliomas are a large and diverse group of primary brain tumors that include those that are diffusely infiltrative and others that are well-circumscribed and low grade. Diffuse gliomas are currently classified by the World Health Organization as astrocytomas, oligodendrogliomas, or oligoastrocytomas and range in grade from II to IV. Glioblastoma (GBM), World Health Organization grade IV, is the highest grade and most common form of astrocytoma. In the past, the diagnosis of gliomas was almost exclusively based on histopathologic features. More recently, improved understanding of molecular genetic underpinnings has led to ancillary molecular studies becoming standard for classification, prognostication, and predicting therapy response. Isocitrate dehydrogenase (IDH) mutations are frequent in grade II and III infiltrating gliomas and secondary GBMs. Infiltrating astrocytomas and secondary GBMs are characterized by IDH, TP53, and ATRX mutations, whereas oligodendrogliomas demonstrate 1p/19q codeletion and mutations in IDH, CIC, FUBP1, and the telomerase reverse transcriptase (TERT) promoter. Primary GBMs typically lack IDH mutations and are instead characterized by EGFR, PTEN, TP53, PDGFRA, NF1, and CDKN2A/B alterations and TERT promoter mutations. Pediatric GBMs differ from those in adults and frequently have mutations in H3F3A, ATRX, and DAXX, but not IDH. In contrast, circumscribed, low-grade gliomas of childhood, such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma, often harbor mutations or activating gene rearrangements in BRAF. Neuropathologic assessment of gliomas increasingly relies on ancillary testing of molecular alterations for proper classification and patient management.

Outcomes and patterns of failure for grade 2 meningioma treated with reduced-margin intensity modulated radiation therapy.

PURPOSE: The purpose of this study was to evaluate intracranial control and patterns of local recurrence (LR) for grade 2 meningiomas treated with intensity modulated radiation therapy (IMRT) with limited total margin expansions of ≤1 cm. METHODS AND MATERIALS: We reviewed records of patients with a neuropathological diagnosis of grade 2 meningioma who underwent IMRT at our institution between 2002 and 2012. Actuarial rates were determined by the Kaplan-Meier method from the end of RT. LR was defined as in-field if ≥90% of the recurrence was within the prescription isodose, out-of-field (marginal) if ≥90% was outside of the prescription isodose, and both if neither criterion was met. RESULTS: Between 2002 and 2012, a total of 54 consecutive patients underwent IMRT for grade 2 meningioma. Eight of these patients had total initial margins >1 cm and were excluded, leaving 46 patients for analysis. The median imaging follow-up period was 26.2 months (range, 7-107 months). The median dose for fractionated IMRT was 59.4 Gy (range, 49.2-61.2 Gy). Median clinical target volume (CTV), planning target volume (PTV), and total margin expansion were 0.5 cm, 0.3 cm, and 0.8 cm, respectively. LR occurred in 8 patients (17%), with 2-year and 3-year actuarial local control (LC) of 92% and 74%, respectively. Six of 8 patients (85%) had a known pattern of failure. Five patients (83%) had in-field LR; no patients had marginal LR; and 1 patient (17%) had both. CONCLUSIONS: The use of IMRT to treat grade 2 meningiomas with total initial margins (CTV + PTV) ≤1 cm did not appear to compromise outcomes or increase marginal failures compared with other modern retrospective series. Of the 46 patients who had margins ≤1 cm, none experienced marginal failure only. These results demonstrate efficacy and low risk of marginal failure after IMRT treatment of grade 2 meningiomas with reduced margins, warranting study within a prospective clinical trial.

Molecular genetics of gliomas.

Diffusely infiltrating gliomas are the most common primary brain tumors and include astrocytomas, oligodendrogliomas, and oligoastrocytomas of grades II and III and glioblastoma (GBM), grade IV. Histologic classification is increasingly aided by molecular genetic studies, which assist in the diagnosis and provide prognostic and predictive value. Mutations in IDH1 are frequent in grades II and III astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as secondary GBMs. IDH1-mutated diffuse gliomas are distinct from their IDH1 wild-type counterparts based on clinical features, growth rates, and concurrent genomic alterations. Grades II and III astrocytomas, as well as secondary GBMs are characterized by IDH1, TP53, and ATRX mutations, whereas oligodendrogliomas most frequently harbor codeletion of 1p/19q and mutations in CIC, FUBP1, and the TERT promoter. Primary GBMs frequently show molecular alterations in EGFR, PDGFRA, PTEN, TP53, NF1, and CDKN2A/B, as well as TERT promoter mutations, but not IDH mutations. Pediatric GBMs have a distinctive molecular pathogenesis, as H3F3A and DAXX mutations are frequent, and their gene expression profile is different than adult GBMs. Other lower-grade gliomas of childhood, such as pilocytic astrocytoma and pleomorphic xanthoastrocytoma, are characterized by BRAF mutations or activating gene rearrangements involving BRAF.

PDGFRA amplification is common in pediatric and adult high-grade astrocytomas and identifies a poor prognostic group in IDH1 mutant glioblastoma.

High-grade astrocytomas (HGAs), corresponding to World Health Organization grades III (anaplastic astrocytoma) and IV (glioblastoma; GBM), are biologically aggressive, and their molecular classification is increasingly relevant to clinical management. PDGFRA amplification is common in HGAs, although its prognostic significance remains unclear. Using fluorescence in situ hybridization (FISH), the most sensitive technique for detecting PDGFRA copy number gains, we determined PDGFRA amplification status in 123 pediatric and 263 adult HGAs. A range of PDGFRA FISH patterns were identified and cases were scored as non-amplified (normal and polysomy) or amplified (low-level and high-level). PDGFRA amplification was frequent in pediatric (29.3%) and adult (20.9%) tumors. Amplification was not prognostic in pediatric HGAs. In adult tumors diagnosed initially as GBM, the presence of combined PDGFRA amplification and isocitrate dehydrogenase 1 (IDH1)(R132H) mutation was a significant independent prognostic factor (P = 0.01). In HGAs, PDGFRA amplification is common and can manifest as high-level and focal or low-level amplifications. Our data indicate that the latter is more prevalent than previously reported with copy number averaging techniques. To our knowledge, this is the largest survey of PDGFRA status in adult and pediatric HGAs and suggests PDGFRA amplification increases with grade and is associated with a less favorable prognosis in IDH1 mutant de novo GBMs.

Glioblastoma with oligodendroglioma component (GBM-O): molecular genetic and clinical characteristics.

Glioblastoma (GBM) is an aggressive primary brain tumor with an average survival of approximately 1 year. A recently recognized subtype, glioblastoma with oligodendroglioma component (GBM-O), was designated by the World Health Organization (WHO) in 2007. We investigated GBM-Os for their clinical and molecular characteristics as compared to other forms of GBM. Tissue samples were used to determine EGFR, PTEN, and 1p and 19q status by fluorescence in situ hybridization (FISH); p53 and mutant IDH1 protein expression by immunohistochemistry (IHC); and MGMT promoter status by methylation-specific polymerase chain reaction (PCR). GBM-Os accounted for 11.9% of all GBMs. GBM-Os arose in younger patients compared to other forms of GBMs (50.7 years vs. 58.7 years, respectively), were more frequently secondary neoplasms, had a higher frequency of IDH1 mutations and had a lower frequency of PTEN deletions. Survival was longer in patients with GBM-Os compared to those with other GBMs, with median survivals of 16.2 and 8.1 months, respectively. Most of the survival advantage for GBM-O appeared to be associated with a younger age at presentation. Among patients with GBM-O, younger age at presentation and 1p deletion were most significant in conferring prolonged survival. Thus, GBM-O represents a subset of GBMs with distinctive morphologic, clinical and molecular characteristics.

Transformation of a Silent Adrencorticotrophic Pituitary Tumor Into Central Nervous System Melanoma.

Silent adrenocorticotrophic pituitary adenomas are nonfunctioning pituitary adenomas that express adrenocorticotrophic hormone (ACTH) but do not cause the clinical or laboratory features of hypercortisolemia. Primary central nervous system (CNS) melanoma is well documented, but rarely originates in the sellar region or pituitary gland. Here we report transformation of an aggressive silent adrenocorticotrophic pituitary adenoma that transformed into CNS melanoma and review other presentations of pituitary melanoma. A 37-year-old woman initially presented with apoplexy and an invasive nonfunctioning pituitary macroadenoma for which she underwent transphenoidal surgery. The patient underwent 3 subsequent surgeries as the tumor continued to progress. Pathology from the first 3 operations showed pituitary adenoma or carcinoma. Pathology from the final surgery showed melanoma and the magnetic resonance imaging characteristics of the tumor had changed to become consistent with CNS melanoma. Dermatologic and ophthalmologic examinations did not identify cutaneous or ocular melanoma. The patient's disease progressed despite aggressive surgical, medical and radiologic treatment. To our knowledge, this is the first report demonstrating transformation of a primary pituitary tumor into melanoma. The mechanism of tumor transformation is unclear, but it is possible that a mutation in the original ACTH-producing tumor lead to increased cleavage of pro-opiomelanocortin or ACTH into α-melanocyte-stimulating hormone, which in turn stimulated the expression of microopthalmia transcription factor, leading to melanocytic phenotype transformation.