Matthew J. Schniederjan

Tumor-infiltrating lymphocytes in glioblastoma are associated with specific genomic alterations and related to transcriptional class

Purpose: Tumor-infiltrating lymphocytes (TIL) have prognostic significance in many cancers, yet their roles in glioblastoma have not been fully defined. We hypothesized that TILs in glioblastoma are associated with molecular alterations, histologies, and survival. Experimental Design: We used data from The Cancer Genome Atlas (TCGA) to investigate molecular, histologic, and clinical correlates of TILs in glioblastomas. Lymphocytes were categorized as absent, present, or abundant in histopathologic images from 171 TCGA glioblastomas. Associations were examined between lymphocytes and histologic features, mutations, copy number alterations, CpG island methylator phenotype, transcriptional class, and survival. We validated histologic findings using CD3G gene expression. Results: We found a positive correlation between TILs and glioblastomas with gemistocytes, sarcomatous cells, epithelioid cells, and giant cells. Lymphocytes were enriched in the mesenchymal transcriptional class and strongly associated with mutations in NF1 and RB1. These mutations are frequent in the mesenchymal class and characteristic of gemistocytic, sarcomatous, epithelioid, and giant cell histologies. Conversely, TILs were rare in glioblastomas with small cells and oligodendroglioma components. Lymphocytes were depleted in the classical transcriptional class and in EGF receptor (EGFR)-amplified and homozygous PTEN-deleted glioblastomas. These alterations are characteristic of glioblastomas with small cells and glioblastomas of the classical transcriptional class. No association with survival was shown. Conclusions: TILs were enriched in glioblastomas of the mesenchymal class, strongly associated with mutations in NF1 and RB1 and typical of histologies characterized by these mutations. Conversely, TILs were depleted in the classical class, EGFR-amplified, and homozygous PTEN-deleted tumors and rare in histologies characterized by these alterations. Clin Cancer Res; 19(18); 4951–60. ©2013 AACR.

High rate of concurrent BRAF-KIAA1549 gene fusion and 1p deletion in disseminated oligodendroglioma-like leptomeningeal neoplasms (DOLN)

Disseminated oligodendroglioma-like leptomeningeal neoplasm (DOLN) is a recently described entity that predominantly affects children, is slowly progressive, and exhibits little, if any, parenchymal involvement. Studies to date have demonstrated some similarities between DOLNs and adult oligodendrogliomas with respect to morphology (infiltrative, monotonous cells with round, regular nuclei and perinuclear clearing), immunohistochemistry (synaptophysin, GFAP, Olig-2 expression), and genetics (high rate of chromosome 1p deletions and some 1p19q co-deletions) [5-8]. In contrast however, no DOLNs have been shown to harbor the isocitrate dehydrogenase-1 (IDH1) R132H mutation. BRAF abnormalities are common in pediatric low-grade CNS tumors, with the BRAF-KIAA1549 fusion/tandem duplication at 7q34 identified in approximately, 60 % of cerebellar and optic pathway pilocytic astrocytomas [4]. Others, like pleomorphic xanthoastrocytoma (PXA), ganglioglioma, and dysembryoplastic neuroepithelial tumor (DNET), have shown variable frequencies of activating BRAF V600E point mutations [1]. Because DOLNs partially overlap with these neoplasms, we assessed a series of 20 cases for BRAF alterations. We examined 23 archival DOLNs by FISH for the BRAF-KIAA1549 fusion, and 17 cases for deletions of 1p and 19q (Abbott, North Chicago, IL, USA). One was additionally interrogated by SNP-array. Testing for BRAF V600E was also performed on nine cases. Of 15 informative cases for BRAF-KIAA1549 by FISH, 11 were fusion positive (Fig. 1). Another case was non-informative by FISH, yet harbored a duplication (ch7:138550993-140509923) by SNP-array consistent with gene fusion, for a total of 12 of 16 cases positive for BRAF-KIAA1549 (75 %). FISH revealed loss of 1p in 10/17 cases (59 %), with 3 of those being co-deleted for 19q (18 %). Of the 12 cases with BRAF fusions, 9 had 1p deletion (75 %) and 2 had 19q co-deletion (17 %). None of 9 tested specimens were positive for BRAF-V600E mutation. Fig. 1 DOLN nuclei with FITC-labeled probe RP11-355D18 corresponding to KIAA1549 (green) and digoxigenin-labeled probe 726N20 corresponding to BRAF (red). Yellow signals indicate fusion These findings indicate a high rate of concurrent BRAF-KIAA1549 gene fusions and 1p deletions in DOLNs. Although BRAF fusions are typical of pilocytic astrocytomas, 1p deletions are not, confirming that DOLNs are pathologically and genetically distinct in most cases. These findings also further separate these oligodendroglioma-like tumors from adult oligodendrogliomas, although some overlap remains since 1p19q co-deletion is occasionally found in DOLN and rare BRAF fusions have been recently reported in otherwise classic, 1p19q co-deleted oligoden-droglioma [3]. DOLNs have also displayed occasional ganglion cell differentiation and areas of richly myxoid stroma, raising related possible link to gangliogliomas or DNETs, yet none of our 9 tested cases showed evidence of a BRAF V600E mutation, suggesting that DOLNs are genetically distinct from those entities as well. First-generation RAF inhibitors have been demonstrated in vitro to paradoxically activate the BRAF-KIAA1549 fusion protein [9]. Instead, MEK or mTOR inhibitors could be considered in the treatment of DOLNs, as in other fusion-positive tumors [2]. This report examines the role of common BRAF abnormalities in DOLNs and establishes a high frequency of concurrent BRAF-KIAA1549 fusions and 1p deletions. Although DOLNs are already clinicopathologically distinct, these findings further demonstrate fundamental genetic differences from other entities and implicate a new potential therapeutic target for patients with these otherwise challenging disseminated tumors.

Machine-Based Morphologic Analysis of Glioblastoma Using Whole-Slide Pathology Images Uncovers Clinically Relevant Molecular Correlates

Pathologic review of tumor morphology in histologic sections is the traditional method for cancer classification and grading, yet human review has limitations that can result in low reproducibility and inter-observer agreement. Computerized image analysis can partially overcome these shortcomings due to its capacity to quantitatively and reproducibly measure histologic structures on a large-scale. In this paper, we present an end-to-end image analysis and data integration pipeline for large-scale morphologic analysis of pathology images and demonstrate the ability to correlate phenotypic groups with molecular data and clinical outcomes. We demonstrate our method in the context of glioblastoma (GBM), with specific focus on the degree of the oligodendroglioma component. Over 200 million nuclei in digitized pathology slides from 117 GBMs in the Cancer Genome Atlas were quantitatively analyzed, followed by multiplatform correlation of nuclear features with molecular and clinical data. For each nucleus, a Nuclear Score (NS) was calculated based on the degree of oligodendroglioma appearance, using a regression model trained from the optimal feature set. Using the frequencies of neoplastic nuclei in low and high NS intervals, we were able to cluster patients into three well-separated disease groups that contained low, medium, or high Oligodendroglioma Component (OC). We showed that machine-based classification of GBMs with high oligodendroglioma component uncovered a set of tumors with strong associations with PDGFRA amplification, proneural transcriptional class, and expression of the oligodendrocyte signature genes MBP, HOXD1, PLP1, MOBP and PDGFRA. Quantitative morphologic features within the GBMs that correlated most strongly with oligodendrocyte gene expression were high nuclear circularity and low eccentricity. These findings highlight the potential of high throughput morphologic analysis to complement and inform human-based pathologic review.

Heterozygosity for Pten Promotes Tumorigenesis in a Mouse Model of Medulloblastoma

Background Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma. Methodology/Principal Findings We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/−; Pten +/− medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/−; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression. Conclusions/Significance This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma.

Diffuse leptomeningeal neuroepithelial tumor: 9 pediatric cases with chromosome 1p/19q deletion status and IDH1 (R132H) immunohistochemistry.

Leptomeningeal dissemination in children is typical of high-grade, and occasionally low-grade, neoplasms. Rare cases of widely disseminated oligodendroglia-like leptomeningeal tumors, sometimes with associated spinal cord lesions, have been described that respond to treatment and follow an indolent course. Whether these lesions represent an established tumor category or are a unique entity remains to be established. We present 9 pediatric cases of such diffuse leptomeningeal neuroepithelial tumors (DLNT), 8 with assessment of 2 common genetic alterations seen in oligodendrogliomas, 1p and 19q chromosomal deletions and isocitrate dehydrogenase-1 (IDH1) R132H mutations. Four patients were male and 5 female, with a mean age at presentation of 4 years (range, 2 to 7 y). All presented with signs of increased intracranial pressure and diffuse contrast enhancement of the leptomeninges by magnetic resonance imaging. Three had a cervical or upper thoracic spinal cord tumor, and another had a small cerebellar lesion. Leptomeningeal biopsies showed a thickened and fibrotic arachnoid infiltrated by monotonous cells with round nuclei and prominent perinuclear clearing. All cases were strongly immunoreactive for S100 protein, and most showed faint granular synaptophysin reactivity. Six of 8 cases showed deletions of chromosome arm 1p by fluorescence in situ hybridization, 2 of which also had loss of 19q. None of the lesions reacted with IDH1-R132H antibodies. Although the clinicopathologic features show overlap of these DLNT lesions with oligodendroglioma and extraventricular neurocytoma, they do not exactly match either one, suggesting that DLNTs are a distinct tumor entity.

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.

Silent corticotroph adenomas: Emory University cohort and comparison with ACTH-negative nonfunctioning pituitary adenomas.

BACKGROUND Silent corticotroph adenomas (SCAs) are clinically nonfunctioning pituitary adenomas (NFPAs) with positive staining for corticotropin (ACTH) by immunohistochemistry. Whether SCAs behave more aggressively than NFPAs without ACTH immunoreactivity (ACTH negative) remains controversial. OBJECTIVE To compare characteristics and outcomes of SCAs with ACTH-negative NFPAs and to identify predictors of aggressive outcome. Primary composite endpoint included the first of any of the following events: progression, recurrence, or death. METHODS We reviewed all cases of SCAs and all ACTH-negative macroadenomas operated on between April 1995 and December 2007 by 1 neurosurgeon. RESULTS Our retrospective cohorts included 33 SCAs followed for 42.5 months (median) (range, 6.7-179.0 months) and 126 ACTH-negative patients followed for 42 months (range, 6-142 months). SCA were younger (mean ± SD; 49.6 ± 14.1) than ACTH-negative patients (55.6 ± 12.8, P = .02). Tumor diameter was similar (2.8 ± 1.0 cm); cavernous sinus invasion was present in 45.5% of SCAs and 30.2% of ACTH-negative NFPAs (P = .09). Postoperative tumor residual was detected in 53.1% of SCAs and 49.6% of ACTH-negative patients. Radiation was administered in 40.6% of SCAs at 16 months (range, 3-149 months) and 33.3% of ACTH-negative patients at 13 months (range, 3-94) postoperatively. Progression of residual tumor occurred in 24.2% of SCAs and 11.1% of ACTH-negative patients (P = .08); recurrence was similar (6.0% SCAs vs 5.5% ACTH-negative patients). Cumulative event-free survival rates were not significantly different between the 2 groups (P = .3). Age, sex, tumor size, cavernous sinus invasion, or SCA subtypes were not associated with outcome. CONCLUSION SCA patients were younger, but exhibited similar postoperative tumor regrowth rates as ACTH-negative macroadenomas while using a similar adjuvant radiation protocol. Long-term follow-up is warranted because predictors of regrowth are currently lacking.

Metastasis infiltration: an investigation of the postoperative brain-tumor interface.

PURPOSE This study aims to evaluate brain infiltration of metastatic tumor cells past the main tumor resection margin to assess the biological basis for the use of stereotactic radiosurgery treatment of the tumor resection cavity and visualized resection edge or clinical target volume. METHODS AND MATERIALS Resection margin tissue was obtained after gross total resection of a small group of metastatic lesions from a variety of primary sources. The tissue at the border of the tumor and brain tissue was carefully oriented and processed to evaluate the presence of tumor cells within brain tissue and their distance from the resection margin. RESULTS Microscopic assessment of the radially oriented tissue samples showed no tumor cells infiltrating the surrounding brain tissue. Among the positive findings were reactive astrocytosis observed on the brain tissue immediately adjacent to the tumor resection bed margin. CONCLUSIONS The lack of evidence of metastatic tumor cell infiltration into surrounding brain suggests the need to target only a narrow depth of the resection cavity margin to minimize normal tissue injury and prevent treatment size-dependent stereotactic radiosurgery complications.

The WIP1 oncogene promotes progression and invasion of aggressive medulloblastoma variants.

Recent studies suggest that medulloblastoma, the most common malignant brain tumor of childhood, is comprised of four disease variants. The WIP1 oncogene is overexpressed in Group 3 and 4 tumors, which contain medulloblastomas with the most aggressive clinical behavior. Our data demonstrate increased WIP1 expression in metastatic medulloblastomas, and inferior progression-free and overall survival of patients with WIP1 high-expressing medulloblastoma. Microarray analysis identified upregulation of genes involved in tumor metastasis, including the G protein-coupled receptor CXCR4, in medulloblastoma cells with high WIP1 expression. Stimulation with the CXCR4 ligand SDF1α activated PI-3 kinase signaling, and promoted growth and invasion of WIP1 high-expressing medulloblastoma cells in a p53-dependent manner. When xenografted into the cerebellum of immunodeficient mice, medulloblastoma cells with stable or endogenous high WIP1 expression exhibited strong expression of CXCR4 and activated AKT in primary and invasive tumor cells. WIP1 or CXCR4 knockdown inhibited medulloblastoma growth and invasion. WIP1 knockdown also improved the survival of mice xenografted with WIP1 high-expressing medulloblastoma cells. WIP1 knockdown inhibited cell surface localization of CXCR4 by suppressing expression of the G protein receptor kinase 5, GRK5. Restoration of wild-type GRK5 promoted Ser339 phosphorylation of CXCR4 and inhibited the growth of WIP1-stable medulloblastoma cells. Conversely, GRK5 knockdown inhibited Ser339 phosphorylation of CXCR4, increased cell surface localization of CXCR4 and promoted the growth of medulloblastoma cells with low WIP1 expression. These results demonstrate crosstalk among WIP1, CXCR4 and GRK5, which may be important for the aggressive phenotype of a subclass of medulloblastomas in children.

Successful Retreatment of a Child with a Refractory Brainstem Ganglioglioma with Vemurafenib.

A child with brainstem ganglioglioma underwent subtotal resection and focal radiation. Magnetic resonance imaging confirmed tumor progression 6 months later. Another partial resection revealed viable BRAF V600E‐positive residual tumor. Vemurafenib (660 mg/m2/dose) was administered twice daily, resulting in >70% tumor reduction with sustained clinical improvement for 1 year. Vemurafenib was then terminated, but significant tumor progression occurred 3 months later. Vemurafenib was restarted, resulting in partial response. Toxicities included Grade I pruritus and Grade II rash. Vemurafenib was effectively crushed and administered in solution via nasogastric tube. We demonstrate benefit from restarting vemurafenib therapy.