Faculty Bibliography

Objectives  AlloDerm is an acellular dermal matrix often used for reconstruction throughout the body. AlloDerm has been shown to undergo revascularization when used to reconstruct soft tissue such as in abdominal wall reconstruction. In this study, the authors review the literature on revascularization of AlloDerm and demonstrate the histologic findings of AlloDerm after implantation during skull base reconstruction. Study Design  Literature review and case reports. Setting  Tertiary Care Institution Participants  Patients from a tertiary care institution Main Outcome Measures  Histologic slides are evaluated and compared with nonimplanted AlloDerm. Methods  The authors review a case of explanted AlloDerm that had been used for skull base reconstruction after endoscopic skull base surgery. Results  Upon reviewing the histologic slides of explanted AlloDerm to nonimplanted AlloDerm, we demonstrate revascularization of AlloDerm when used in skull base reconstruction. Representative slides will be included. Conclusions  AlloDerm undergoes revascularization when used for skull base reconstruction.

PURPOSE/OBJECTIVE:Isocitrate dehydrogenase (IDH) mutant gliomas are a distinct glioma molecular subtype for which no effective molecularly-directed therapy exists. Low-grade gliomas, which are 80-90% IDH mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by immunohistochemistry in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH mutant glioma. EXPERIMENTAL DESIGN/METHODS:We evaluated DLL3 expression by RNA using TCGA data and by immunohistochemistry in a discovery set of 63 gliomas and 20 non-tumor brain tissues and a validation set of 62 known IDH wildtype and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH mutant glioma tumorspheres was determined by cell viability assay. RESULTS:Compared to IDH wildtype glioblastoma, IDH mutant gliomas have significantly higher DLL3 RNA (P<1x10-15) and protein by immunohistochemistry (P=0.0014 and P<4.3x10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 non-tumor brains. Patient-derived IDH mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. CONCLUSIONS:DLL3 is selectively and homogeneously expressed in IDH mutant gliomas and can be targeted with Rova-T in patient-derived IDH mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

BACKGROUND: Tumor heterogeneity presents a major challenge to cancer diagnosis and treatment. In addition to interpatient tumor variability, intratumoral heterogeneity characterized by distinct molecular and phenotypic profiles is increasingly recognized as a major cause of therapy resistance and cancer recurrence. Because DNA methylation patterns are largely responsible for determining cell-type-specific functioning, we hypothesized that distinct DNA methylation and proteomic alterations could be identified in histologically-defined invasive and proliferative tumor cell populations in human isocitrate dehydrogenase 1 (IDH1)- mutated and wild-type glioblastoma (GBM).
METHOD(S): Formalin-fixed paraffin-embedded tissue sections of human adult IDH1-mutated and wild-type GBM were laser-microdissected (LM) into perinecrotic pseudopalisading tumor cells (PPCs), non-pseudopalisading tumor core cells (NPPCs), invasive subpial spread (SPS) and perivascular satellitosis tumor cells and brain adjacent to tumor cells prior to analysis and compared to non-microdissected tumor (NMT) and/or germline DNA. Genomewide DNA methylation and chromosomal copy numbers were determined with Infinium MethylationEPIC 850K BeadChip and intratumoral DNA methylation patterns compared by unsupervised hierarchical clustering. Label-free quantitative liquid chromatography-mass spectrometry of proteins was performed and proteins differentially expressed across LM areas subjected to pathway enrichment analysis.
RESULT(S): Unsupervised hierarchical classification of DNA methylation patterns for each LM area and NMT demonstrated remarkable clustering for all patients, based on methylation probe and methylated gene patterns. Proteomics analysis showed upregulation of hypoxia-inducible factor-1 inducible proteins in hypoxic PPCs. Out of 1819 proteins quantified, 5 were overexpressed and 9 underexpressed more than 10-fold in SPS compared with NPPCs and associated with alterations in metabolism, transport, extracellular matrix and apoptosis. Correlation of protein expression and DNA methylation patterns was noted.
CONCLUSION(S): Compared to NPPCs, SPS cells migrating toward the invasive edge share a relatively consistent epigenetic and proteomic signature, suggesting potentially targetable common mechanism(s) of invasion shared among GBM

Epigenetic patterns on the level of DNA methylation have already shown to separate clinically relevant subgroups of meningiomas. Based on a reference set (Sahm et al., Lancet Oncol 2017), an epigenetic meningioma classifier employing DNA methylation patterns is made available through molecularneurpathology.org. We now set out to identify prognostic implications of epigenetic modification on the proteome level, particularly modifications of histones. First focus was on H3K27 trimethylation (H3K27me3). H3K27me3 was assessed by immunohistochemistry on 232 meningiomas. In 194 cases, trimethylation was detected in tumor cells. In 25 cases, staining was limited to vessels while all tumor cells were negative. Finally, 13 cases yielded equivocal staining patterns. Reduced abundance of H3K27me3 in cases with staining limited to vessels was confirmed by mass spectrometry on a subset of cases. Lack of staining for H3K27me3 in all tumor cells was significantly associated with more rapid progression (p=0.009). In line, H3K27me3 negative cases were associated with a DNA methylation pattern of the more aggressive types among the recently introduced DNA methylation groups. Also, NF2 and SUFU mutations were enriched among cases with lack of H3K27me3 in tumor cells (p<0.0001 and p=0.029, respectively). H3K27me3 staining pattern added significant prognostic insight in WHO grade II cases and in the compound subset of WHO grade I and II cases (p=0.04 and p=0.007, respectively). However, it did not further stratify within WHO grade III cases. Collectively, this data indicate that epigenetic modifications beyond DNA methylation are involved in the aggressiveness of meningioma. It also suggests that H3K27me3 immunohistochemistry might be a useful adjunct in meningioma diagnostics, particularly for cases with WHO grade II histology or at the borderline between WHO grade I and II. Ongoing studies evaluate the role of histone marks other than H3K27me3 and consequences on the proteomic composition of meningioma cells by high-throughput mass spectrometry

Isocitrate dehydrogenase (IDH) mutation have been reported to impose in gliomas a shortage of NADPH required to maintain a redox state and may rely on cysteine (Cys) availability for biosynthesis of glutathione (GSH) to ensure antioxidant levels. Cys may be replenished via extracellular intake or by de novo intracellular synthesis via transsulfuration (TS) pathway. The aim of this study was to investigate alterations of Cys metabolism in genetic variants of high-grade gliomas (HGG). Seventeen tumor samples from 15 adult patients (11 M / 4 F; average age 57 years, range 25 - 81 years), who underwent surgical resection for newly diagnosed or recurrent HGG were analyzed by HPLC. Levels of Cys, homocysteine and GSH were correlated with the genetic signature of HGG (wild-types vs. IDH1 mutation, PTEN deletion, EGFR amplification and MGMT methylation). Cys levels were significantly higher (2.1 fold increase; p=0.0038) in IDH1-mut (n=4) vs. IDH1-wt HGG (n=13), with comparable homocysteine and GSH levels. PTEN deletion and EGFR amplification did not significantly alter Cys metabolites with comparable levels of Cys, homocysteine and GSH detected in PTEN-del (n=7) and PTEN-intact (n=6) HGG, as well as in EGFR-amp (n=7) and EGFR-non amp (n=9) HGG. Significantly higher Cys levels (3.2 fold increase; p=0.0186) were also found in MGMT methylated (n=4) vs. non-methylated (n=3) HGG, with comparable levels of homocysteine and GSH. Increased Cys levels detected in IDH1-mut and MGMT methylated HGG support the hypothesis that these tumors may preferentially use the TS pathway for GSH synthesis. These findings are consistent with our report of increased TS pathway enzyme cystathionine B-synthase (CBS) in HGG, but concurrent increased intake of Cys cannot be excluded. Our results suggest utilizing Cys metabolites as potential markers and/or therapeutic targets in some genetic variants of HGG, a hypothesis that should be further explored in larger translational trials

Background/UNASSIGNED:While effective for the repair of large skull base defects, the Hadad-Bassagasteguy nasoseptal flap increases operative time and can result in a several-week period of postoperative crusting during re-mucosalization of the denuded nasal septum. Endoscopic transsphenoidal surgery for pituitary adenoma resection is generally not associated with large dural defects and high-flow cerebrospinal fluid (CSF) leaks requiring extensive reconstruction. Here, we present the posterior nasoseptal flap as a novel technique for closure of skull defects following endoscopic resection of pituitary adenomas. This flap is raised in all surgeries during the transnasal exposure using septal mucoperiosteum that would otherwise be discarded during the posterior septectomy performed in binostril approaches. Methods/UNASSIGNED:We present a retrospective, consecutive case series of 43 patients undergoing endoscopic transsphenoidal resection of a pituitary adenoma followed by posterior nasoseptal flap placement and closure. Main outcome measures were extent of resection and postoperative CSF leak. Results/UNASSIGNED:The mean extent of resection was 97.16 ± 1.03%. Radiographic measurement showed flap length to be adequate. While a defect in the diaphragma sellae and CSF leak were identified in 21 patients during surgery, postoperative CSF leak occurred in only one patient. Conclusions/UNASSIGNED:The posterior nasoseptal flap provides adequate coverage of the surgical defect and is nearly always successful in preventing postoperative CSF leak following endoscopic transsphenoidal resection of pituitary adenomas. The flap is raised from mucoperiosteum lining the posterior nasal septum, which is otherwise resected during posterior septectomy. Because the anterior septal cartilage is not denuded, raising such flaps avoids the postoperative morbidity associated with the larger Hadad-Bassagasteguy nasoseptal flap.

BACKGROUND AND PURPOSE/OBJECTIVE:Few studies have shown MR imaging features and ADC correlating with molecular markers and survival in patients with glioma. Our purpose was to correlate MR imaging features and ADC with molecular subtyping and survival in adult diffuse gliomas. MATERIALS AND METHODS/METHODS:promoter methylation, and overall survival. RESULTS:wild-type glioma. Other MR imaging features were not statistically significant predictors of survival. CONCLUSIONS:wild-type gliomas.

BACKGROUND: Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. METHODS: We analyzed pediatric and adult pineoblastoma samples (n=23) using integrated genomic studies, including genome-wide DNA methylation profiling, whole-exome or whole-genome sequencing, and whole-transcriptome analysis. RESULTS: Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower grade pineal tumors and normal pineal gland. Recurrent somatic mutations were found in genes involved in PKA-and NF-kappaB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expression of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain. Whole-transcriptome analysis showed that homozygous loss of DROSHA led to distinct changes in RNA expression profile. Disruption of the DROSHA locus in human neural stem cells using the CRISPR/Cas9 system, led to decrease of the DROSHA protein, and massive loss of miRNAs. CONCLUSION: We identified recurrent homozygous deletions of DROSHA in pineoblastoma, suggesting that different mechanisms disrupting miRNA processing are involved in the pathogenesis of familial versus sporadic pineoblastoma. Furthermore, a novel microduplication of PDE4DIP leading to upregulation of DUF1220 protein suggests DUF1220 as a novel oncogenic driver in pineoblastoma

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain.