Genome-wide loss of heterozygosity predicts aggressive, treatment-refractory behavior in pituitary neuroendocrine tumors.

Pituitary neuroendocrine tumors (PitNETs) exhibiting aggressive, treatment-refractory behavior are the rare subset that progress after surgery, conventional medical therapies, and an initial course of radiation and are characterized by unrelenting growth and/or metastatic dissemination. Two groups of patients with PitNETs were sequenced: a prospective group of patients (n = 66) who consented to sequencing prior to surgery and a retrospective group (n = 26) comprised of aggressive/higher risk PitNETs. A higher mutational burden and fraction of loss of heterozygosity (LOH) was found in the aggressive, treatment-refractory PitNETs compared to the benign tumors (p = 1.3 × 10-10 and p = 8.5 × 10-9, respectively). Within the corticotroph lineage, a characteristic pattern of recurrent chromosomal LOH in 12 specific chromosomes was associated with treatment-refractoriness (occurring in 11 of 14 treatment-refractory versus 1 of 14 benign corticotroph PitNETs, p = 1.7 × 10-4). Across the cohort, a higher fraction of LOH was identified in tumors with TP53 mutations (p = 3.3 × 10-8). A machine learning approach identified loss of heterozygosity as the most predictive variable for aggressive, treatment-refractory behavior, outperforming the most common gene-level alteration, TP53, with an accuracy of 0.88 (95% CI: 0.70-0.96). Aggressive, treatment-refractory PitNETs are characterized by significant aneuploidy due to widespread chromosomal LOH, most prominently in the corticotroph tumors. This LOH predicts treatment-refractoriness with high accuracy and represents a novel biomarker for this poorly defined PitNET category.

Virtual multi-institutional tumor board: a strategy for personalized diagnoses and management of rare CNS tumors.

PURPOSE: Multidisciplinary tumor boards (MTBs) integrate clinical, molecular, and radiological information and facilitate coordination of neuro-oncology care. During the COVID-19 pandemic, our MTB transitioned to a virtual and multi-institutional format. We hypothesized that this expansion would allow expert review of challenging neuro-oncology cases and contribute to the care of patients with limited access to specialized centers. METHODS: We retrospectively reviewed records from virtual MTBs held between 04/2020-03/2021. Data collected included measures of potential clinical impact, including referrals to observational or therapeutic studies, referrals for specialized neuropathology analysis, and whether molecular findings led to a change in diagnosis and/or guided management suggestions. RESULTS: During 25 meetings, 32 presenters discussed 44 cases. Approximately half (n = 20; 48%) involved a rare central nervous system (CNS) tumor. In 21% (n = 9) the diagnosis was changed or refined based on molecular profiling obtained at the NIH and in 36% (n = 15) molecular findings guided management. Clinical trial suggestions were offered to 31% (n = 13), enrollment in the observational NCI Natural History Study to 21% (n = 9), neuropathology review and molecular testing at the NIH to 17% (n = 7), and all received management suggestions. CONCLUSION: Virtual multi-institutional MTBs enable remote expert review of CNS tumors. We propose them as a strategy to facilitate expert opinions from specialized centers, especially for rare CNS tumors, helping mitigate geographic barriers to patient care and serving as a pre-screening tool for studies. Advanced molecular testing is key to obtaining a precise diagnosis, discovering potentially actionable targets, and guiding management.

Capturing evolving definitions of 12 select rare CNS tumors: a timely report from CBTRUS and NCI-CONNECT.

PURPOSE: Incidence, prevalence, and survival are population-based statistics describing cancer burden. The National Cancer Institute's (NCI) Comprehensive Oncology Network Evaluating Rare CNS Tumors (NCI-CONNECT) specializes in tumor biology and outcomes for 12 rare CNS tumor types selected for their importance in adults, research interest, or potential for targeted treatment. The aim of this study was to update incidence, prevalence, and survival statistics for these tumors. METHODS: The Central Brain Tumor Registry of the United States (CBTRUS) database, a combined dataset of Centers for Disease Control and Prevention's (CDC) National Program of Cancer Registries (NPCR) and NCI's Surveillance, Epidemiology and End Results (SEER) data, was used to calculate average annual age-adjusted incidence rates (AAAIR) per 100,000 population overall and by sex, race-ethnicity, and age for diagnosis years 2008-2019. Incidence time trends were calculated for diagnosis years 2004-2019. NPCR data were used to calculate relative survival rates. Point prevalence on December 31, 2019 was estimated using annual age-specific incidence and survival. RESULTS: AAAIR was 1.47 per 100,000 for these tumors combined, with highest incidence in ependymomas (AAAIR = 0.41/100,000). Most tumor types were more common in males, adults (ages 40 + years) or children (ages < 15 years), and non-Hispanic White individuals. Ependymomas were the most prevalent tumor type (19,320 cases) followed by oligodendrogliomas (14,900 cases). Ependymomas had the highest five-year survival (90.6%) and primary CNS sarcomas the lowest (7.7%). CONCLUSIONS: These data provide means to measure the impact of clinical care and evaluate new therapies and the evolving histopathology definitions in rare CNS tumor types.

High-grade glioma therapy: adding flexibility in trial design to improve patient outcomes.

INTRODUCTION: Outcomes for patients with high grade gliomas have changed little over the past thirty years. This realization prompted renewed efforts to increase flexibility in the design and conduct of clinical brain tumor trials. AREAS COVERED: This manuscript reviews the development of clinical trial methods, challenges and considerations of flexible clinical trial designs, approaches to improve identification and testing of active agents for high grade gliomas, and evaluation of their delivery to the central nervous system. EXPERT OPINION: Flexibility can be introduced in clinical trials in several ways. Flexible designs tout smaller sample sizes, adaptive modifications, fewer control arms, and inclusion of multiple arms in one study. Unfortunately, modifications in study designs cannot address two challenges that are largely responsible for the lack of progress in treating high grade gliomas: 1) the identification of active pharmaceutical agents and 2) the delivery of these agents to brain tumor tissue in therapeutic concentrations. To improve the outcomes of patients with high grade gliomas efforts must be focused on the pre-clinical screening of drugs for activity, the ability of these agents to achieve therapeutic concentrations in non-enhancing tumors, and a willingness to introduce novel compounds in minimally pre-treated patient populations.

Correlation of commercially available quantitative MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation scores and GBM patient survival.

BACKGROUND: Between 2011 and 2016, O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation testing at University of California Los Angeles (UCLA) was performed through LabCorp, using a threshold of 2 to distinguish MGMT methylated from unmethylated tumors. In this study, we sought to determine whether the magnitude of the methylation score correlated with outcome. METHODS: We identified 165 newly diagnosed glioblastoma (GBM) isocitrate dehydrogenase (IDH) wild-type and temozolomide-treated upfront patients at UCLA and Kaiser Permanente Los Angeles with LabCorp-derived quantitative MGMT scores obtained on pretreatment tissue samples. Using LabCorp's threshold, we found 102 unmethylated and 63 methylated patients. We then further substratified each group based on the magnitude of the score, and performed Kaplan-Meier and Cox regression analyses of overall survival (OS) and progression-free survival (PFS). RESULTS: We validated that the standard LabCorp threshold of 2 could separate our cohort by survival, showing longer OS and PFS for MGMT methylated patients vs unmethylated patients. Cox regression analysis confirmed that MGMT (<1) patients had worse outcome, with OS and PFS hazard ratios of 2.375 (P = .053) and 2.463 (P = .023), respectively, when compared to the MGMT (1-1.99) patients. Contrary to our expectation, when we substratified the ≥2 (methylated) group, we did not find a dose-dependent relationship between the magnitude of MGMT methylation and improved survival. CONCLUSIONS: The MGMT unmethylated group contains a partially methylated group (greater than 1) that shares survival benefits similar to the methylated group. However, we did not demonstrate an association of very high methylation scores with increased survival. These findings will require validation in additional independent clinical data sets.

D-2-Hydroxyglutarate Is Necessary and Sufficient for Isocitrate Dehydrogenase 1 Mutant-Induced MIR148A Promoter Methylation.

Mutant isocitrate dehydrogenase (IDH) 1/2 converts α-ketoglutarate (α-KG) to D-2 hydroxyglutarate (D-2-HG), a putative oncometabolite that can inhibit α-KG-dependent enzymes, including ten-eleven translocation methylcytosine dioxygenase (TET) DNA demethylases. We recently established that miRNAs are components of the IDH1 mutant-associated glioma CpG island methylator phenotype (G-CIMP) and specifically identified MIR148A as a tumor-suppressive miRNA within G-CIMP. However, the precise mechanism by which mutant IDH induces hypermethylation of MIR148A and other G-CIMP promoters remains to be elucidated. In this study, we demonstrate that treatment with exogenous D-2-HG induces MIR148A promoter methylation and transcriptional silencing in human embryonic kidney 293T (293T) cells and primary normal human astrocytes. Conversely, we show that the development of MIR148A promoter methylation in mutant IDH1-overexpressing 293T cells is abrogated via treatment with C227, an inhibitor of mutant IDH1 generation of D-2-HG. Using dot blot assays for global assessment of 5-hydroxymethylcytosine (5-hmC), we show that D-2-HG treatment reduces 5-hmC levels, whereas C227 treatment increases 5-hmC levels, strongly suggesting TET inhibition by D-2-HG. Moreover, we show that withdrawal of D-2-HG treatment reverses methylation with an associated increase in MIR148A transcript levels and transient generation of 5-hmC. We also demonstrate that RNA polymerase II binds endogenously to the predicted promoter region of MIR148A, validating the hypothesis that its transcription is driven by an independent promoter.Implications: Establishment of D-2-HG as a necessary and sufficient intermediate by which mutant IDH1 induces CpG island methylation of MIR148A will help with understanding the efficacy of selective mutant IDH1 inhibitors in the clinic. Mol Cancer Res; 16(6); 947-60. ©2018 AACR.

Design of surfaces for liquid crystal-based bioanalytical assays.

Surface-induced ordering of liquid crystals (LCs) offers the basis of a label-free analytical technique for the detection of surface-bound biomolecules. The orientation-dependent energy of interaction of a LC with a surface (anchoring energy of LC), in particular, is both sensitive to the presence of surface-bound molecules and easily quantified. Herein, we report a study that analyzes a simple model of twisted nematic LC systems and thereby identifies surfaces with LC anchoring energies in the range of 0.5 microJ/m(2) to 2.0 microJ/m(2) to be optimal for use with LC-based analytical methods. Guided by these predictions, we demonstrate that analytic surfaces possessing anchoring energies within this range can be fabricated with a high level of precision (< 0.1 microJ/m(2)) through formation of monolayers of organothiols (with omega-functional groups corresponding to oligoethyleneglycols and amines) on gold films deposited by physical vapor deposition at oblique angles of incidence. Finally, by using the human epidermal growth factor receptor (EGFR) as a model protein analyte, we have characterized the influence of the anchoring energies of the surfaces on the response of the LC to the presence of surface-bound EGFR. These results, when combined with (32)P-radiolabeling of the EGFR to independently quantify the surface concentration of EGFR, permit identification of surfaces that allow use of LCs to report surface densities of EGFR of 30-40 pg/mm(2). Overall, the results reported in this paper guide the design of surfaces for use in LC-based analytical systems.

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein from the PDMS surface was found to be 75-81%. However, the transfer of non-specifically bound protein was substantially less than 75%, thus leading to the important finding that mechanical transfer of the EGF receptor leads to an approximately four-fold increase in signal-to-background from 20 : 1 to 88 : 1. The signal-to-background obtained following mechanical transfer is also better than that obtained using ELISA plates and stripping buffer (<48 : 1). The EGF receptor is a clinically important protein and the target of numerous anticancer agents and thus these results, when combined, provide guidance for the design of PDMS-based microanalytical systems for the capture and isolation of complex and clinically important transmembrane proteins.

DNA-analogous structures from deoxynucleophosphates and polylysine by ionic self-assembly.

Here we show that ionic self-assembly of simple biological tectons can be used to synthesize stable and highly ordered molecular structures. In particular, nucleotides and charged polypeptides can be assembled to form a complex analogous to DNA under relatively benign conditions. The combination of polylysine and pure dGMP leads to a fourfold ladder structure stabilizing an interior G-quartet structure by four polypeptide scaffolds. Making use of the Watson-Crick G∶C base pairing motive leads to double-stranded complexes. Interestingly, these complexes show stable DNA-like organization in aqueous solutions, as proven by gel electrophoresis and intercalation experiments.