Stewart G. Neill

Beyond the World Health Organization grading of infiltrating gliomas: advances in the molecular genetics of glioma classification

BACKGROUND Traditional classification of diffuse infiltrating gliomas (DIGs) as World Health Organization (WHO) grades II-IV is based on histological features of a heterogeneous population of tumors with varying prognoses and treatments. Over the last decade, research efforts have resulted in a better understanding of the molecular basis of glioma formation as well as the genetic alterations commonly identified in diffuse gliomas. METHODS A systematic review of the current literature related to advances in molecular phenotypes, mutations, and genomic analysis of gliomas was carried out using a PubMed search for these key terms. Data was studied and synthesized to generate a comprehensive review of glioma subclassification. RESULTS This new data helps supplement the existing WHO grading scale by subtyping gliomas into specific molecular groups. The emerging molecular profile of diffuse gliomas includes the studies of gene expression and DNA methylation in different glioma subtypes. The discovery of novel mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) provides new biomarkers as points of stratification of gliomas based on prognosis and treatment response. Gliomas that harbor CpG island hypermethylator phenotypes constitute a subtype of glioma with improved survival. The difficulty of classifying oligodendroglial lineage of tumors can be aided with identification of 1p/19q codeletion. Glioblastomas (GBMs) previously described as primary or secondary can now be divided based on gene expression into proneural, mesenchymal, and classical subtypes and the identification of mutations in the promoter region of the telomerase reverse transcriptase (TERTp) have been correlated with poor prognosis in GBMs. CONCLUSIONS Incorporation of new molecular and genomic changes into the existing WHO grading of DIGs may provide better patient prognostication as well as advance the development of patient-specific treatments and clinical trials.

Whole-brain spectroscopic MRI biomarkers identify infiltrating margins in glioblastoma patients

BACKGROUND The standard of care for glioblastoma (GBM) is maximal safe resection followed by radiation therapy with chemotherapy. Currently, contrast-enhanced MRI is used to define primary treatment volumes for surgery and radiation therapy. However, enhancement does not identify the tumor entirely, resulting in limited local control. Proton spectroscopic MRI (sMRI), a method reporting endogenous metabolism, may better define the tumor margin. Here, we develop a whole-brain sMRI pipeline and validate sMRI metrics with quantitative measures of tumor infiltration. METHODS Whole-brain sMRI metabolite maps were coregistered with surgical planning MRI and imported into a neuronavigation system to guide tissue sampling in GBM patients receiving 5-aminolevulinic acid fluorescence-guided surgery. Samples were collected from regions with metabolic abnormalities in a biopsy-like fashion before bulk resection. Tissue fluorescence was measured ex vivo using a hand-held spectrometer. Tissue samples were immunostained for Sox2 and analyzed to quantify the density of staining cells using a novel digital pathology image analysis tool. Correlations among sMRI markers, Sox2 density, and ex vivo fluorescence were evaluated. RESULTS Spectroscopic MRI biomarkers exhibit significant correlations with Sox2-positive cell density and ex vivo fluorescence. The choline to N-acetylaspartate ratio showed significant associations with each quantitative marker (Pearsons ρ = 0.82, P < .001 and ρ = 0.36, P < .0001, respectively). Clinically, sMRI metabolic abnormalities predated contrast enhancement at sites of tumor recurrence and exhibited an inverse relationship with progression-free survival. CONCLUSIONS As it identifies tumor infiltration and regions at high risk for recurrence, sMRI could complement conventional MRI to improve local control in GBM patients.

Immunohistochemical Investigation of BRAF p.V600E Mutations in Thyroid Carcinoma Using 2 Separate BRAF Antibodies

Background:Approximately 45% of papillary thyroid carcinomas harbor BRAF p.V600E mutations and current practice algorithms endorse molecular testing for BRAF p.V600E. We assessed the utility of immunohistochemistry to detect BRAF p.V600E mutations in thyroid carcinomas using 2 separate BRAF monoclonal antibodies: one that detects both mutant and wild-type protein (pan-BRAF) and another that detects only the mutant protein (mut-BRAF). Methods:We selected 41 formalin-fixed paraffin-embedded thyroid carcinomas (29 papillary, 1 follicular, 7 medullary, and 4 anaplastic) from 37 thyroidectomies and 4 fine-needle aspirations. Immunohistochemistry was performed using a pan-BRAF (clone EP152Y) or a mut-BRAF (clone VE1) monoclonal antibody. Tumors were considered positive if >10% of neoplastic cells showed moderate (2+) or strong (3+) cytoplasmic staining. BRAF p.V600E mutations were confirmed by molecular pyrosequencing, the gold standard for statistical analysis. Results:pan-BRAF reactivity was observed in 80.5% (n=33) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 46.3% (n=19) were wild type. mut-BRAF reactivity was observed in 46.3% (n=19) of cases: 34.1% (n=14) harbored BRAF p.V600E mutations and 12.2% (n=5) were wild type. The pan-BRAF antibody detected 14 more false positives (specificity: 29.6%, PPV: 42.4%) compared with the mut-BRAF antibody (specificity: 61.5%, PPV: 73.7%), but both antibodies detected the same 5 false positives. No false negatives were detected with either antibody (sensitivity and NPV 100.0% for both). Conclusions:The suboptimal specificity and PPV limits the diagnostic utility of both antibodies to reliably detect BRAF p.V600E mutations in thyroid carcinoma. However, both antibodies provide excellent sensitivity and NPV and either could be used to exclude BRAF wild-type thyroid carcinomas before molecular testing.

Section III: molecular diagnostics in neuro-oncology.

As is true in other areas of pathology, the relationship between the surgical neuropathologist, the neurosurgeon, the neuro-oncologist, and molecular diagnostician is evolving at a rapid pace. This pace is commensurate with the almost daily revelations regarding the nature of neoplastic diseases attributable to molecular methods and investigations. The task of integrating the findings of molecular diagnostics into the time-honored practices of traditional diagnostics may seem daunting. Some may say that the field of molecular diagnostics is still in its infancy and that meaningful changes to the clinical practice of surgical neuropathology based on molecular findings should be delayed. However, recent molecular genetic data related to central nervous system (CNS) neoplasia carry critical implications for disease diagnosis and management. The question has become not whether but how to incorporate molecular diagnostic findings into the treatment of patients with CNS neoplasia. Here, we present elements of a testing strategy that is both (1) currently clinically relevant or actionable and (2) reasonable to pursue in almost any health care environment.

A systematic pipeline for the objective comparison of whole-brain spectroscopic MRI with histology in biopsy specimens from grade III glioma

The diagnosis, prognosis, and management of patients with gliomas are largely dictated by the pathological analysis of tissue biopsied from a selected region within the lesion. However, the heterogeneous and infiltrative nature of gliomas make it difficult to identify the optimal region for biopsy with conventional magnetic resonance imaging (MRI). This is particularly true for low-grade gliomas, which are often nonenhancing tumors. To improve the management of patients with such tumors, neuro-oncology requires an imaging modality that can specifically identify a tumors most anaplastic/aggressive region(s) for biopsy targeting. The addition of metabolic mapping using spectroscopic MRI (sMRI) to supplement conventional MRI could improve biopsy targeting and, ultimately, diagnostic accuracy. Here, we describe a pipeline for the integration of state-of-the-art, high-resolution, whole-brain 3-dimensional sMRI maps into a stereotactic neuronavigation system for guiding biopsies in gliomas with nonenhancing components. We also outline a machine-learning method for automated histological analysis that generates normalized, quantitative metrics describing tumor infiltration in immunohistochemically stained tissue specimens. As a proof of concept, we describe the combination of these 2 techniques in a small cohort of patients with grade 3 glioma. With this work, we aim to present a systematic pipeline to stimulate histopathological image validation of advanced MRI techniques, such as sMRI.

Chordoid glioma of the third ventricle: report of a rapidly progressive case

Chordoid gliomas are slowly growing third ventricular tumors that can be challenging to manage clinically. Rapid progression causing death has not been previously reported for this tumor type. We present and discuss a case of chordoid glioma that arose in a 46-year-old female who presented with progressive fatigue, headache, and altered mental status, attributable to severe hydrocephalus caused by a third ventricular mass. She underwent urgent subtotal resection and ventriculo-peritoneal shunt placements. Post-operative MRI noted residual tumor in the anterior resection cavity. An MRI performed 9 weeks later showed substantial progression, with marked tumor enlargement and compression of adjacent hypothalamic structures and the optic chiasm. Despite a course of radiation therapy, the tumor continued to enlarge, and the patient died from tumor progression 7 months after initial presentation. Post-mortem exam demonstrated a mass that expanded the third ventricle and compressed adjacent hypothalamic, thalamic and suprasellar structures. Histologic and immunohistochemical studies confirmed a chordoid glioma and revealed multifocal coagulative necrosis and intravascular thrombosis, which are unusual in this tumor type. Cytogenomic microarray testing revealed numerous DNA copy number abnormalities, many of which had not previously been reported in this tumor. The pathologic and cytogenetic changes may correlate with the aggressive behavior of this chordoid glioma and can be pursued by future investigation of additional cases.

Meningioma With Tyrosine-Rich Crystalloids: A Case Report and Review of the Literature

We report a case of fibrous meningioma with tyrosine-rich crystalloid in the frontal lobe of a middle-aged woman. The patient presented with a history of several years of worsening headaches and blurry vision, which progressed to include syncopal episodes and right-sided weakness. Imaging demonstrated a dural-based extra-axial mass arising from the right orbital roof and extending superiorly along the right frontal convexity causing right-to-left midline shift. The patient underwent a craniotomy and operative resection. Tumor architecture and cytology was similar to that of a Schwannian neoplasm, with spindled cells arranged in a fascicular architecture and displaying focal nuclear palisading. Immunohistochemical stains confirmed a diagnosis of fibrous meningioma. Light microscopy demonstrated extracellular deposits of eosinophilic crystalline material parallel to the spindled tumor cells, reminiscent of “tyrosine-rich” crystals described in salivary gland neoplasms. This is the third meningioma featuring tyrosine-rich crystalloid reported in the literature; we also summarize the previous 2 reports.

Genomic analysis in the practice of surgical neuropathology: The emory experience

The evaluation of central nervous system tumors increasingly relies on molecular genetic methods to aid in classification, offer prognostic information, and predict response to therapy. Available assays make it possible to assess genetic losses, amplifications, translocations, mutations, or the expression levels of specific gene transcripts or proteins. Current molecular diagnostics frequently use a panel-based approach and whole genome analysis, and generally rely either on DNA sequencing or on hybridization-based methodologies, such as those used in cytogenomic microarrays. In some cases, immunohistochemistry can be used as a surrogate for genetic analysis when the mutation of interest consistently results in overexpression or underexpression of a known protein product. In surgical neuropathology practice, the diagnostic workup of diffuse gliomas, medulloblastomas, low-grade circumscribed gliomas, as well as other diseases, now routinely incorporates the results of genomic studies. Here we summarize our institutions current approach to diagnostic surgical neuropathology, using these contemporary molecular diagnostic applications.

Adult Intramedullary Teratoma of the Spinal Cord: A Case Report and Review of Literature

BACKGROUND Teratomas of the spinal cord constitute 0.1% of all spinal tumors, and these lesions are extremely rare in adults. The authors describe a rare case of intradural intramedullary teratoma of the conus medullaris and perform review of literature of intramedullary teratomas seen in the thoracolumbar region. CASE DESCRIPTION A 48-year-old man presented with fasciculations in the bilateral upper and lower extremities. Radiologic findings revealed an L2-L3 level intradural, nonenhancing, extramedullary cystic mass measuring 15 × 13 mm with a 6-mm enhancing nodule at the level of the conus medullaris. The patient was followed up for 1 year, during which time enlargement of the lesion with new areas of patchy contrast enhancement were observed. L1-L2 decompressive laminectomies were performed, and gross total resection of the lesion was achieved. Histopathologic examination confirmed the diagnosis of benign mature cystic teratoma. A literature review revealed no incidence difference in intramedullary teratomas between males and females (P > 0.05). The mean age at the time of diagnosis was 36.4 ± 12.3 years for men and 41.3 ± 11.6 for women (P < 0.05). The mean symptom duration before treatment was 64.6 ± 79.4 months for females and 20.7 ± 13.8 months for men (P < 0.05). Complete resection was achieved in 48.1% of the cases. CONCLUSIONS Teratomas should be taken into consideration in the differential diagnosis of intramedullary lesions when the imaging reveals variable signal intensity because of tissue heterogeneity. A partial resection is a viable treatment option when the lesion is attached to vital structures because of the low recurrence rates reported in the literature.

Abstract 3607: An evaluation of NGS to identify gene fusions using RNA from FFPE solid tumor samples

Gene fusions have long been considered strong drivers of cellular transformation, making the accurate and precise assessment of these variants a necessity for any tumor profiling assay. Recent studies have indicated the utility of next-generation sequencing (NGS) for tumor profiling due to increasing data output and decreasing costs of the technology. Unfortunately, because a critical facet of NGS is the evaluation of short DNA fragments, sufficiently covering all possible breakpoint regions (many of which are intronic) has proven difficult and costly. Recent studies have indicated that NGS may prove better at detecting gene fusions using RNA instead of DNA, given the higher probability of breakpoint-spanning reads. This allows for de-novo discovery of fusion partners without knowing the precise breakpoint and guarantees expression of the fusion transcript. To that end, Illumina is developing a novel method for simultaneous library preparation from low input amounts of degraded DNA and RNA from a single FFPE tumor sample. With a turnaround time from nucleic acid to data of less than 4 days, this enrichment-based assay surveys 170 genes for single nucleotide variants and small indels, 57 genes for gene amplifications, 55 genes for fusions and four genes for splice variants. To determine the limit of detection for gene fusions, a panel of different synthetic RNA transcripts were prepared in vitro, pooled at equal molar amounts, and spiked into 20ng of cell line RNA (MCF-7). Fusions were detected over several orders of magnitude down to 1×10-8 picomoles, equivalent to 3 to 15 fusion transcripts per cell. In addition, a similar range of fusion detection was observed when RNA from two different cell lines were mixed, as when RNA from a cell line with high expression of an FGFR2-COL14A1 fusion was mixed in proportional amounts with RNA from a different cell line where FGFR2 is minimally expressed. Importantly, our method allowed for fusion detection from as little as 100 picograms of cell line RNA. We then tested our new method on previously characterized FFPE solid tumor samples harboring known gene rearrangements identified by FISH and other methods. Not only was the NGS method able to detect the majority of previously characterized variants, including EML4-ALK and SDC4-ROS1, it also identified the gene fusions and their uncharacterized fusions partners by combining the non-targeted sequence information gained from using an enrichment-based assay with novel fusion calling algorithms. From this information, we were able to glean new insights into the structure of the rearrangements and how the gene fusions may be involved in tumorigenesis. These results indicate that NGS can identify fusions from the low amounts of degraded RNA from solid tumor samples, identify fusion partners not uncovered by current technologies, and further emphasizes the advantage of NGS in solid tumor profiling. Citation Format: Julianna Tdr Parks, Luo Byron, Brian Crain, Snedecor June, Zhao Chen, Tingting Du, Gabriel L. Sica, Taofee K. Owonikoko, Stewart G. Neill, Scott Newman, Debra F. Saxe, Jennifer S. LoCoco, Han-Yu Chuang, Charles Lin, Kathryn M. Stephens, Michael R. Rossi, Matthew C. Friedenberg. An evaluation of NGS to identify gene fusions using RNA from FFPE solid tumor samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3607.