Jonathan Serrano

Polymorphous low-grade neuroepithelial tumor of the young (PLNTY): an epileptogenic neoplasm with oligodendroglioma-like components, aberrant CD34 expression, and genetic alterations involving the MAP kinase pathway.

Epileptogenic tumors affecting children and young adults are a morphologically diverse collection of neuroepithelial neoplasms that, as a group, exhibit varying levels of glial and/or neuronal differentiation. Recent advances in molecular profiling technology, including comprehensive DNA sequencing and methylation analysis, have enabled the application of more precise and biologically relevant classification schemes to these tumors. In this report, we describe a morphologically and molecularly distinct epileptogenic neoplasm, the polymorphous low-grade neuroepithelial tumor of the young (PLNTY), which likely accounts for a sizable portion of oligodendroglioma-like tumors affecting the pediatric population. Characteristic microscopic findings most notably include infiltrative growth, the invariable presence of oligodendroglioma-like cellular components, and intense immunolabeling for cluster of differentiation 34 (CD34). Moreover, integrative molecular profiling reveals a distinct DNA methylation signature for PLNTYs, along with frequent genetic abnormalities involving either B-Raf proto-oncogene (BRAF) or fibroblast growth factor receptors 2 and 3 (FGFR2, FGFR3). These findings suggest that PLNTY represents a distinct biological entity within the larger spectrum of pediatric, low-grade neuroepithelial tumors.

IDH2 Mutations Define a Unique Subtype of Breast Cancer with Altered Nuclear Polarity.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1 One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118-29. ©2016 AACR.

Notch signaling regulates metabolic heterogeneity in glioblastoma stem cells

Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.Glioblastoma (GBM) stem cells (GSCs) reside in both hypoxic and vascular microenvironments within tumors. The molecular mechanisms that allow GSCs to occupy such contrasting niches are not understood. We used patient-derived GBM cultures to identify GSC subtypes with differential activation of Notch signaling, which co-exist in tumors but occupy distinct niches and match their metabolism accordingly. Multipotent GSCs with Notch pathway activation reside in perivascular niches, and are unable to entrain anaerobic glycolysis during hypoxia. In contrast, most CD133-expressing GSCs do not depend on canonical Notch signaling, populate tumors regardless of local vascularity and selectively utilize anaerobic glycolysis to expand in hypoxia. Ectopic activation of Notch signaling in CD133-expressing GSCs is sufficient to suppress anaerobic glycolysis and resistance to hypoxia. These findings demonstrate a novel role for Notch signaling in regulating GSC metabolism and suggest intratumoral GSC heterogeneity ensures metabolic adaptations to support tumor growth in diverse tumor microenvironments.

Whole Genome DNA Methylation Analysis of Human Glioblastoma Using Illumina BeadArrays

In this chapter, we describe the use of Illumina® Infinium® HD Assay in conjunction with Illuminas EPIC Methylation 8-sample array platform to obtain glioblastoma molecular profiles. The procedure spans four days, and can be performed by a single laboratory technician. Starting with as little as 250 ng of DNA input, this method allows the flexibility to begin with DNA derived from either formalin-fixed, paraffin-embedded (FFPE) or fresh tissue and is compatible with an Illumina iScan or HiScan system.

Abstract 11: Advancing methylation profiling in neuropathology: Diagnosis and clinical management

Introduction: Although molecular profiling is increasingly being applied to improve subgroup classification and to provide novel prognostic and predictive biomarkers, clinical neuropathology practice is largely based on morphology and immunohistochemistry. Current molecular methods play only a small role in determining the diagnosis itself. Methods: For molecular subclassification of tumors at NYU neuropathology and to improve diagnostic accuracy, we introduced genome-wide methylation profiling through Illumina Infinium HumanMethylation 450k array that can detect methylation marks from the DNA extracted from formalin-fixed paraffin embedded tissues. To this effort, an in-house pipeline was established in-house pipeline, which includes morphologic review, sample preparation, molecular profiling and bioinformatics analysis. We compared the methylation profiles to a reference cohort of 2150 cases from 77 tumor entities previously profiled and analyzed at German Cancer Research Center using a random forest algorithm and customized bioinformatics packages, which were shared between our institutions. Selected copy number variants (CNV) and mutations were confirmed by Fluorescence in situ Hybridization (FISH) or sequencing, and mutation specific immunohistochemistry, respectively. Results: We profiled 60 difficult in-house or consult adult and pediatric brain tumors where diagnosis, grade and/or molecular subtype were not conclusive by morphology, immunohistochemistry or standard molecular studies alone. There was 100% concordance with concurrently performed molecular tests such as 1p/19q, EGFR/BRAF CNV, MGMT promoter methylation or IDH1 status testing when these tests were performed for clinical care. Methylation profiling provided additional, relevant information in 30 of 60 (50%) cases, leading to a change of diagnosis in 9 (15%), clarification of the diagnosis in 7 (12%) cases, and further molecular subgroup refinement in 14 (23%) of cases, helping to direct further molecular testing and clinical management. Conclusion: The 450k methylation array platform represents a cost-efficient method to obtain molecular profiles of brain tumors to identify biologically relevant diagnostic subgroups, thereby improving diagnostic accuracy, and helping inform appropriate clinical management decisions. Citation Format: Kasthuri S. Kannan, Aristotelis Tsirigos, Jonathan Serrano, Lynn Ann Forrester, Arline Faustin, Cheddhi Thomas, David Capper, Volker Hovestadt, Stefan M. Pfister, David T. W Jones, Martin Sill, Daniel Schrimpf, Andreas von Deimling, Adriana Heguy, Sharon L. Gardner, Jeffrey Allen, Cyrus Hedvat, David Zagzag, Matija Snuderl, Matthias A. Karajannis. Advancing methylation profiling in neuropathology: Diagnosis and clinical management. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 11.

Cardiac arrhythmia and neuroexcitability gene variants in resected brain tissue from patients with sudden unexpected death in epilepsy (SUDEP)

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related mortality in young adults. The exact mechanisms are unknown but death often follows a generalized tonic–clonic seizure. Proposed mechanisms include seizure-related respiratory, cardiac, autonomic, and arousal dysfunction. Genetic drivers underlying SUDEP risk are largely unknown. To identify potential SUDEP risk genes, we compared whole-exome sequences (WES) derived from formalin-fixed paraffin embedded surgical brain specimens of eight epilepsy patients who died from SUDEP with seven living controls matched for age at surgery, sex, year of surgery and lobe of resection. We compared identified variants from both groups filtering known polymorphisms from publicly available data as well as scanned for epilepsy and candidate SUDEP genes. In the SUDEP cohort, we identified mutually exclusive variants in genes involved in µ-opiod signaling, gamma-aminobutyric acid (GABA) and glutamate-mediated synaptic signaling, including ARRB2, ITPR1, GABRR2, SSTR5, GRIK1, CTNAP2, GRM8, GNAI2 and GRIK5. In SUDEP patients we also identified variants in genes associated with cardiac arrhythmia, including KCNMB1, KCNIP1, DPP6, JUP, F2, and TUBA3D, which were not present in living epilepsy controls. Our data shows that genomic analysis of brain tissue resected for seizure control can identify potential genetic biomarkers of SUDEP risk.Epilepsy: Spotting genetic drivers of sudden deathGene variants associated with abnormal heart rhythm and neuronal excitability may increase the risk of Sudden Unexpected Death in Epilepsy (SUDEP). SUDEP is the most common cause of death directly related to epilepsy, but little is known about the risk factors and mechanisms through which seizures can lead to death. Daniel Friedman, Orrin Devinsky and colleagues at New York University Langone Medical Center, US, compared whole-exome sequences from brain tissue belonging to eight epilepsy patients who died from SUDEP and seven matched living controls who had brain tissue removed for epilepsy treatment. In the SUDEP cases they identified 13 rare gene variants involved in cardiac arrhythmia and excitatory neurotransmission as potential genetic biomarkers of SUDEP risk. Further understanding the genetic contribution to epilepsy-related mortality will help develop effective preventive strategies.

Recurrent homozygous deletion of DROSHA and microduplication of PDE4DIP 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.Pineoblastoma is a highly aggressive and rare childhood brain cancer, and the genetic drivers of sporadic pineoblastoma are unknown. Here, the authors genomically interrogated pediatric and adult pineoblastomas and found novel variants including recurrent homozygous deletions of DROSHA.

A DNA methylation-based classifier for accurate molecular diagnosis of bone sarcomas.

Purpose Pediatric sarcomas provide a unique diagnostic challenge. There is considerable morphologic overlap between entities, increasing the importance of molecular studies in the diagnosis, treatment, and identification of therapeutic targets. We developed and validated a genome-wide DNA methylation based classifier to differentiate between osteosarcoma, Ewings sarcoma, and synovial sarcoma. Materials and Methods DNA methylation status of 482,421 CpG sites in 10 Ewings sarcoma, 11 synovial sarcoma, and 15 osteosarcoma samples were determined using the Illumina Infinium HumanMethylation450 array. We developed a random forest classifier trained from the 400 most differentially methylated CpG sites within the training set of 36 sarcoma samples. This classifier was validated on data drawn from The Cancer Genome Atlas (TCGA) synovial sarcoma, TARGET Osteosarcoma, and a recently published series of Ewings sarcoma. Results Methylation profiling revealed three distinct patterns, each enriched with a single sarcoma subtype. Within the validation cohorts, all samples from TCGA were accurately classified as synovial sarcoma (10/10, sensitivity and specificity 100%), all but one sample from TARGET-OS were classified as osteosarcoma (85/86, sensitivity 98%, specificity 100%) and 14/15 Ewings sarcoma samples classified correctly (sensitivity 93%, specificity 100%). The single misclassified osteosarcoma sample demonstrated high EWSR1 and ETV1 expression on RNA-seq although no fusion was found on manual curation of the transcript sequence. Two additional clinical samples, that were difficult to classify by morphology and molecular methods, were classified as osteosarcoma when previously suspected to be a synovial sarcoma and Ewings sarcoma on initial diagnosis, respectively. Conclusion Osteosarcoma, synovial sarcoma, and Ewings sarcoma have distinct epigenetic profiles. Our validated methylation-based classifier can be used to provide diagnostic assistance when histological and standard techniques are inconclusive.