Nervous System Primitive Neuroectodermal Tumors Research Articles

ETMR-15. RECLASSIFICATION OF CHILDHOOD CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR: CLINICAL FEATURES AND OUTCOME

Abstract BACKGROUND The “central nervous system primitive neuroectodermal tumor” (CNS-PNET) nosology was removed from the World Health Organization (WHO) classification after DNA methylation profiling and new tumor entities characterized by specific recurrent genetic alterations were described. The aim of this study was to re-evaluate the diagnosis of CNS-PNET and to describe their clinical characteristics. METHODS Patients aged < 18 years with CNS-PNET or unclassified tumor, who were treated at Gustave Roussy between 1999 and 2019 or enrolled in the PNET HR trial were retrospectively identified. The study included all patients with sufficient archival material for diagnosis re-evaluation. RESULTS Sixty-two patients were eligible for the current analysis. The central histopathological review proposed a diagnosis according to the WHO classification in 58 cases. Out of these cases, 34 embryonal tumors were identified: 22 Embryonal Tumors with Multilayered Rosettes (ETMR), 4 CNS neuroblastomas with FOXR2 activation (CNS NB-FOXR2), 4 CNS high-grade neuroepithelial tumors with BCOR alteration (CNS HGNET-BCOR), 4 medulloblastomas. Another 24 tumors were classified as non embryonal tumors: 11 high-grade gliomas, 5 ependymomas, 4 pineoblastomas, 2 teratomas, one desmoplastic small round cell tumor, and one pilocytic astrocytoma. Four tumors without suspected diagnosis after first analysis remain unclassified after DNA methylation profiling. The 5-year event-free survival and overall survival for patients with ETMR were 14% (95% CI, 4.7-39%) and 27% (95% CI, 14-54%) respectively. For patients with CNS HGNET-BCOR and CNS NB-FOXR2 the median time to relapse was respectively 11 months (range, 0.5 to 1.3 year) and 3.1 years (range 1.7 to 4.4 years). CONCLUSIONS This study confirmed that the former nosology of CNS PNET does not correspond to a homogenous class but encompasses a diversity of rare histomolecular entities with specific clinical characteristics and outcomes.

ETMR-09.In vitro modelling of embryonal tumors with multilayered rosettes (ETMR) and other novel brain tumor types.

Over the last decade, molecular characterization has resulted in many tumors previously classified as central nervous system primitive neuroectodermal tumors (CNS-PNETs) now being classified into their own distinct tumor types. These novel types are often characterized by very specific genomic aberrations. For instance, embryonal tumors with multilayered rosettes (ETMR) harbor amplifications of miRNA cluster C19MC or complex DICER1 mutations, while in CNS neuroblastoma with FOXR2 activation structural aberrations result in aberrant FOXR2 expression. Despite the presence of distinct oncodrivers, our understanding of these tumors is still limited. To elucidate tumor biology and to discover tumor specific treatments, we need to uncover how these oncodrivers contribute to tumorigenesis. However, a bottleneck in basic and translational research of these novel tumor types, is the lack of representative preclinical models, especially in vitro. To overcome this hurdle, we aim to mimic tumor development in genetically modified brain organoids. Human brain organoids derived from pluripotent stem cells are generated to represent either the developing forebrain or cerebellum. To mimic oncodriving events, DNA plasmids are introduced via electroporation into the proposed cell-of-origin populations to knockout tumor suppressor genes or overexpress oncogenes. By detecting fluorescent proteins encoded by the plasmids, electroporated cells are followed over time. Based on our preliminary data, for instance, overexpression of C19MC results in ectopic expansion of the electroporated cells. Ongoing histological and molecular characterizations, including (single cell) transcriptomic and epigenomic analyses, will reveal to which extend these organoid models resemble the specific human tumor types. Although further validation is required, these organoid models provide a novel avenue to study especially brain tumor types with distinct oncodriving events for which patient-derived models have not yet been established. They also allow for in-depth analyses of the potential cells of origin and the contribution of different mutations to tumor biology.

Nimotuzumab therapy in the treatment of pediatric central nervous system tumors: single-center experience.

Relapsed or refractory central nervous system (CNS) tumors still have poor prognosis, and, therefore, new treatment options are required. We retrospectively researched treatment results of patients with CNS tumors treated with nimotuzumab from 2010 to 2015. The study included nine patients with the diffuse intrinsic pontine glioma; eight with medulloblastoma; three each with anaplastic ependymoma, glioblastoma multiforme, and central nervous system primitive neuroectodermal tumor (CNS PNET); two patients with gliomatosis cerebri; and one patient each with other tumor types, including atypical teratoid rhabdoid tumor, thalamic astrocytoma, low-grade glial tumor, high-grade glial tumor, and cribriform neuroepithelial tumor. An objective response was observed in 10 of 33 patients with four patients showing a complete response, three a partial response, and three patients had stable disease. The 2-year overall survival (OS) and progression-free survival (PFS) rates were 35 ±9% and 19 ±8%, respectively. Due to the objective response in medulloblastoma, CNS PNET, and anaplastic ependymoma (MED group), survival rates of this group were analyzed. The 2-year OS and PFS for the MED group were 71 ±12% and 30 ±13%, respectively. The treatment was well tolerated. The treatment responses for medulloblastoma, CNS PNET, and anaplastic ependymoma have been promising. Likewise, some patients with relapsed or progressive CNS tumors may benefit through nimotuzumab-containing regimen.

Delta-24-RGD, an Oncolytic Adenovirus, Increases Survival and Promotes Proinflammatory Immune Landscape Remodeling in Models of AT/RT and CNS-PNET.

Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells. Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in vitro in a battery of CNS-PNET and AT/RT cell lines. In vivo, efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34+-NSG-SGM3). Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC50 value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. In vivo, a single intratumoral Delta-24-RGD injection (107 or 108 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD-treated tumors revealed increased CD8+ T-cell infiltration. Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.

Is Ovarian Tissue Transplantation Safe in Patients with Central Nervous System Primitive Neuroectodermal Tumors?

The risk of reseeding malignancy harbored in cryopreserved and transplanted ovarian tissue has been a source of concern. This study aimed to determine the potential relationship between frozen–thawed ovarian tissue transplantation and primary cancer recurrence. Three patients with cerebral primitive neuroectodermal tumors (PNET) were included in this study. One woman gave birth to three healthy babies following reimplantation of her cryopreserved ovarian tissue, but subsequently died due to cancer relapse six years after ovarian tissue transplantation. The second subject died from progressive cancer, while the third is still alive and awaiting reimplantation of her ovarian tissue in due course. Frozen ovarian cortex from all three patients was analyzed and xenotransplanted to immunodeficient mice for five months. Main outcomes were the presence of cancer cells in the thawed and xenografted ovarian tissue at histology, immunostaining (expression of neuron-specific enolase and glial fibrillary acidic protein (GFAP)), and reverse-transcription droplet digital polymerase chain reaction (RT-ddPCR) (levels of enolase 2 and GFAP). In conclusion, no malignant cells were detected in ovarian tissue from patients with PNET, even in those who experienced recurrence of the disease, meaning that the risk of reseeding cancer cells with ovarian tissue transplantation in these patients can be considered low.

LINC-07. PREVALENCE AND SPECTRUM OF EARLY ENDOCRINE DISORDERS IN SURVIVORS OF PEDIATRIC EMBRYONAL BRAIN TUMORS (PEBT): EXPERIENCE FROM INDIA

BACKGROUNDSurvivors of pediatric brain tumors are at high risk of developing endocrine disorders, potentially impacting growth,development and quality of life.METHODSetrospective audit of 2-year survivors of PEBT(3-18years at diagnosis)viz. medulloblastoma(MB),Central nervous system Primitive neuro-ectodermal tumors(CNS-PNET) and atypical teratoid/rhabdoid tumor(ATRT) treated January 2006-December 2017 at Tata Memorial Centre,Mumbai, with surgery, cranio-spinal irradiation(CSI; 35Gy in high-risk MB,CNS-PNET,ATRT and 23.4Gy in average-risk MB with tumor boost 19.8Gy)and six cycles of adjuvant chemotherapy(cyclophosphamide,cisplatin and vincristine).Patients were followed up by a paediatric endocrinology team specialized in management of PEBT.RESULTSOf 249 PEBT treated during this period,88 are alive in remission >2 years (69-MB, 15-CNS PNET,4-ATRT),median age at diagnosis 6 years. At a median follow-up of 5.6 years (range 3- 12.5years),63 patients(72%) had at least one endocrine disorder,26(29.%)≥2 hormonal deficiencies. The most common endocrine disorders were central hypothyroidism(57%),growth hormone deficiency(40%), central hypogonadism(5%)and central hypoadrenalism (3.5%).The median time to develop hypothyroidism was 2.8 years(range 5months to 8.5 years) from CSI. Growth hormone replacement therapy began after a median period of 4.2 years(range-1.5 to 11.5years) from CSI. Higher dose of CSI was associated with development of endocrine disorder (odds ratio [OR] 2.71; 95% CI, 1.03 to 7.04,p-0.04).CONCLUSIONSThe high incidence of endocrine deficits in survivors of PEBT necessitates early and lifelong monitoring. Early and appropriate management is crucial to achieve full growth potential.

PATH-08. THE IMPORTANCE OF RE-DIAGNOSIS OF TUMORS PREVIOUSLY CLASSIFIED AS CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMORS

BACKGROUNDThe recent molecular analyses have revealed that central nervous system primitive neuroectodermal tumors (CNS PNETs) those having clusters of small round tumor cells are genetically different tumors. However, the concepts of CNS PNET are complicated, and it is difficult to diagnose them appropriately in clinical field. To overcome this difficulty, we reviewed previous studies associated with CNS PNETs, and carried out several approaches, those are relatively easy access to use in clinics, for our 8 samples of embryonal brain tumors diagnosed CNS PNETs in our institution, initially.METHODSWe used in combination with immunohistochemistry (IHC), Sanger sequence, Pyrosequence, polymerase chain reaction (PCR), real time PCR and copy number analysis referring recent reports.RESULTSIn terms of the diagnosis three out of 8 cases were changed based on the results in this study from previous diagnoses.CONCLUSIONIn this review, it seemed that either the histopathological evaluation or molecular analyses would be not enough to make accurate diagnosis of CNS embryonal brain tumors, and it is essential to combine both of them including recent comprehensive analysis methods.

C19MC amplification and expression of Lin28A and Olig2 in the classification of embryonal tumors of the central nervous system: A 14-year retrospective study from a tertiary care center.

CNS embryonal tumors (CET) other than medulloblastomas (MB) and atypical teratoid/rhabdoid tumors (AT/RTs), previously designated as 'central nervous system primitive neuroectodermal tumors' ('CNS PNETs'), are a heterogenous subset of tumors with poorly defined diagnostic criteria. Other than the subset of embryonal tumor with multilayered rosettes (ETMR) defined by C19MC amplification, most CETs are diagnosed by exclusion of other molecularly defined entities and histological mimics including MB, AT/RTs, and high-grade gliomas, and termed as CET, not otherwise specified (NOS) in the 2016 WHO classification. To reclassify 'CNS PNETs' as per WHO 2016, and estimate the true proportion of CET, NOS in a tertiary healthcare setting, and to evaluate the diagnostic utility of C19MC amplification, Lin28A and Olig2 expression in the subclassification of CETs. Previously diagnosed cases of 'CNS PNETs' (2002-2016) were first evaluated by immunohistochemistry (IHC) for MIC2, RelaA, L1CAM, IDH1R132H, H3K27M, H3G34R, H3G34V, INI1, and BRG1 proteins and by fluorescence in-situ hybridization (FISH) for EWSR1 translocation to exclude histological mimics. The selected CETs (case cohort) and 79 histological mimics (comparison cohort) comprising of MB, AT/RT, pineal parenchymal tumors, Ewing sarcoma, esthesioneuroblastoma, intraocular medulloepithelioma, and H3G34R mutant high-grade glioma were subject to IHC for Olig2 and Lin28A, and FISH for C19MC amplification. Twenty-two cases of 'CNS PNETs' were retrieved, all of which were negative for the first panel of markers and showed retained INI-1/BRG1 expression. Three of them (3/22, 13.6%) showed C19MC amplification (ETMR, C19MC-altered) with ETMR histology, Lin28A positivity, and Olig2 negativity. Among the remaining 19 CETs, one showed medulloepithelioma histology (Medulloepithelioma, NOS) and remaining were non-descript small round cell tumors (CET, NOS), all negative for Lin28A. Olig2 was positive in only 3 CETs (13.6%), all being CET, NOS. All tumors in the comparison cohort were negative for C19MC amplification, Lin28A and Olig2 except for 27% of ATRTs that were Lin28A positive. ETMR, C19MC-altered constitute less than 14% of CETs, with majority remaining uncharacterized as CET, NOS. Lin28A is 100% sensitive for the detection of C19MC amplification; however, its specificity is limited by its expression in ATRTs. Olig2 expression is seen only in a small subset of CET, NOS and is of limited diagnostic utility.

Primary sellar neuroblastoma pretending pituitary adenoma: A new case report and review of literature

Introduction Sellar neuroblastoma is hugely rare, only ten cases having been described in the literature. Primary intracranial neuroblastoma mainly occurs in children and arises in the supratentorial parenchyma. These tumors belong to embryonal central nervous system primitive neuroectodermal tumors (PNET). Magnetic resonance imaging (MRI) appearance is non-specific. Management is primary based on surgical resection. Diagnosis relied on histopathological examination. Through this case, it seems relevant to consider neuroblastoma face to MRI atypical aspects of pituitary adenoma. Clinical presentation We report the case of a 58-year-old-man with a 3-year old history of a rapidly growing sellar lesion diagnosed preoperatively as a pituitary non-functioning adenoma. He developed progressive bitemporal hemianopsia and visual field loss. Endocrinal investigations disclosed a functional hyperprolactinemia due to pituitary stalk effect. MRI displayed a sellar and supra-seller mass extension compressing the optic chiasm, with intra-sphenoidal sinus development, skull bone lysis and bilateral cavernous sinus invasion. He underwent near complete surgical removal through an endoscopic transseptal transsphenoidal approach. Histological and immunohistochemical studies yielded a diagnosis of neuroblastoma. Postoperatively his visual deficit improved. Neither endrocinal disturbance nor rhinorrhea occurred. He underwent fractioned radiotherapy a few weeks later. Discussion Primary sellar neuroblastoma could be easily misdiagnosed as pituitary adenoma. Unusual MRI features could lead to consider this diagnosis. The optimal management seems to be surgical resection followed by radiation therapy. Some authors suggested adjuvant chemotherapy in patients with subtotal resection or recurrence however this is no consensual.

Prolongation of radiotherapy duration is associated with inferior overall survival in patients with pediatric medulloblastoma and central nervous system primitive neuroectodermal tumors.

The importance of radiotherapy (RT) duration in medulloblastoma in the modern era of chemotherapy has not been well elucidated. The aim of this study was to determine the impact of RT treatment duration on overall survival (OS) in pediatric medulloblastoma and cenral nervous system neuroectodermal tumors (PNETs). The National Cancer Database (NCDB) was queried to identify patients with newly diagnosed medulloblastoma and CNS PNETs diagnosed between 2004 and 2014. Patients were excluded if they had extraneural metastasis, did not receive standard craniospinal irradiation dose, had a nonstandard total dose outside of 54 or 55.8 Gy, did not receive adjuvant chemotherapy, or if the RT duration was outside of the expected range of 37 to 80 days. The Kaplan-Meier estimator was used to estimate the association between RT duration (≤45 days or >45 days) and OS. Multivariate Cox regression was used to assess other confounders of OS. Six-hundred twenty-five patients met inclusion criteria, of which 181 were assigned to the "RT long" (>45 days) cohort (29.0%) and 444 (71.0%) to the "RT short" group (≤45 days). The five-year OS for the "RT short" compared with "RT long" cohort was 82.2% versus 70.9%, respectively (log-rank, P < 0.0037). For average risk patients, the five-year OS was 84.6% versus 86.4% for "RT short" and "RT long," respectively (log-rank, P = 0.40). However, for high-risk patients, five-year OS was 77.7% versus 51.0% (log-rank, P < 0.0001) in the "RT short" and "RT long" cohorts. For patients with high-risk medulloblastoma and CNS PNETs, RT duration >45 days was associated with inferior OS.

PDTM-19. MYC AND FAK/SRC COMBINATION TREATMENT OF FOXR2-HIGH BRAIN TUMORS

Abstract Brain tumors are the leading cause of cancer-related deaths in children. To identify novel therapeutic targets in two types of pediatric brain tumors, medulloblastoma (MB) and central nervous system primitive neuroectodermal tumors (CNS-PNETs), we performed a Sleeping Beauty transposon mutagenesis screen in mice. In doing this, we identified FOXR2 (Forkhead box R2) as a candidate oncogene in MB. FOXR2 is expressed at high levels in a subset of human MB and increased FOXR2 expression drives MB formation in mice. FOXR2 has been implicated as an oncogene in several cancers of neural original, including CNS-PNET, adult and pediatric high-grade glioma, and malignant peripheral nerve sheath tumors. We have also found that FOXR2 is upregulated in non-MYCN amplified high-risk neuroblastoma. Therefore, we propose that FOXR2 represents a strong candidate for targeted therapy across multiple brain tumor entities. We have uncovered a dual role of FOXR2 in stabilization of CMYC protein and activation of the FAK/SRC signaling pathway. We are working to further define the mechanism of FOXR2-mediated MYC stabilization and FAK/SRC activation. We plan to treat FOXR2-high tumors with combinations of drugs effective against tumors with high MYC, FAK, or SRC in clinical development. Inhibitors or combinations of that score well in vitro will be used to treat mice with orthotopically injected FOXR2-high tumor cells (MB, neuroblastoma, and glioma). In addition, we are working to construct mouse models of FOXR2-driven neural tumors for use in drug testing. We hypothesize that FOXR2-high tumors will be sensitive to MYC and FAK/SRC inhibitor combination therapy, establishing FOXR2 as a molecular marker for treatment success of MYC and FAK/SRC inhibitors in patients.

RARE-12. EARLY FOCAL RADIOTHERAPY AND TEMOZOLOMIDE FOLLOWING COMPLETE RESECTION APPEAR SUPERIOR TO INTENSIVE CHEMOTHERAPY IN CHILDREN WITH EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES (ETMR)

Abstract BACKGROUND Embryonal tumor with multilayered rosettes (ETMR) is an extremely rare entity characterized by LIN28A expression and alterations in the C19MC locus. ETMRs predominantly occur in young children and have a dismal prognosis. We report on our single institution experience in seven consecutive patients. METHODS Between 2006 and 2016, seven patients were diagnosed with ETMR. Diagnosis was established prospectively by characteristic histopathologic features, LIN28A immunostaining and amplification of the C19MC region in five patients and retrospectively confirmed in two additional patients originally diagnosed as central nervous system primitive neuroectodermal tumor. Tumor location was supratentorial in all patients. Median age at diagnosis was 25 months (range 5–38). Male to female ratio was 1:6. RESULTS All patients received gross total tumor resection and four patients were treated with intensive chemotherapy regimens including high-dose chemotherapy in three patients. Four patients recurred after a median of 6 months (range 2–11 months) and all except one patient who died after high-dose chemotherapy, succumbed to their disease after a median of 13 months (range 7–28 months). Consequently, the two most recent patients were treated with gross total tumor resection, primary focal radiotherapy and concomitant temozolomide followed by temozolomide for one year. Due to the high risk of leptomeningeal dissemination intraventricular chemotherapy was administered to all except one patient. Both patients who were treated with primary focal radiotherapy are in continuous complete remission 41 and 36 months after diagnosis. CONCLUSION Gross total resection followed by early focal radiotherapy, temozolomide, and intrathecal chemotherapy seem to be superior to intensive chemotherapy including high-dose chemotherapy.

Sleeping Beauty Insertional Mutagenesis Reveals Important Genetic Drivers of Central Nervous System Embryonal Tumors.

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Clinicopathologic Features of the Non-CNS Primary Ewing Sarcoma Family of Tumors in the Head and Neck Region.

Ewing sarcoma family of tumor (ESFT) is a group of malignant neoplasms that affect children and young adults. Primary ESFT does not commonly arise from the head and neck region. This study aimed to elucidate the clinicopathologic characteristics of ESFT of the head and neck region except for central nervous system primitive neuroectodermal tumors. Among the 207 cases of ESFT of the bone and soft tissue, diagnosed at Asan Medical Center during a 20-year period, 25 (12.1%) involved the head and neck region. Of those, 21 were available for histologic, immunohistochemical, and molecular studies. EWSR1 rearrangement was detected in 19 cases by reverse transcriptase-polymerase chain reaction and/or fluorescence in situ hybridization. Primary sites included the cranial area (6 cases, 31.6%), sinonasal tract (6 cases, 31.6%), paraspinal space (4 cases, 21.0%), and other spaces (3 cases, 15.8%). The 5-year overall survival and disease-free survival rates for all cases were 69.7% and 33.6%, respectively. A large tumor size (>5 cm) correlated significantly with overall survival (P=0.009), but not with disease-free survival (P=0.210). Microscopically, 8 cases (42.1%) showed nested growth pattern. Clear and/or eosinophilic cytoplasm was observed in 68.4% cases. Immunopositivity for CD99, Friend leukaemia integration-1 (FLI-1), CD57, and caveloin-1 were detected in 100%, 88.9%, 83.3%, and 50% cases, respectively. ESFT in the head and neck region had a favorable prognosis and frequent atypical and epithelioid features. An awareness of these histologic and immunophenotypic characteristics will improve the diagnostic accuracy for head and neck round cell malignancies.

Molecular Classification and Management of Rare Pediatric Embryonal Brain Tumors.

Malignant embryonal brain tumors (EBTs) of childhood span a wide clinical spectrum but can share remarkably similar morphologic features. This overlap presents significant diagnostic challenges, particularly for tumor entities that are rarely encountered in clinical practice and for which diagnostic criteria were poorly defined. This review will provide an update on the evolving characterization and treatment of rare EBTs. Rapid advances in genomic tools have led to the discovery of robust molecular markers, and identification of novel tumor types and subtypes for almost all major categories of pediatric brain tumors. These developments have had significant impact on improving the diagnostic classification of the rare EBTs, particularly for tumors with newly recognized C19MC alterations, central nervous system primitive neuroectodermal tumors (CNS-PNET), and pineoblastoma (PB). These important developments in the clinical and molecular understanding of rare EBTs are paving the way for novel therapeutic strategies and improved clinical management.

Abstract 2264: RNA sequencing based analysis of transposon-induced tumors reveals novel insights into cancer pathogenesis and progression

Abstract Current challenges in oncology include the discovery of drivers suitable for targeting using small molecules or antibodies and predictive animal models. We're addressing these challenges in several ways. We have developed mouse models using the Sleeping Beauty (SB) transposon system to perform unbiased, forward genetic screens to define strong candidate cancer genes. Further, our studies and others have utilized transposon mutagenesis to derive cancers from all three germ layers including carcinomas, sarcomas, neuroectodermal tumors, and hematopoietic malignancies. We hypothesize that data from these screens will help to identify genetic drivers of human cancer that are altered at the gene copy number or epigenetic levels. Our T2/Onc SB transposons create fusions with endogenous gene transcripts and RNA sequencing (RNA-seq) reveals the genes targeted in the tumor. From these results, correlations can be drawn between the alteration of specific genes and changes in tumor gene expression patterns. Thus, the goals of our recent research have been to discover novel associations between SB-induced tumor phenotypes and specific driver gene alterations (i.e. the tumor genotype). Moreover, RNA-seq has also revealed tumor molecular subtypes, in many cases with correlating transposon insertion mutations. Several examples will be described: mammary tumors, osteosarcoma (OS), medulloblastoma and central nervous system primitive neuro-ectodermal tumors. In each case, RNA-seq has revealed novel genotype-phenotype correlations including drivers of high cell cycle activity, metastasis, white blood cell exclusion from the tumor, SHH pathway activation, and hormone receptor signaling. These models provide a source of genetically heterogenous tumors with the same initiating mutation useful for identifying cooperating pathways and drivers of specific tumor phenotypes. Secondly, we are using SB transposon mutagenesis to understand the genetic basis of chemotherapy resistance in cancer including OS. In our OS model, SB mutagenesis is an ongoing process due to the constitutive expression of SB transposase, which permits the continued mobilization of transposons. We hypothesize that in vivo treatment with chemotherapy agents will allow tumor cells that contain transposon mutations in chemoresistance genes to persist. Preliminary studies with primary tumor cells derived from SB-enhanced OS tumors demonstrate resistance to combination chemotherapy treatment in vivo. Further analysis to identify candidate chemotherapy resistance genes are currently underway. Citation Format: Emily T. Camilleri, Pauli J. Beckmann, Jon D. Larson, Morito Kurata, Jingmin Shu, Emily Pope, Wendy A. Hudson, Nuri A. Temiz, Susan K. Rathe, Rebecca S. LaRue, Anne E. Sarver, Milcah C. Scott, Jyotika Varshney, Jaime F. Modiano, Branden S. Moriarity, Aaron L. Sarver, Somasekar Seshagiri, David A. Largaespada. RNA sequencing based analysis of transposon-induced tumors reveals novel insights into cancer pathogenesis and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2264.

Epigenetic regulators Rbbp4 and Hdac1 are overexpressed in a zebrafish model of RB1 embryonal brain tumor, and are required for neural progenitor survival and proliferation.

ABSTRACTIn this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells.This article has an associated First Person interview with the first author of the paper.

The case for DNA methylation based molecular profiling to improve diagnostic accuracy for central nervous system embryonal tumors (not otherwise specified) in adults

Central nervous system primitive neuro-ectodermal tumors (CNS-PNETs), have recently been re-classified in the most recent 2016 WHO Classification into a standby catch all category, “CNS Embryonal Tumor, not otherwise specified” (CNS embryonal tumor, NOS) based on epigenetic, biologic and histopathologic criteria. CNS embryonal tumors (NOS) are a rare, histologically and molecularly heterogeneous group of tumors that predominantly affect children, and occasionally adults. Diagnosis of this entity continues to be challenging and the ramifications of misdiagnosis of this aggressive class of brain tumors are significant. We report the case of a 45-year-old woman who was diagnosed with a central nervous system embryonal tumor (NOS) based on immunohistochemical analysis of the patient’s tumor at diagnosis. However, later genome-wide methylation profiling of the diagnostic tumor undertaken to guide treatment, revealed characteristics most consistent with IDH-mutant astrocytoma. DNA sequencing and immunohistochemistry confirmed the presence of IDH1 and ATRX mutations resulting in a revised diagnosis of high-grade small cell astrocytoma, and the implementation of a less aggressive treatment regime tailored more appropriately to the patient’s tumor type. This case highlights the inadequacy of histology alone for the diagnosis of brain tumours and the utility of methylation profiling and integrated genomic analysis for the diagnostic verification of adults with suspected CNS embryonal tumor (NOS), and is consistent with the increasing realization in the field that a combined diagnostic approach based on clinical, histopathological and molecular data is required to more accurately distinguish brain tumor subtypes and inform more effective therapy.

Diagnostic Advances Made for CNS-PNET

Abstract A recent study found that a majority of pediatric patients diagnosed with central nervous system primitive neuroectodermal tumors may have other types of central nervous system cancers instead. Researchers also concluded that these rare brain tumors fall into four distinct molecular subtypes. The findings may lead to more accurate diagnoses and more-tailored treatment plans.

Pediatric Primitive Neuroectodermal Tumors of the Central Nervous System Differentially Express Granzyme Inhibitors.

BackgroundCentral nervous system (CNS) primitive neuroectodermal tumors (PNETs) are malignant primary brain tumors that occur in young infants. Using current standard therapy, up to 80% of the children still dies from recurrent disease. Cellular immunotherapy might be key to improve overall survival. To achieve efficient killing of tumor cells, however, immunotherapy has to overcome cancer-associated strategies to evade the cytotoxic immune response. Whether CNS-PNETs can evade the immune response remains unknown.MethodsWe examined by immunohistochemistry the immune response and immune evasion strategies in pediatric CNS-PNETs.ResultsHere, we show that CD4+, CD8+, γδ-T-cells, and Tregs can infiltrate pediatric CNS-PNETs, although the activation status of cytotoxic cells is variable. Pediatric CNS-PNETs evade immune recognition by downregulating cell surface MHC-I and CD1d expression. Intriguingly, expression of SERPINB9, SERPINB1, and SERPINB4 is acquired during tumorigenesis in 29%, 29%, and 57% of the tumors, respectively.ConclusionWe show for the first time that brain tumors express direct granzyme inhibitors (serpins) as a potential mechanism to overcome cellular cytotoxicity, which may have consequences for cellular immunotherapy.