C19MC Amplification Research Articles

ETMR-16. PEDIATRIC SPINAL EMBRYONAL TUMORS; PATHOLOGICAL INSIGHTS INTO THIS RARE ENTITY

Abstract BACKGROUND Spinal cord is an extremely uncommon primary location for the CNS embryonal group of tumours. This study aims to collate the immunohistological spectrum of these rare spinal CNS embryonal tumours, diagnosed in the institute during the last 20 years. METHODS Of the total 446 spinal tumours, 15 were embryonal tumours. Of these, 5 were metastases, four from medulloblastoma and one from AT/RT (diagnosed subsequently) which were excluded and thus the study cohort comprised 10 cases. Eight were atypical teratoid/rhabdoid tumour (AT/RT; 80%), and 2 were embryonal tumour with multilayered rosettes (ETMR; 20%). RESULTS Six AT/RT cases were of ≤2 years age; while other two were 6 and 11 years of age with a M:F ratio of 1.3:1 (thoracic: 4; cervical: 2; lumbar: 2). Histologically, rhabdoid-like morphology (87.5%), necrosis (100%), and calcification (25%) were noted. All of them were of INI1 protein (SMARCB1)-deficient., all of them were negative for brachyury and all were variably positive for AE1/AE3. Two ETMR cases were of <2 years of age, females and at lumbar and cervical levels respectively. C19MC amplification by FISH was done for one case which was not amplified. Histologically, one of them showed a medulloepithelioma-like pattern; while the other showed multilayered rosettes and neuropil-like matrix. LIN28A strong positivity, retained nuclear staining for INI1 protein and negativity for AE1/AE3 was noted in both. Significant BCOR positivity was seen in one but was negative for SATB2. CONCLUSIONS Spinal embryonal tumours are extremely rare and metastases needs to be ruled out before considering as primary. AT/RT is the most common embryonal tumour in the spine, commonly SMARCB1(INI1)-deficient. ETMR can also encountered in the spinal location.

ETMR-05. DEVELOPMENT OF NOVEL PRECLINICAL MODELS AND THERAPEUTIC STRATEGIES FOR ETMR

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a very rare, aggressive pediatric brain tumor with a dismal 5-year overall survival rate. Few genetic aberrations in miRNA clusters and processing machinery have been detected in ETMRs with 90% of ETMR patients having the C19MC amplification. ETMRs show unique histopathological features which, together with the detection of C19MC or DICER alterations, are necessary for diagnosis. The cellular and molecular mechanisms underlying ETMR development are poorly understood, and targeted therapeutics have not been developed due to scarcity of preclinical models. To expand knowledge of ETMR biology and accelerate the design of novel and therapies, it is thus essential to develop preclinical models. METHODS We have generated two novel patient-derived ETMR cell lines from resected patient derived tumor samples (CTBP-03A and CBTP-139) and created three patient-derived xenograft (PDX) models (CTBP-03A, CBTP-139 and CBTP-38). We have used these unique patient-derived cell lines to identify mechanisms driving ETMR development and progression. To identify novel therapeutic targets, we conducted high-throughput drug screening (HTDS) utilizing 2480 approved and investigational drugs, against the CBTP-03A and CBTP-139 lines. RESULTS Among the candidate drugs showing an inhibitory effect on both lines, compounds were selected based on their activity, CNS penetration ability, mechanism of action, and bioavailability, if known. The top selected hits were screened in an all-versus-all format, totaling 1953 combinations. The rapidity of action and efficacy of the top choice compounds used as single agent or in double and triple combination was assessed by caspase glow and cell titer glow assays. This initial combination screen identified DNA replication stress, retinoid receptor agonist and FGF signaling as potential combinatorial targets. Data are currently being validated in vivo. CONCLUSIONS This is the first combinatorial HTDS on two novel patient-derived cell lines which highlighted potential drug combination strategies for the treatment of ETMR.

ATRT-11. DEVELOPMENT OF NOVEL PRECLINICAL MODELS AND THERAPEUTIC STRATEGIES FOR ETMR

Abstract Embryonal Tumor with Multilayered Rosettes (ETMR) is a very rare, aggressive pediatric brain tumor with a dismal 5-year overall survival rate. Few genetic aberrations in miRNA clusters and processing machinery have been detected in ETMRs with 90% of ETMR patients having the C19MC amplification. ETMRs show heterogenous histopathological features, including regions of abundant neuropil, hypercellular regions with multilayered rosettes, and papillary structures resembling primitive neuroectoderm. This unique histology, together with the detection of C19MC or DICER alterations, are necessary for diagnosis. The cellular and molecular mechanisms underlying ETMR development is poorly understood, and targeted therapeutics have not been developed due to scarcity of preclinical models. To expand knowledge of ETMR biology and accelerate the design of novel, it is thus essential to develop preclinical models. We have generated a novel patient-derived ETMR cell line from a resected patient tumor sample (CTBP-03A) and created two patient-derived xenograft (PDX) models (CTBP-03A and CBTP-38). We have used these unique patient-derived cell lines to identify mechanisms driving ETMR development and progression. To identify novel therapeutic targets, we conducted high-throughput drug screening (HTDS) utilizing 2480 approved and investigational drugs, against the CBTP-03A cell line. Among the candidate drugs showing an inhibitory effect on CBTP-03, 63 compounds were selected based on their activity, CNS penetration ability, mechanism of action, and bioavailability, if known. The 63 potency-selected hits were screened in an all-versus-all format, totaling 1953 combinations. The rapidity of action of the top choice compounds used as single agent or in double and triple combination was assessed by caspase glow assay. This initial combination screen identified DNA replication stress and FGF signaling as potential new drug combinations for ETMR. We will thus describe the first combinatorial HTDS on two novel ETMR patient-derived cell lines and potential drug combination strategies for the treatment of ETMR.

ATRT-19. RARE EMBRYONAL TUMORS INCLUDING EMBRYONAL TUMOR MULTILAYER ROSETTE, OUTCOME AND PATTERN OF TREATMENT FAILURE: MD ANDERSON CANCER CENTER EXPERIENCE

Abstract Rare embryonal tumors (excluding medulloblastoma and ATRT) are aggressive pediatric cancers that include embryonal tumor with multilayer rosettes (ETMR), embryonal tumor with abundant neuropil and true rosettes (ETANTR), and embryonal tumors not otherwise specified (ET-NOS). The presence of driver events like C19MC amplification, DICER1 mutations and other microRNA-related aberrations are vital in diagnostic classification of these tumors. The prognosis of these tumors is dismal despite intensive multimodal treatments with the 5-year overall survival being less than 30%. We retrospectively reviewed 12 patients with rare embryonal tumors who were treated at MD Anderson Cancer Center from 2010 to 2022. Of the 12 patients identified, the mean age at diagnosis was 3.6 years (range: 1-12 years) with no sex differences. Histologically, they were classified into ETMR (4/12), ETANTR (3/12) and ET-NOS (5/12) with C19MC found positive in 2/4 ETMR cases. All patients presented with large infiltrative tumors to the supratentorial (6/12), brainstem (4/12), and spinal (2/12) regions. The majority of patients (10/12) were treated by gross/subtotal resection. Treatment at diagnosis included surgery alone (3/12), surgery with chemotherapy (4/12), surgery with chemotherapy plus radiation (4/12),and chemotherapy plus radiation without surgery (1/12). Five patients reported with de-novo metastasis and 10/12 patients had disease progression at 4.1 months of median follow-up (range: 1.2-10.6 months). Six patients had local recurrence and three patients had local with distant disease at progression. Eight patients are still alive, with 5/8 of them surviving more than 5 years. In conclusion, these rare embryonal tumors are hard to diagnose and treat effectively. Even with successful surgery and intense chemotherapy, almost all of them progress/relapse but combined treatments seem to be effective in a subset of patients. Collaborative multi-institutional efforts are required to define standard treatment approaches and future patient stratification.

Embryonal tumors with multilayered rosettes, C19MC-altered or not elsewhere classified: Clinicopathological characteristics, prognostic factors, and outcomes of 17 children from 2018 to 2022.

Embryonal tumors with multilayered rosettes (ETMRs) are a histologically heterogeneous entity and gather embryonal tumors with abundant neuropil and true rosettes (ETANTRs), ependymoblastoma, and medulloepithelioma. ETMRs are highly aggressive and associated with poorer clinical courses. However, cases of this entity are rare, and advances in molecular genetics and therapy are minor. The purpose of our study was to retrospectively analyze the clinical, pathological features, and prognostic factors of ETMRs. Our cohort consisted of 17 patients diagnosed with ETMRs in our hospital from 2018 to 2022, and two of them were lost to follow-up. Clinical data were retrieved, and immunohistochemistry and genetic analyses were performed. Among 17 cases, 16 were ETANTRs, and one was medulloepithelioma. Morphologically, tumor cells of ETANTRs could transform into anaplasia and lose the biphasic architecture during tumor progression. Immunohistochemistry of LIN28A revealed positive expression in 17 cases, and the expression of LIN28A was more intense and diffuse in the recurrent lesions than in primaries. The increased N-MYC copy numbers were detected in the primary tumor and recurrence of patient 8. Moreover, the incidence of metastatic disease was 100% in patients aged > 4 years and 18% in the younger group. For patients receiving chemotherapy, the median overall survival time was 7.4 months, while that of those who didn't receive it was 1.2 months. Nevertheless, surgical approaches, radiotherapy, age at presentation, gender, tumor location, and metastatic status were not associated with independent prognosis. ETANTR might not present as the typical morphologies during tumor progression, so analyses of C19MC amplification and Lin28A antibody are indispensable for diagnosing ETMRs accurately. Children aged > 4 years tend to have a higher rate of metastasis in ETMRs. Chemotherapy is the only prognostic factor for ETMRs patients with a favorable prognosis. The biological nature and clinical patterns for recurrent diseases need to be further demonstrated to predict prognosis and guide treatment.

Germ cell tumors with neuroglial differentiation do not show molecular features akin to their central nervous system counterpart: experience from extra-gynecological sites.

Teratomas with secondary somatic malignancy showing neuroglial differentiation (central nervous system (CNS)-type tumors) arising from a glial or neuroepithelial component is a very uncommon event and primarily described in the ovary. We aimed to describe the morphological spectrum and molecular features of CNS type of neuroepithelial tumors arising from the germ cell tumors (GCT) in the extra-gynecological sites. All cases of teratoma and mixed GCT arising from the non-gynecological sites over 7years were screened for CNS type of neuroepithelial tumors. Detailed histological and immunohistochemical analysis was performed. IDH1 and 2 sequencings were performed in the glial tumors. Fluorescent in situ hybridization (FISH) was performed for EWSR1 rearrangement, 19/19q co-deletion, CDKN2A homozygous deletion, EGFR amplification, and C19MC amplification, wherever required. Out of 302 GCTs examined, the neuroglial tumor was detected in 15 cases. It included nine cases of glial tumors (including one pilocytic astrocytoma (grade I), two diffuse astrocytomas (grade II), one oligodendroglioma (grade II), one gemistocytic astrocytoma (grade II), three anaplastic astrocytomas (grade III), and one case of glioblastoma (grade IV)) and six cases of the embryonal tumor with multilayered rosettes (ETMR). None of the gliomas showed IDH mutation by immunohistochemistry or sequencing. The ETMR cases did not show Lin28 expression or C19MC amplification. To conclude, the spectrum of neuroglial tumors arising from teratoma in the extragonadal sites is vast and most commonly includes glial neoplasms and embryonal tumors. Our findings indicate that the genotype and pathogenesis of tumors with neuroglial differentiation in teratoma are distinct from their CNS counterpart.

ETMR-13. Embryonal Tumor with Multilayered Rosettes in an Infant: Case Report

Embryonal tumor with multilayered rosettes (ETMR) is a highly malignant tumor (WHO grade 4) seen predominantly in infants. It includes morphologically distinct embryonal tumors namely, embryonal tumor with abundant neuropil and true rosettes, ependymoblastoma, and medulloepithelioma. The presence of multilayered rosettes and C19MC amplification at chromosome 19q13.42 confirms the diagnosis. The median overall survival is less than a year and the prognosis is generally poor. We report the case of a 1-year-old girl who presented with vomiting, lethargy, and increasing head circumference over a period of six months. On admission, she was drowsy and irritable. Verbal output was limited to moans and motor response was localizing. She was macrocephalic with a head circumference of 51 cm. MRI showed a large 5 x 5 x 6.5cm contrast-enhancing cerebellar vermian tumor with obstructive hydrocephalus. There was no evidence of leptomeningeal disease or spinal metastasis at this time. She underwent a right frontal ventriculoperitoneal shunt insertion, followed by suboccipital craniotomy and subtotal tumor resection one week later. Her shunt was ligated two days after tumor excision, due to development of bilateral subdural hygromas. The patient regained full consciousness, but still had spastic lower extremities and inability to swallow at the time of discharge. Histopathology and immunostains were consistent with an embryonal tumor, possibly ETMR, and the patient was for advised chemotherapy. Before initation of chemotherapy, the patient was admitted in another instution because of alteration in sensorium. Repeat imaging showed progression of the patient’s subdural hygromas, requiring insertion of a subduroperitoneal shunt. The patient died seven weeks after tumor resection due to progression of her tumor residual. Management options for ETMR are limited, especially in low- and middle-income countries. International linkages may help facilitate the accurate diagnosis and early treatment of these patients with rare but aggressive brain tumors.

RARE-06. Expanding the clinical and molecular spectrum of pituitary blastoma

Pituitary blastomas (PitB) are rare DICER1-associated tumors that occur exclusively in children < 2 years of age. They are locally aggressive tumors that are driven by a combination of germline and somatic alterations involving DICER1. Here, we report two patients with pituitary neoplasms that expand the clinical and molecular spectrum of PitB. Patient 1 presented at 10 months with diabetes insipidus and was initially diagnosed with pituitary ependymoblastoma. She received debulking, chemotherapy and focal radiation with complete response achieved, but unfortunately died at the age of 8 years due to cerebral edema. Patient 2 was a survivor of infant leukemia who was treated with chemotherapy and then further chemotherapy with cranial irradiation at relapse. The patient was then diagnosed at the age of 8 years with pituitary CNS-PNET, which was treated with craniospinal irradiation and chemotherapy, and had remained in remission for 6 years. Review of histology in both cases indicates presence of neuroendocrine lobules, primitive cells, and Rathke pouch-like glandular structures, with high Ki67, ACTH and PRAME positivity compatible with PitB. Next-generation sequencing revealed presence of two DICER1 mutations (germline frameshifting and somatic missense) in Patient 1, and one somatic missense DICER1 mutation plus loss of heterozygosity in Patient 2 (no germline alteration). Surprisingly, C19MC amplification was also detected in Patient 1. Methylation profiling confirms clustering among our samples and PitB references, but not ETMR references. MicroRNA array revealed decrease in mature microRNA expression and preferential down-regulation of 5p/3p species in tumor compared to control pituitary tissue. In all, PitBs may present with clinical and molecular characteristics not conforming with the classical descriptions. It might be prudent to consider sequencing for DICER1 alteration in pediatric pituitary tumors to facilitate diagnosis of this increasingly heterogeneous rare entity.

ETMR-04. EMBRYONAL TUMOR WITH MULTILAYERED ROSETTES: THE MD ANDERSON CANCER CENTER EXPERIENCE

BACKGROUNDEmbryonal Tumor with Multilayered Rosettes (ETMR) are rare tumors that are molecularly diagnosed by C19MC amplification. Rarity of this tumor has precluded profiling uniform therapeutic strategy.METHODSRetrospective review after institutional approval, identified 10 pediatric case of ETMR, treated at MD Anderson Cancer Center during the period of 2005 to 2019.RESULTSMedian age of at diagnosis was 4.6 years. Tumor sites include frontal or parietal lobes (3), spine (3) and posterior fossa involving the brainstem (4). All patients received a combination of chemotherapy and radiation. 4 patients had metastasis at the presentation. 9 patients received focal radiation but only 6 of them received Craniospinal irradiation (CSI). Average dose of radiation was 50 Gy. Surgical resection was performed in all cases except the brainstem tumors. 7 children had recurrence including all the patients with metastasis at diagnosis (median time: 9.4 months), 1 passed away secondary to hemorrhage in brainstem and data was not available for 2 patients. 5/6 patients who received CSI had recurrence.CONCLUSIONSTo-date no well-defined treatment regimens exists for these neoplasms, resulting in poor overall survival. Preclinical drug screen have shown the efficacy of topotecan, actinomycin D, and volasertib as potential new therapeutic candidates, though this has not translated successfully into the clinical arena. Given the limited success with current conventional therapeutic methods, molecular interrogation in addition to histopathological diagnosis are essential upfront, as it could provide clues to targeted therapy. Defining molecularly-based treatment with less toxicities and increased survival are warranted.

ETMR-18. TARGETING Lin28 IN ETMR WITH ODC1 INHIBITOR DFMO

Embryonal tumor with multilayered rosettes (ETMR), is an aggressive brain tumor primarily occurring in young patients (<4 years of age) and characterized by C19MC amplification and Lin28 overexpression. These genetic hallmarks have been shown to participate in driving ETMR in a C19MC-Lin28-MYCN circuit. Reducing Lin28 disrupts this circuit and reduces cell viability in ETMR models. Investigation of therapeutic agents targeting this pathway is required to provide new treatment options for this deadly disease. We present data showing the effect of DFMO (α-difluoromethylornithine) in ETMR, an ODC1 inhibitor known to reduce Lin28 in neuroblastoma. DFMO treatment of the ETMR cell line BT-183 resulted in a significant reduction of intracellular Lin28 protein levels (P<0.05) as indicated by flow cytometry. In concert with this reduction in Lin28, there was a as significant reduction in viable cells (P<0.05), and the number of CD133+ cells were reduced 2-fold (P<0.05). High throughput drug testing of BT-183 identified a number of additional therapeutic agents with potential therapeutic efficacy for ETMR and combining these with cytostatic agent DFMO demonstrated the potential use of these drugs in combination. These in vitro data were complemented by testing of DFMO in an in vivo stereotaxic xenograft ETMR model, with inhibition of tumor burden monitored by bioluminescent imaging of the tumors. Together this work shows that Lin28 targeting agents such as DFMO merit further examination and integrating these types of agents into treatment strategies for ETMR may lead to better outcomes.

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.

Embryonal tumors with multi-layered rosettes: a disease of dysregulated miRNAs.

ETMRs are highly lethal, pediatric embryonal brain tumors, previously classified as various histologic diagnoses including supratentorial primitive neuroectodermal tumors (sPNET) and CNS PNET. With recognition that these tumors harbor recurrent amplification of a novel oncogenic miRNA cluster on chr19, C19MC, ETMRs were designated as a distinct biological and molecular entity with a spectrum of histologic and clinical manifestations. We reviewed published literature describing clinical presentation, the genetic and epigenetic drivers of oncogenesis, and recent therapeutic strategies adopted to combat these aggressive tumors. As a consequence of C19MC amplification, ETMRs upregulate several oncogenic and pluripotency proteins, including LIN28A, DNMT3B and MYCN, that confer a unique epigenetic signature reminiscent of nascent embryonic stem cells. In this review, we focus on the dysregulation of miRNAs in ETMR, the major pathogenic mechanism identified in this disease. Despite the use of multi-modal therapeutic regimens, ETMR patients have dismal survival. Understanding the unique biology of these tumors has provided new insights towards novel therapeutic targets.

Abstract A39: Molecular characterization of ETMRs reveals role for R-loop mediated genomic instability and new treatment options

Abstract Introduction: Embryonal tumors with multilayered rosettes (ETMRs) are aggressive brain tumors that occur mainly in infants. Patients face a very poor prognosis with a median overall survival of ~12 months after diagnosis. The tumors harbor in ~90% of all cases amplification of a miRNA cluster on chromosome 19 (C19MC) that is thought to be the driver of the disease. However, current treatment options are lacking as (a) the mechanisms downstream of C19MC are poorly understood and (b) the drivers in cases lacking the C19MC aberration are unknown. To develop better treatment protocols for ETMR patients, more insight is needed in what is driving these tumors and how that can be targeted. Materials and Methods: To investigate the genomic and epigenomic landscape of ETMR in depth, we collected 193 ETMR samples and 23 matched relapses and performed DNA methylation profiling on all and DNA (whole genome, whole exome, and panel) sequencing and mRNA and miRNA transcriptome analysis on a subset of them. The BT183 ETMR cell line was used for drug treatments. Results: Among the 22 tumors without C19MC amplification, we identified 8 cases with truncating DICER1 germline mutations in one allele and somatic missense mutations in the RNASE III domain in the other allele. No DICER1 mutations were identified in C19MC amplified cases. In addition, structural variations (SVs) affecting C19MC were found in 3 other C19MC nonamplified cases and amplification of another miRNA cluster, miR-17-92, in 2 other cases. However, despite the presence of different genetic aberrations, based on DNA methylation and transcriptome profiling no molecular subgrouping was observed within our cohort. Whole-genome sequencing revealed an overall low recurrence and conservation of SNVs but strong conservation of SVs from primary tumors to relapses, especially surrounding C19MC. Moreover, many newly acquired SNVs in the relapses are associated to a new cisplatin treatment-related mutational signature. SVs detected in ETMRs significantly colocalized with R-loops, structures that form upon a collision of replication and transcription and are associated to increased levels of chromosomal instability, which is frequently observed in ETMRs. Using a DICER1 KO model, we found that global deregulation of miRNAs led to increased levels of R-loops and R-loop associated chromosomal instability. Finally, we show that a combination of topoisomerase and PARP inhibitors is highly synergistic and strongly increased the levels of both R-loops and DNA damage in ETMR cells and effectively killed the cells. Conclusions: Our results show that genomically instable ETMR cells are vulnerable to further increases in chromosomal instability, knowledge that may lead to new treatment strategies for ETMR patients and possibly other cancers with high levels of R-loops. Citation Format: Sander Lambo, Susanne Grübner, Tobias Rausch, Sebastian Waszak, Christin Schmidt, Sonja Krausert, Loreen Weichert, Aparna Gorthi, Carolina Romero, Annie Huang, Julia Schueler, Jan Korbel, Alexander Bishop, Stefan Pfister, Andrey Korshunov, Marcel Kool. Molecular characterization of ETMRs reveals role for R-loop mediated genomic instability and new treatment options [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A39.

Abstract 3172: Targeting genomic instability in embryonal tumors with multilayered rosettes (ETMR)

Abstract Embryonal Tumors with Multilayered Rosettes (ETMRs) are pediatric brain tumors mainly occurring in infants. Characteristic to ETMRs is the highly recurrent (~90%) amplification of the C19MC miRNA cluster fused to TTYH1 that drives the expression of this cluster. As the overall survival of these patients is very poor, there is an urgent need for a better understanding of these tumors that may lead to other treatment strategies. Whole genome and panel sequencing data have been generated for 60 ETMRs and matching germline when available. Data have been complemented with DNA methylation profiling and m(i)RNA sequencing data. Our results show that ETMR is a single disease entity without molecular subgroups. ETMRs lacking the C19MC amplification (~10%) are highly similar to tumors with C19MC amplification, based on methylation and m(i)RNA profiling, indicating that they do not represent a distinct subgroup. Germline sequencing revealed mutations in genes involved in DNA repair or miRNA processing, while tumor specific mutations included genes involved in the TP53-, SHH-, WNT-, or miRNA processing pathways. These pathways are also highly upregulated compared to other pediatric brain tumors. Mutations in DNA repair, miRNA processing, structural variations (SVs) and mutations in close proximity of SVs occur at high allele frequencies and are conserved in recurrent tumors while many other SNVs are lost. These data suggest that C19MC amplification/fusion, miRNA processing and DNA repair defects are the early (driving) events in tumor formation while aberrations involving for instance the SHH and WNT pathways are later (passenger) events. Aside from frequent and recurrent copy number changes, ETMRs show pluriploidy, complex rearrangements and strong presence of R-loops suggesting that ETMR genomes are highly unstable. We identified a high number of R-loops in the region forming the C19MC aberration and an enrichment of breakpoints in other R-loop forming regions. This may suggest a role for R-loops in both tumor progression and initiation. Finally, we tested whether further inducing the number of R-loops in these tumors may increase replication stress and cell death. Indeed, topoisomerase inhibition coupled to PARP inhibition increased the amount of R-loops and acted synergistically in killing ETMR cells. These data show that targeting the genomic instability in ETMRs could be a viable treatment option for treating ETMR patients. Citation Format: Sander Lambo, Andrey Korshunov, Christin Schmidt, Carolina Romero, Aparna Gorthi, Sonja Krausert, Tobias Rausch, Susanne Gröbner, Sebastian Brabetz, Sebastian Waszak, Alexander J. Bishop, Stefan Pfister, Marcel Kool. Targeting genomic instability in embryonal tumors with multilayered rosettes (ETMR) [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 3172.

Practice-Changing Abstracts From the 2016 Society for Neuro-Oncology Annual Scientific Meeting.

The most relevant practice-changing presentations at the 2016 Society for Neuro-Oncology (SNO) Annual Scientific Meeting revolved around the topic of the new 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors. The most notable change in this new classification is the introduction of molecular markers into the morphologic classification of diffuse gliomas (isocitrate dehydrogenase [IDH] mutation, 1p19q codeletion, and H3K27M mutation), ependymomas (RELA fusion), medulloblastomas (WNT- and sonic hedgehog-activated), and other embryonal tumors (C19MC amplification), thus allowing for more precise diagnosis of these entities compared with the use of morphologic features alone. Among the clinical trials presented, only one phase III trial evaluating a device therapy for treatment of newly diagnosed glioblastoma (EF14; tumor-treating fields) met prespecified statistical criteria for success, showing a modest benefit in progression-free survival and overall survival in patients without progression after radiation and concurrent temozolomide. Other topics of interest included the spatial and temporal heterogeneity of primary brain tumors and the prevalence of burnout among neuro-oncologists.

Practice-Changing Abstracts From the 2016 Society for Neuro-Oncology Annual Scientific Meeting

The most relevant practice-changing presentations at the 2016 Society for Neuro-Oncology (SNO) Annual Scientific Meeting revolved around the topic of the new 2016 World Health Organization (WHO) classification of central nervous system (CNS) tumors. The most notable change in this new classification is the introduction of molecular markers into the morphologic classification of diffuse gliomas (isocitrate dehydrogenase [IDH] mutation, 1p19q codeletion, and H3K27M mutation), ependymomas (RELA fusion), medulloblastomas (WNT- and sonic hedgehog-activated), and other embryonal tumors (C19MC amplification), thus allowing for more precise diagnosis of these entities compared with the use of morphologic features alone. Among the clinical trials presented, only one phase III trial evaluating a device therapy for treatment of newly diagnosed glioblastoma (EF14; tumor-treating fields) met prespecified statistical criteria for success, showing a modest benefit in progression-free survival and overall survival in patients without progression after radiation and concurrent temozolomide. Other topics of interest included the spatial and temporal heterogeneity of primary brain tumors and the prevalence of burnout among neuro-oncologists.

Tissue Factor Regulation by miR-520g in Primitive Neuronal Brain Tumor Cells: A Possible Link between Oncomirs and the Vascular Tumor Microenvironment

Pediatric embryonal brain tumors with multilayered rosettes demonstrate a unique oncogenic amplification of the chromosome 19 miRNA cluster, C19MC. Because oncogenic lesions often cause deregulation of vascular effectors, including procoagulant tissue factor (TF), this study explores whether there is a link between C19MC oncogenic miRNAs (oncomirs) and the coagulant properties of cancer cells, a question previously not studied. In a pediatric embryonal brain tumor tissue microarray, we observed an association between C19MC amplification and reduced fibrin content and TF expression, indicative of reduced procoagulant activity. In medulloblastoma cell lines (DAOY and UW228) engineered to express miR-520g, a biologically active constituent of the C19MC cluster, we observed reduced TF expression, procoagulant and TF signaling activities (responses to factor VIIa stimulation), and diminished TF emission as cargo of extracellular vesicles. Antimir and luciferase reporter assays revealed a specific and direct effect of miR-520g on the TF 3' untranslated region. Although the endogenous MIR520G locus is methylated in differentiated cells, exposure of DAOY cells to 5-aza-2'-deoxycytidine or their growth as stem cell-like spheres up-regulated endogenous miR-520g with a coincident reduction in TF expression. We propose that the properties of tumors harboring oncomirs may include unique alterations of the vascular microenvironment, including deregulation of TF, with a possible impact on the biology, therapy, and hemostatic adverse effects of both disease progression and treatment.

OT-10 * IMPLEMENTATION OF NEXT GENERATION COPY NUMBER AND MUTATIONAL ANALYSIS IN ROUTINE NEUROPATHOLOGY: MOLECULAR INVERSION PROFILING IS A HELPFUL TOOL IN DIFFERENTIAL DIAGNOSTICS AND PROGNOSTIFICATION OF PEDIATRIC BRAIN TUMORS

Brain tumor entities are characterized by specific copy number alterations (CNA), allelic losses/disbalances and recurrend mutations. Emerging technologies including SNP arrays, whole exome and whole genome sequencing are not suitable for degraded DNA derived from formalin-fixed, paraffin embedded (FFPE) samples. The aim of our study was to analyse the sensitivity and robustness of molecular inversion profiling (MIP) as a tool to identify these alterations in brain tumors and to compare this method to FISH and multiplex ligation probe analysis (MLPA). Genomic DNA extracted from up to 20 years old FFPE materials from more than 1300 brain tumors covering most WHO entities were analyzed by MIP profiling (Oncoscan V2/V3, Affymetrix). MIP revealed genome-wide copy number information from as little as 20 ng of degraded DNA; drop-out rate was <5%. In contrast to FISH and MLPA, MIP allowed a genome-wide analysis, adding significant information to the differential diagnosis. Characteristic CNA were detected, including BRAF duplications in pilocytic astrocytomas, chromosome 22 loss in ATRT, chromosome 10 loss, 7 gain and amplifications of PDGFR or EGFR in glioblastoma, 1p19q codeletion in oligodendroglial tumors, chromosome 2 gain and C19MC amplification in ependymoblastoma. Novel prognostic markers including MYCN amplification in CNS-PNET were identified. Established prognostic markers including MYC/MYCN amplifications in medulloblastomas and chromosome 1q gain in ependymomas were easily detected and validated by orthogonal methods. Extended chromosomal alterations including widespread hyperploidy in plexus papillomas or complex alterations such as chromosomal scattering of whole chromosomes (chromotripsis) in TP53 deficient medulloblastomas were easily uncovered. MIP also detected copy-neutral LOH and recurrent tumor-associated mutations (e.g BRAFV600E, IDH gene mutations). Our data indicate that MIP is a sensitive and robust method to assess CNA, allelic imbalances/losses and specific recurrent point mutations in FFPE tumor material, and therefore represents a novel tool for precise neuropathological tumor diagnostics and prognostic stratification.

GE-25 * NEXT GENERATION COPY NUMBER ANALYSIS BY MOLECULAR INVERSION PROFILING - A HELPFUL TOOL IN DIAGNOSTICS AND PROGNOSTIFICATION OF TUMORS OF THE CNS

Brain tumor entities are characterized by specific copy number alterations (CNA). Emerging technologies including SNP arrays, whole exome and whole genome sequencing are not suitable for degraded DNA derived from formalin-fixed, paraffin embedded (FFPE) samples. The aim of our study was to analyse the sensitivity and robustness of molecular inversion profiling (MIP) as a tool to identify CNA in brain tumor diagnostics and to compare this method to FISH and multiplex ligation probe analysis (MLPA). Genomic DNA was extracted from up to 20 years old FFPE materials and analyzed by MIP profiling (Oncoscan V2, Affymetrix) with more than 330,000 copy number probes. More than 1100 brain tumors covering most WHO entities were analysed. MIP revealed genome-wide copy number information from as little as 20 ng of degraded DNA; the drop-out rate was lower than 5%. In contrast to FISH and MLPA which enabled the analysis of only single or few genomic loci, MIP allowed a genome-wide analysis. It added significant information to the differential diagnosis as characteristic CNA were detected even with low input DNA, including BRAF duplications in pilocytic astrocytomas, chromosome 22 loss in ATRT, chromosome 10 loss and EGFR amplification in glioblastoma, 1p19q codeletion in oligodendroglial tumors, and chromosome 2 gain, C19MC amplification in ependymoblastoma. Prognostic markers including MYCC and MYCN copy number status in medulloblastomas and chromosome 1q gain in ependymomas were easily detected and validated by orthogonal methods. MIP also detected copy-neutral LOH and tumor-associated point mutations. Our data indicate that MIP is a sensitive and robust method to assess CNA in archival FFPE tumor material.

CNS-PNETs with C19MC amplification and/or LIN28 expression comprise a distinct histogenetic diagnostic and therapeutic entity.

Amplification of the C19MC oncogenic miRNA cluster and high LIN28 expression has been linked to a distinctly aggressive group of cerebral CNS-PNETs (group 1 CNS-PNETs) arising in young children. In this study, we sought to evaluate the diagnostic specificity of C19MC and LIN28, and the clinical and biological spectra of C19MC amplified and/or LIN28+ CNS-PNETs. We interrogated 450 pediatric brain tumors using FISH and IHC analyses and demonstrate that C19MC alteration is restricted to a sub-group of CNS-PNETs with high LIN28 expression; however, LIN28 immunopositivity was not exclusive to CNS-PNETs but was also detected in a proportion of other malignant pediatric brain tumors including rhabdoid brain tumors and malignant gliomas. C19MC amplified/LIN28+ group 1 CNS-PNETs arose predominantly in children <4 years old; a majority arose in the cerebrum but 24 % (13/54) of tumors had extra-cerebral origins. Notably, group 1 CNS-PNETs encompassed several histologic classes including embryonal tumor with abundant neuropil and true rosettes (ETANTR), medulloepithelioma, ependymoblastoma and CNS-PNETs with variable differentiation. Strikingly, gene expression and methylation profiling analyses revealed a common molecular signature enriched for primitive neural features, high LIN28/LIN28B and DNMT3B expression for all group 1 CNS-PNETs regardless of location or tumor histology. Our collective findings suggest that current known histologic categories of CNS-PNETs which include ETANTRs, medulloepitheliomas, ependymoblastomas in various CNS locations, comprise a common molecular and diagnostic entity and identify inhibitors of the LIN28/let7/PI3K/mTOR axis and DNMT3B as promising therapeutics for this distinct histogenetic entity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1291-1) contains supplementary material, which is available to authorized users.