A retrospective analysis of surgical resection of large ear keloids

Australasian Journal of Dermatology, EarlyView.

Multinuclear and Ground Glass-Formed Cells Detected in the Peritoneal Dialysate

(See pages 312–3 for the Answer to the Photo Quiz.)

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Multinuclear and Ground Glass-Formed Cells Detected in the Peritoneal Dialysate

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In the Literature

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Copy number profiling across glioblastoma populations has implications for clinical trial design

Abstract

Background

Copy number alterations form prognostic molecular subtypes of glioblastoma with clear differences in median overall survival. In this study, we leverage molecular data from several glioblastoma cohorts to define the distribution of copy number subtypes across random cohorts as well as cohorts with selection biases for patients with inherently better outcome.

Methods

Copy number subtype frequency was established for four glioblastoma patient cohorts. Two randomly selected cohorts include The Cancer Genome Atlas (TCGA) and German Glioma Network (GGN). Two more selective cohorts include the phase II trial ARTE in elderly patients with newly diagnosed glioblastoma and a multi-institutional cohort focused on paired resected initial/recurrence glioblastoma. The paired initial/recurrence cohort also had exome data available, which allowed for evaluation of multidimensional scaling analysis.

Results

Smaller selective glioblastoma cohorts are enriched for copy number subtypes that are associated with better survival, reflecting the selection of patients who do well enough to enter a clinical trial or who are deemed well enough to undergo resection at recurrence. Adding exome data to copy number data provides additional data reflective of outcome.

Conclusions

The overall outcome for diffuse glioma patients is predicted by DNA structure at initial tumor resection. Molecular signature shifts across glioblastoma populations reflect the inherent bias of patient selection towards longer survival in clinical trials. Therefore it may be important to include molecular profiling, including copy number, when enrolling patients for clinical trials in order to balance arms and extrapolate relevance to the general glioblastoma population.

Differences of Cd uptake and expression of MT family genes and NRAMP2 in two varieties of ryegrasses

Abstract

In order to understand the mechanism of the difference of Cd absorption and Cd enrichment in different ryegrass varieties, pot experiment was conducted to study on the response of two varieties of ryegrass (Bond and Abbott) to Cd stress as well as the differences of Cd uptake and expression of MT family genes and NRAMP2. Results showed that root dry weights of two varieties and shoot dry weights of Abbott increased first and then decreased with the increase of Cd level in soil. When exposed to 75 mg kg⁻¹ Cd, shoot dry weight and plant dry weight of Abbott both reached maximum values (10.92 and 12.03 g pot⁻¹), which increased by 11.09 and 10.67% compared with the control, respectively. Shoot dry weight and plant dry weight of Bond decreased with the increase of Cd level in soil. When the Cd level in soil was 75 mg kg⁻¹, shoot Cd concentrations of the two varieties were 111.19 mg kg⁻¹ (Bond) and 133.69 mg kg⁻¹ (Abbott), respectively, both of which exceeded the critical value of Cd hyperaccumulator (100 mg kg⁻¹). The expression of MT gene family and NRAMP2 in the leaf of Bond variety significantly increased at the Cd level of 75 mg kg⁻¹ and reached maximum value (except MT2C) at Cd level of 150 mg kg⁻¹. The expression of MT gene family in the stem of Bond variety showed a double-peak pattern, while the expression of NRAMP2 was a single-peak pattern. The expression of MT gene family and NRAMP2 in Abbott variety was consistent with single-peak pattern. The expression of MT gene family and NRAMP2 in leaf both significantly increased at Cd level of 150 mg kg⁻¹, while that in stem and root significantly increased at Cd level of 75 mg kg⁻¹. For both varieties of ryegrass, the expression amount of MT family genes and Nramp2 in leaf was higher than that in root and stem, indicating the Cd tolerance of ryegrass can be improved by increasing the expression levels of MT family genes and Nramp2 in stem and root. There was significant genotypic difference in the expression of MT gene family and NRAMP2 between the two varieties of ryegrass, and the expression of MT gene family and NRAMP2 in leaves and stems of Bond variety was higher than that in Abbott variety, while the expression of MT gene family and NRAMP2 in roots of Abbott variety was higher than that in Bond variety. The two gene families investigated in this study may be closely related to Cd uptake, but not related to Cd transport from root to leaf and Cd enrichment in shoot.

Drug-induced Anaphylaxis Documented in Electronic Health Records

Publication date: Available online 30 June 2018
Source:The Journal of Allergy and Clinical Immunology: In Practice
Author(s): Neil Dhopeshwarkar, Aziz Sheikh, Raymond Doan, Maxim Topaz, David W. Bates, Kimberly G. Blumenthal, Li Zhou
BackgroundAlthough drugs represent a common cause of anaphylaxis, few large studies of drug-induced anaphylaxis have been performed.ObjectiveTo describe the epidemiology and validity of reported drug-induced anaphylaxis in the electronic health records (EHRs) of a large United States healthcare system.MethodsUsing EHR drug allergy data from 1995-2013, we determined the population prevalence of anaphylaxis including anaphylaxis prevalences over time, and the most commonly implicated drugs/drug classes reported to cause anaphylaxis. Patient risk factors for drug-induced anaphylaxis were assessed using a logistic regression model. Serum tryptase and allergist visits were used to assess the validity and follow up of EHR-reported anaphylaxis.ResultsAmong 1,756,481 patients, 19,836 (1.1%) reported drug-induced anaphylaxis: Penicillins (45.9 per 10,000), sulfonamide antibiotics (15.1 per 10,000), and nonsteroidal anti-inflammatory drugs (NSAIDs) (13.0 per 10,000) were most commonly implicated. Patients with white race (odds ratio [OR] 2.38, 95% CI 2.27-2.49), female sex (OR 2.20, 95% CI 2.13-2.28), systemic mastocytosis (OR 4.60, 95% CI 2.66-7.94), Sjögren's syndrome (OR 1.94, 95% CI 1.47-2.56), and asthma (OR 1.50, 95% CI 1.43-1.59) had an increased odds of drug-induced anaphylaxis. Serum tryptase was performed in 135 (<1%) anaphylaxis cases and 1,587 patients (8.0%) saw an allergist for follow-up.ConclusionEHR-reported anaphylaxis occurred in approximately 1% of patients, most commonly from penicillins, sulfonamide antibiotics, and NSAIDs. Females, whites, and patients with mastocytosis, Sjögren's syndrome, and asthma had increased odds of reporting drug-induced anaphylaxis. The low observed frequency of tryptase testing and specialist evaluation emphasize the importance of educating providers on anaphylaxis management.

LTBK-01. UPDATES ON THE PHASE II AND RE-TREATMENT STUDY OF AZD6244 (SELUMETINIB) FOR CHILDREN WITH RECURRENT OR REFRACTORY PEDIATRIC LOW GRADE GLIOMA: A PEDIATRIC BRAIN TUMOR CONSORTIUM (PBTC) STUDY

Abstract

The PBTC is conducting a phase II study (NCT01089101) evaluating selumetinib (AZD6244, ARRY-142886), a MEK I/II inhibitor, in children with recurrent/refractory LGG assigned to 6 strata. We present the updated data on Stratum 2 and 5. Also, data on subsequent progression after treatment completion in patients enrolled on Stratum 1 and 3 will be discussed. Finally, we present details on the re-treatment study (PBTC-029C). Both stratum 2 (pilocytic astrocytoma [PA] without common BRAF aberrations) and Stratum 5 (non-pilocytic LGG with BRAF aberrations) met response criteria for expansion (> 2 objective responses in 16 patients), and accrual to a total of 25 patients on each stratum is ongoing. Among 50 patients treated on Stratum 1 (PA with BRAF aberrations) or Stratum 3 (NF-associated LGG), 21 have progressed. Thirteen of 21 have progressed after stopping therapy. The median time to progression for these 13 patients is 119 days (10–928). The re-treatment study has enrolled 25 patients who received a median of 12 re-treatment courses (2–36). The most common attributable toxicities after re-treatment were grade 1 CPK elevation (44%), diarrhea (44%), hypoalbuminemia (40%), elevated AST (36%), rash (36%) and fatigue (32%). The most common grade 3/4 attributable toxicities were grade 3 paronychia (8%), CPK elevation (4%), AST elevation (4%), decreased ejection fraction (4%), neutropenia (4%), elevated triglycerides (4%), peripheral neuropathy (4%) and grade 4 CPK elevation (4%). There is not a significant difference between the toxicities observed during original therapy versus re-treatment. The most current response and patient demographic data will be presented.

CRAN-34. TRANSCRIPTOMIC AND PROTEOMIC COMPARISON OF PEDIATRIC AND ADULT ADAMANTINOMATOUS CRANIOPHARYNGIOMA

Abstract

Adamantinomatous craniopharyngioma (ACP) is a biologically benign but clinically aggressive lesion that has a significant impact on quality of life. The incidence of the disease has a bimodal distribution with peaks occurring in children and adults. Our group previously published the results of a transcriptome analysis of pediatric ACPs that identified several genes that were consistently overexpressed relative to other brain tumors. We now present the results of a transcriptome analysis of both pediatric and adult ACP to identify biological differences between these groups that may provide novel therapeutic insights, or support the assertion that potential therapies identified through the study of pediatric ACP may also have a role in adult ACP. Following bulk RNA sequencing, we explored the differential expression of adult ACP versus pediatric ACPs via geneset enrichment analysis. Preliminary studies identified a decreased cytokine gene expression profile in adult ACP that may correspond to a reduced senescence-associated secretory phenotype (SASP), which has recently been described in pediatric ACP. Additionally, our results corroborate our previous work demonstrating an increase in IL-6 expression and overall inflammatory response in pediatric tumors. To further characterize age-associated transcriptomic differences, we performed immunohistochemistry, immunoblotting, and other proteomic studies. Our results demonstrate phenotypic similarities and differences between pediatric and adult ACP.

IMMU-08. PHASE I TRIAL (NCT02457845) SAFETY, TOLERABILITY AND PRELIMINARY EFFICACY OF IMMUNOVIROTHERAPY WITH HSV G207 IN CHILDREN WITH PROGRESSIVE MALIGNANT SUPRATENTORIAL BRAIN TUMORS

Abstract

BACKGROUND

Outcomes for children with progressive malignant supratentorial brain tumors are dismal. Preclinical evidence indicates that pediatric brain tumors are highly sensitive to genetically engineered oncolytic HSV-1 G207, which lacks genes essential for replication in normal brain. We report on this first-in-children oncolytic virotherapy brain tumor trial of G207.

DESIGN/METHODS

A 3 + 3 design was used to evaluate the safety, tolerability and preliminary efficacy of G207 at two doses (10⁷ and 10⁸ plaque-forming units). After informed consent, patients underwent biopsy to confirm viable tumor followed by placement of up to four silastic intratumoral catheters. The following day, G207 was infused by convection enhanced delivery over 6 hours. Patients were assessed for viral shedding and viremia by PCR and response by serial MRIs.

RESULTS

Six subjects have been treated (5 glioblastoma; 1 anaplastic astrocytoma). No dose limiting toxicities, serious adverse events, or ≥ grade 2 toxicities related to G207 have occurred. No G207 shedding or viremia was detected in the saliva, conjunctiva or blood at any time. Evidence of radiographic responses to G207 were seen in 5 of 6 patients including a patient >12 months post-G207 with an ongoing response and improvement in performance score from 80 to 100.

CONCLUSION

G207 delivered intratumorally is safe and tolerable in children with progressive malignant supratentorial brain tumors. Preliminary evidence of efficacy is very promising to date. The next phase of the study (NCT02457845) will test the safety of G207 combined with a single 5 Gy dose of radiation within 24 hours of virus inoculation.

EMBR-14. RECLASSIFICATION OF CENTRAL NERVOUS SYSTEM PRIMITIVE NEUROECTODERMAL TUMOR (CNS-PNET) INTO ENTITIES REFLECTS OUTCOME: RESULTS FROM THE PROSPECTIVE SJYC07 AND SJMB03 TRIALS

Abstract

BACKGROUND

Central nervous system primitive neuroectodermal tumor (CNS-PNET) was removed from the WHO classification after DNA-methylation profiling unveiled its underlying heterogeneity. Here we describe the makeup and outcome of patients histopathologically diagnosed as CNS-PNET treated on 2 multi-center, prospective trials.

METHODS

Patients <3yr received chemotherapy with/without focal irradiation (SJYC07). Patients ≥3yr received risk-adapted craniospinal-irradiation (23.4Gy for localized disease, 36–39.6Gy for metastatic) and chemotherapy (SJMB03). DNA-methylation was performed using Infinium MethylationEPIC BeadChip and profiled on DKFZ molecularneuropathology2.0 classifier.

RESULTS

Fifty-six patients were enrolled (SJYC07=32; SJMB03=24) with median age 2.86yr (range: 0.64–19.47). Sixteen were stratified as high-risk including 10 with metastasis. Histopathological diagnosis upon central review was CNS-PNET (n=34), ETMR (n=17), CNS-neuroblastoma/ganglioneuroblastoma (n=3) and HGNET (n=2). 42/56 cases were methylation-profiled into ETMR (N=13), GBM (N=3), MB_group3 (N=2), CNS-NB-FOXR2 (N=3), CNS-EFT-CIC (N=1), EPN-RELA (N=1), choroid plexus tumor (pediatric B) (N=1), PB_groupB (N=1), normal tissue (N=3), and "no match" (calibrated scores <0.9) (N=14). Median duration of follow-up was 2.24yr (range: 0.06–12.7). 5yr-OS/PFS in SJMB03-AR and -HR patients were 46.7 ± 12.9%/46.7 ± 12.9% and 33.3 ± 15.7%/33.3 ± 15.7% (OS p=0.503; PFS p=0.494). 5yr-OS/PFS in SJYC07-IR and -HR patients were 46.8 ± 10.6%/44.7 ± 10.4% and 57.1 ± 18.7%/14.3 ± 13.2% respectively (OS p=0.973; PFS p=0.046). Patients methylation-profiled as ETMR/GBM/MB had 5yr-OS/PFS of 24.1 ± 10.4%/14.8 ± 8.9% compared to 90.0 ± 9.5%/80.0 ± 12.6% in the other entities (OS p=0.001; PFS p<0.001). Age, metastasis, extent of surgery and treatment protocol did not significantly impact outcome. Classification by DNA-methylation profiling (p=0.037), histopathological diagnosis (p=0.019) and radiation use (p<0.001) were predictive of PFS.

CONCLUSION

Outcome of pediatric CNS-PNET varied but better conformed to convention when assigned a new entity.

ATRT-07. MURINE SOX2-POSITIVE EARLY PRECURSOR CELLS GIVE RISE TO RHABDOID TUMORS WITH FEATURES OF THE HUMAN ATRT-MYC GROUP

Abstract

PURPOSE

Atypical teratoid rhabdoid tumors (ATRT), characterized by SMARCB1 loss, have been classified by DNA methylation and gene expression profiling into three distinct molecular subgroups (ATRT-SHH, -MYC, -TYR). We hypothesize that ATRT from distinct subgroups have a different cell of origin.

METHODS

Multiple precursor cell specific, constitutive and inducible Smarcb1 knockout mouse strains were established by using a Cre-loxP system. Murine tumors were analyzed by histology and gene expression profiling. Unsupervised hierarchical clustering and differential expression analyses were performed.

RESULTS

Rhabdoid tumor (RT) development was detected only when Smarcb1 abrogation occurred in a very restricted time frame during embryonic development and only under the control of an ubiquitous (Rosa26) or Sox2 promoter. Unsupervised hierarchical clustering of Affymetrix gene expression profiles of these murine and of published human ATRT classified tumors of the Rosa26cre^ERT2::Smarcb1^Fl/Fl model either as ATRT-MYC or as ATRT-SHH. In contrast, ATRT of Sox2cre^ERT2::Smarcb1^Fl/Fl mice were assigned to the ATRT-MYC subgroup only. Mouse strains in which the Smarcb1 knockout was driven by Nestin-, hGFAP-, Math1-, Olig1- Cre recombinase presented other phenotypes but not RT.

CONCLUSION

Subgroup-specific RT genesis depends on the cell of origin. Here we identify early Sox2-positive progenitors as precursor cells of ATRT-MYC subgroup. We further demonstrate that Smarcb1 abrogation during a specific, short time period and in a specific targeted cell population is crucial for induction of ATRT-MYC.

DIPG-41. IDENTIFICATION OF BIRC5 AS A NOVEL THERAPEUTIC TARGET FOR DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract

Diffuse Intrinsic Pontine Glioma (DIPG) remains to be a lethal type of pediatric brain tumor to date, indicating an urgent need of finding novel therapeutic strategy. Through screening of a collection of anti-tumor agents against a patient-derived DIPG primary tumor cell line, we identified a few novel therapeutic candidates for treating DIPG, among which BIRC5 inhibitor YM155 was the top potent agent. Next, we confirmed that YM155 could selectively inhibit multiple DIPG primary cell lines with IC50 less than 10nM. Moreover, our gene expression analyses showed that BIRC5 was significantly upregulated in DIPG primary tumor tissues and paired normal cortex tissues from the same patient. Genetically targeting of BIRC5 with CRISPR-Cas9 or RNAi approach could also effectively disrupt the growth of multiple DIPG cell lines, confirming BIRC5 as a valid therapeutic target for treating DIPG. Mechanistically, BIRC5 inhibition could cause cell cycle arrest, shut off proliferation and induce massive apoptosis. Furthermore, we tested the combinatory inhibitory effects of YM155 plus chemotherapy or targeted therapy drugs with proven anti-DIPG activity, including THZ1, JQ1, Gemcitabine, Panobinostat, GSK-J4 and et al. The preliminary results demonstrated YM155 could work synergistically with a few of above-mentioned drugs in vitro. We are also testing the in vivo inhibitory effects of targeting BIRC5 against DIPG preclinical PDX models. Together, our study identified BIRC5 inhibition as a novel therapeutic strategy against DIPG.

GERM-15. A PHASE 2 TRIAL OF RESPONSE-BASED RADIATION THERAPY FOR PATIENTS WITH LOCALIZED CENTRAL NERVOUS SYSTEM GERM CELL TUMORS (CNS GCT): A CHILDREN’S ONCOLOGY GROUP (COG) STUDY

Abstract

Intracranial germ cell tumors represent 3-5% of pediatric CNS tumors. Non-germinomatous germ cell tumors (NGGCT) have worse outcomes compared to germinomas, however, improved survivals were achieved on COG ACNS0122 utilizing a combination of chemotherapy and craniospinal irradiation (CSI). Since CSI is associated with significant late effects, a Phase 2 study was undertaken to determine whether irradiation could be reduced without impacting survival in a subgroup of NGGCT patients. Patients with localized disease who achieved a complete (CR) or partial response (PR) to chemotherapy were eligible for reduced irradiation to 30.6Gy whole ventricular field (WVI) and 54Gy tumor-bed boost as compared to 36Gy CSI plus tumor-bed boost as used on ACNS0122. Between 5/2012 and 11/2016, 107 eligible patients were accrued. Median age was 11 years (range: 4-22) and 75% were male. Tumor location was pineal in 58, suprasellar in 37, ventricular in 6 and bifocal in 6 patients. Sixty-six patients achieved CR/PR post-induction and received reduced irradiation. The 2-year progression-free survival (PFS) was 89% (95% CI: 81%-97%) and overall survival was 92% (95% CI: 86%- 99%). Eight patients progressed; seven had a distant relapse (outside the irradiation field) and one patient had a local plus distant relapse. Alpha-fetoprotein and beta-human chorionic gonadotropin levels were not associated with PFS. There were no unexpected treatment-related adverse events or deaths. Although these survival data are encouraging, distant relapses noted in all of the patients who progressed are concerning. Longer follow-up of the ACNS1123 cohort may better inform our recommendations in the future.

CRAN-01. QUALITY OF LIFE AND GROWTH AFTER CHILDHOOD CRANIOPHARYNGIOMA: RESULTS OF THE MULTINATIONAL TRIAL KRANIOPHARYNGEOM 2007

Abstract

CONTEXT

Quality of life (QoL) after childhood-onset craniopharyngioma (CP) is frequently impaired due to tumor and/or treatment-related factors such as endocrine deficits and hypothalamic involvement/lesions. PATIENTS AND

METHODS

In a multinational trial, we prospectively analyzed parental and self-assessment of CP patient QoL at 3 months, one and 3 years after CP diagnosis related to growth hormone (GH) substitution. 47 of 194 CP recruited between 2007 and 2015 in KRANIOPHARYNGEOM 2007 were analyzed for QoL one and 3 years after CP diagnosis. QoL was assessed by Pediatric Quality of Life (PEDQOL) questionnaire and PEDQOL scores of parental and self-assessed QoL during 3 years follow-up after CP diagnosis were analyzed.

RESULTS

Parents estimated QoL of their children worse than patients did themselves. GH substitution had no relevant effect on short-term weight and height development. CP patients GH-treated at 3 years follow-up presented at baseline (one year after diagnosis, before GH substitution) with reduced self-assessed QoL when compared with GH non-treated CP. QoL stabilized during 1–3 years of follow-up in GH-treated patients, whereas non GH-treated patients experienced decreases in autonomy (p=0.03), cognition (p=0.01), and physical function (p=0.04).

CONCLUSIONS

Parents assess QoL in CP survivors worse than their children. GH substitution should be considered as a therapeutic option to ameliorate imminent impairments of QoL after CP.

DIPG-09. HDAC INHIBITION IN THALAMIC AND SPINAL CORD H3K27M+ DIFFUSE MIDLINE GLIOMA

Abstract

BACKGROUND

Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of brain tumor-related deaths in children. There are no effective treatments and median survival remains dismal. Genomics identified a mutation in the majority of DMGs, a lysine to methionine substitution (K27M) in histones 3.1 and 3.3, which causes changes in gene expression that promote gliomagenesis. Panobinostat, a multiple histone deacetylase (HDAC) inhibitor, was one of the most effective agents against patient-derived DIPG cell cultures and xenograft models in previous studies and is presently in clinical trial for DIPG. HDAC inhibition with panobinostat may also exhibit activity against H3K27M+ DMG of the thalamus and spinal cord.

METHODS

Patient-derived thalamic and spinal cord H3K27M+ DMG cell cultures were treated with single agent panobinostat at a range of concentrations. Cell viability was evaluated using the CellTiter-Glo assay. Panobinostat was systemically administered to murine models of luciferase-expressing spinal cord H3K27M+ DMG. Response to panobinostat was evaluated with IVIS in vivo imaging.

RESULTS

HDAC inhibition with panobinostat significantly decreases cell proliferation with an IC50 of 30 nM and 41 nM in the spinal cord and thalamic glioma patient-derived cell cultures respectively. Panobinostat slowed tumor growth in spinal cord glioma orthotopic xenografts compared to vehicle controls.

CONCLUSION

This study suggests that HDAC inhibition with panobinostat may also be beneficial for thalamic and spinal cord H3K27M+ DMG.

DIPG-74. DNA METHYLATION STOCHASTICITY IN DIPG

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a dismal prognosis and no effective treatment. Recent studies point to a critical role for epigenetic dysregulation in this disease. Nearly 80% of DIPGs harbor mutations in histone H3 encoding replacement of lysine 27 with methionine (K27M), leading to global loss of the repressive histone H3K27 trimethylation mark, global DNA hypomethylation, and a unique gene expression profile. Recent studies across diverse cancers have highlighted the role of epigenetic variability as a driving force in tumor evolution. Epigenetic variability may underlie the heterogeneity and phenotypic plasticity of cancer cells and allow for the selection of cellular traits that promote survival and resistance to therapy. Our group has recently described a novel mathematical framework for analyzing variability of DNA methylation directly from whole-genome bisulfite sequencing data by modeling stochastic Markov processes, allowing rigorous computation of DNA methylation entropy at precise genomic locations. We have carried out genome-wide characterization of DNA methylation at single-base resolution in DIPG in order to generate comprehensive genome-wide maps of DNA methylation entropy. We find a global increase in DNA methylation entropy in DIPG. Dissection of the targets of this perturbation highlights specific genes and regulatory regions under epigenetic control in DIPG cells. We assess the effect of pharmacologic DNA methyltransferase inhibition on the DNA methylation landscape and on gene expression in DIPG patient-derived neurosphere cell lines. We find that DNA methyltransferase inhibition alters expression of genes involved in cell death, differentiation, cellular defense, and immune signaling.

EPEN-30. HISTONE H3 LYSINE 4 TRIMETHYLATION IS A POTENTIAL TARGET TO IMPROVE CHEMOTHERAPEUTIC EFFICACY FOR PEDIATRIC PRIMARY EPENDYMOMAS

Abstract

Ependymomas are the third most common form of brain tumors in children. These tumors are resistant to chemotherapy and despite genomic sequencing, there is a lack of effective molecular treatment targets. Efficient treatment targets need to be identified. Increasing evidence shows epigenetic alterations including posttranslational histone modifications (PTMs), associated with malignancy, chemotherapeutic resistance and prognosis in pediatric ependymomas. In this study we examined histone PTMs in ependymomas and identified potential targets to improve chemotherapeutic efficacy. Global levels of trimethylation at lysine 4 on histone H3 (H3K4me3), detected with immunohistochemistry and western blots, positively correlated with malignancy in pediatric primary ependymomas. Micro-array analysis of 22 pediatric ependymomoas identified upregulated candidate drug resistance genes. Promoter H3K4me3 occupancy was examined for two of these genes, CCND1 and ERBB2. Chromatin-immunoprecipitation with H3K4me3 coupled with real-time PCR, showed higher levels of H3K4me3 at these genes in grade III tumors, in comparison to grade II ones. When H3K4me3 levels were reduced in primary cultured ependymoma cells in vitro, cell response to chemotherapy increased. In summary, these results indicate that H3K4me3 levels are high at promoters of genes which confer chemotherapeutic resistance. Consequently, a novel treatment regimen which targets H3K4me3 in combination with traditional protocols may increase chemotherapeutic efficacy and improve prognosis for children who present with ependymoma.

TBIO-04. A CENTRALIZED MOLECULAR DIAGNOSTIC SERVICE FOR PEDIATRIC BRAIN TUMORS IN JAPAN

Abstract

Molecular tests have now become an essential part of clinical management of pediatric brain tumor patients. We have established a central diagnosis system for pediatric brain tumors in Japan to provide standardized molecular diagnostic service. Tumor specimen are accepted from centers participating Japan Children's Cancer Group (JCCG). The JCCG Datacenter at National Center for Child Health and Development (NCCHD) manages all patients' specimen and information. Histology is centrally reviewed at Gunma University Hospital. Molecular tests are performed at either Osaka National Hospital (medulloblastoma), Gunma University Hospital (ETMR) or National Cancer Center (all the others). Medulloblastomas are classified by expression profiling using the nanostring technology, and mutations of CTNNB1, TP53 and TERT determined. Gliomas are examined for mutations of BRAF, H3F3A, HIST1H3B, FGFR1, IDH1/2 and TERT using pyrosequencing, as well as for BRAF fusion by RT-PCR as necessary. Supratentorial ependymomas are examined for the presence of RELA fusion by RT-PCR, and posterior fossa ependymomas are classified as PFA or PFB by DNA methylation analysis. Those tumors negative for the routine analyses may be subjected to RNA sequencing or targeted sequencing for 93 genes known to be mutated in brain tumors. As of August 2017, over 200 tumors have been molecularly diagnosed and the integrated diagnosis reported to each center. Our system is affordable utilizing minimum amount of high throughput technologies, and will hopefully improve the standard of diagnosis for pediatric brain tumors.

HGG-22. PHASE 1b STUDY POLIO VACCINE SABIN-RHINOVIRUS POLIOVIRUS (PVSRIPO) FOR RECURRENT MALIGNANT GLIOMA IN CHILDREN

Abstract

BACKGROUND

Prognosis of recurrent World Health Organization (WHO) grade IV malignant glioma (GBM) in children is dismal with no effective therapy. We have recently performed a dose-finding and toxicity study within adults with GBM, evaluating the recombinant non-pathogenic polio/rhinovirus chimera (PVSRIPO) by intratumoral convection-enhanced delivery (CED). PVSRIPO recognizes the poliovirus receptor CD155, which is widely expressed in neoplastic cells of solid tumors and in major components of tumor stroma. We have commenced a Phase Ib study to evaluate feasibility, safety, and preliminary evidence of efficacy of the optimal adult dose in a pediatric population.

METHODS

Patients with a recurrent supratentorial WHO Grade III malignant glioma (anaplastic astrocytoma, anaplastic oligoastrocytoma, anaplastic oligodendroglioma, anaplastic pleomorphic xanthoastrocytoma, ependymoma) or WHO Grade IV malignant glioma (glioblastoma, gliosarcoma) based on imaging studies with measurable disease (≥ 1 cm and ≤ 5.5 cm of contrast-enhancing tumor) will be enrolled. A stereotactic biopsy will be performed prior to virus administration. Immediately following the stereotactically-guided tumor biopsy, a catheter will be implanted in the operating room. PVSRIPO will then be delivered intratumorally by CED, using the catheter placed within the enhancing portion of the tumor, at a dose of 5 x 10⁷ TCID over 6.5 hours.

CONCLUSION

We will confirm the activity of intratumoral PVSRIPO infusion in recurrent WHO grade III or IV malignant glioma in the absence of neurovirulent potential in children.

TBIO-21. ANALYSIS OF PAIRED BRAFV600E MUTANT GLIOMA PATIENT SAMPLES IDENTIFIES NOVEL RESISTANCE MECHANISMS TO TARGETED BRAF INHIBITION

Abstract

BRAF^V600E mutations occur in a variety of gliomas and BRAF^V600E targeted therapies have provided new treatment options. Trials in melanoma and colorectal cancer, as well as early results in CNS tumors, indicate a high risk for the development of resistance. To date there is little data available for resistance mechanisms in glioma patients treated with BRAF^V600E inhibition. To identify molecular and pathway alterations driving resistance, we performed targeted next-generation DNA sequencing, RNA sequencing, and Ingenuity Pathway Analysis (IPA) on paired pre-treatment and post-resistance primary pediatric glioma samples (n=10) and cell lines (n=2). Unique genetic alterations likely driving tumor recurrence and resistance to BRAF inhibition were identified. Novel mutations in post-resistance samples included a de novo mutation in the CBL gene within the RING finger domain of the E3 ubiquitin ligase protein that has been recurrently found in myeloid neoplasms, a frameshift mutation in the RB1 tumor suppressor gene, and a nonsense mutation in ERRFI1, which encodes a negative regulator of EGFR signaling. IPA analysis demonstrated significant pathway activation differences in post-resistance samples. There was a predominance of upregulated receptor tyrosine kinase pathways including EGFR, NTRK2, TGFB1, estrogen receptor, ERBB2, and PI3K. Expected up-regulation of MEK, MAPK1, MAP2k1 and ERK1/2 pathways were found. The altered pathways were unique for each paired set. These data suggest that pediatric gliomas develop resistance to BRAF inhibition using a multitude of genetic and transcriptional mechanisms that are potentially distinct in each tumor, and with novel resistance mutations not found in resistant melanoma or colorectal cancers.

ATRT-24. CHROMATIN SEGMENTATION IN ATRT REVEALS AN IMPORTANT ROLE FOR RESIDUAL SWI/SNF MEMBERS

Abstract

Although SMARCB1 mutations represent the only recurrent genetic aberration in atypical teratoid/rhabdoid tumors (ATRT), a comprehensive assessment of the epigenetic effect of SMARCB1 loss has not been attempted so far. To achieve this, we performed ChIP-sequencing for six histone marks (H3K4me1, H3K4me3, H3K27me3, H3K27Ac, H3K9me3, H3K36me3) and EZH2 in 11 primary ATRTs, encompassing all three molecular subgroups of ATRT.Chromatin segmentation analyses revealed that across all three subgroups a high percentage of ATRT epigenomes resides in a quiescent state and active enhancer states are significantly underrepresented as compared to non-neoplastic brain. We validated these findings in a SMARCB1-reexpressing ATRT cell line and also found an overall deincrease of H3K27Ac and H3K27me3 upon SMARCB1 reexpression. Interestingly, by comparing the genome wide distribution of EZH2 and H3K27me3, we also identified classes of ATRT-specific active genes bound by EZH2, but lackingH3K27me3, pointing at a non-canonical role of EZH2 in gene activation. ChIP-Sequencing of SMARCA4 in these samples revealed that many of these „EZH2 only" genes display a high signal of of this protein. Many of these genes (such as e.g. CCND2) were downregulated upon knock-down of SMARCA4 in ATRT cell lines. Adding to the overall characterization of the epigenome, we have shown that a number of genes - despite being bound by EZH2- are maintained in an active state by residual SWI/SNF complex binding. This sheds new light on the role of residual SWI/SNFSMARCA4 still being present in AT/RT and elucidates a mechanism by which AT/RT keep important tumor-genes in an active state.

CRAN-18. CASE SERIES OF INTRACRANIAL NON-GERMINOMATOUS GERM CELL TUMOR: A SINGLE INSTITUTION EXPERIENCE

Abstract

Intracranial non-germinomatous germ cell tumors are a rare, heterogenous group of neoplasms, commonly occurring in the pineal and/or suprasellar regions. The subtypes include yolk sac tumor (YST) or endodermal sinus tumor (EST), embryonal carcinoma (EC), choriocarcinoma (CHC), and mixed malignant subtypes. Diagnosis is usually made without a biopsy, through detection of elevated levels of Alpha-fetoprotein (AFP) and/or beta-human chorionic gonadotropin (βHCG) in the serum and/or cerebrospinal fluid. Three patients with intracranial NGGCT were treated at our institution, using a combination of surgery, chemotherapy, and/or radiation therapy. The clinical presentation of all cases included signs and symptoms of obstructive hydrocephalus. Patient #1 was diagnosed with malignant teratoma at birth via neuroimaging; she died from complications due to surgical resection. Patient #2 was diagnosed with choriocarcinoma of the pituitary gland at age 5 years via elevated AFP in serum and CSF; he was treated with alternating cycles of carboplatin/etoposide and ifosfamide/etoposide, followed by whole ventricle radiation therapy. He is now 28 months off-therapy, without evidence of disease. Patient #3 was diagnosed with choriocarcinoma of the pineal and suprasellar regions at the age 7 years, via elevated beta-HCG in serum and CSF, with evidence of bimodal disease. Treatment included 6 cycles of risk-adapted chemotherapy followed by whole ventricle radiation therapy. Neuroimaging continues to show no evidence of disease with normalization of tumor markers, at 24 months off-therapy. Patients #2 and #3 have residual neuroendocrine and neuropsychological deficits, but remain neurodevelopmentally appropriate. Combination of chemotherapy and radiation therapy is required for optimal prognosis.

DEV-11. OUTCOMES OF MALIGNANT BRAIN TUMORS IN YOUNG CHILDREN TREATED WITH CHEMOTHERAPY AND DELAYED RADIOTHERAPY IN A RESOURCE LIMITED SETTING

Abstract

METHODOLOGY

A retrospective analysis of children less than 3 years diagnosed with malignant brain tumors was conducted from January 2011 to December 2016. Children with malignant brain tumors were treated with cyclophosphamide, etoposide and carboplatin (CEJ) upto 36months of age or till disease progression. Low grade gliomas were excluded from the analysis. An intention to treat analysis was performed to determine the outcomes and delay in radiotherapy (RT).

RESULTS

ninety four children (median age: 26 months, median follow-up: 18.9 months) were diagnosed with malignant brain tumors of which 59 received the CEJ regimen. Twenty six children (44.0%) underwent a complete resection, 23 (38.9%) partial resection, and 10 (16.9%) debulking/biopsy. Progression free survival (PFS) and overall survival (OS) at 24months was 53.8% (95% CI: 38.6% - 66.9%) and 64.0% (95% CI: 48.7%- 78.8%) respectively.41 children (69.5%) received RT with a median time to delaying RT of 9.3months (inter-quartile range 6.5 -13.4 months). Among 39 cases of medulloblastoma (MB), molecular data was available in 24 (wnt-0, shh-11, group 3 and group4 – 13). The outcomes with shh and non-shh were similar.

CONCLUSIONS

Chemotherapy regimens like CEJ are effective in delaying radiotherapy in young children with malignant brain tumors. This approach may be used in resource limited countries to improve the outcomes of young children with malignant brain tumors.

DIPG-25. A KINOME-WIDE shRNA SCREEN UNCOVERS VRK3 AS AN ESSENTIAL GENE FOR DIPG SURVIVAL

Abstract

Diffuse Intrinsic Pontine Glioma (DIPG) remains the most fatal form of pediatric brain tumor. The specific somatic mutation (H3K27M) in the H3 histone gene was found in around 95% of patients making this alteration as the initial oncogenic event. However, until now this alteration cannot be targeted. Consequently, we conducted a RNAi-based loss-of-function screen targeting the human kinome to decipher vulnerabilities in DIPG, unravel their biology and provide therapeutic opportunities in this devastating disease. An integrative lentiviral pooled library of 7450 shRNAs against 770 kinases was used to transduce four GSC (glioma stem cells) cultures harboring either H3.1 (n=2) or H3.3-mutation (n=2) in order to cover the diversity observed in patients, and 2 control NSC (neural stem cell) cultures. The entire set of shRNAs was identified by sequencing 40 hours and 22 days after transduction. Genes required for cell expansion over 22 days of outgrowth in GSC but with limited deleterious effect on NSC proliferation were identified. Among those, we selected the target genes with at least 3 distinct shRNAs presenting a significant decrease in their abundance and identified in particular VRK3 (Vaccinia-Related Kinase 3) as one of the top scoring kinases whose extinction was lethal to DIPG. This serine-threonine kinase was recently linked to cell cycle progression, DNA repair and neuronal differentiation. The role of this protein in DIPG biology is currently assessed especially to identify actionable downstream targets. Drugs mimicking the effect of VRK3 extinction will be evaluated in our vitro and vivo models of DIPG.

DIPG-57. A COMPREHENSIVE GENE/PROTEIN INVESTIGATION OF THE TUMOUR MICROENVIRONMENT IN DIFFUSE MIDLINE GLIOMA IN CHILDREN

Abstract

Pediatric diffuse midline glioma (pDMG) is a rare aggressive childhood malignancy. While much is known concerning molecular genomics, our focus is on understanding the tumour microenvironment and how this influences cancer growth. We hope to learn how interaction and communication between host cells, tumour cells and extracellular matrix proteins (ECM) promote the pDMG growth. We conducted bioinformatic analysis using data obtained from R2: Genomics Analysis and Visualization Platform of pDMG tissue samples (GSE26576). In parallel, fixed-formalin paraffin embedded tissue (FFPE) was obtained from post-mortem brain. RNA was extracted from tumour core, adjacent and 'normal' tissue. Following quality control experiments to ensure RNA integrity, RNA was then used for a focused ECM array. Eighty ECM related genes were chosen for bioinformatics analyses to make indirect comparisons to the ECM focused RT2 profiler. Preliminary bioinformatic analyses indicate that 15 of the 80 genes were significantly up-regulated in pDMG tumours compared to normal brainstem (p<0.05). The most significantly differentially expressed include, Matrix Metalloproteinase1 (MMP1), Collagen type XI alpha 1 (COL11A1), and Matrix Metalloproteinase 16 (MMP16) (p<0.0001). In preliminary analyses of data obtained from FFPE tissue, tumour adjacent RNA was compared to 'normal' brain. Out of 80 genes, 7 were differentially expressed and include COL11A1, Collagen type VI alpha 2 (COL6A2) and Laminin alpha 3 (LAMA3). We believe that by identifying key host/tumour interactions that drive pDMG growth, this knowledge will help lead to improved therapeutics for this devastating childhood brain cancer.

EAPH-13. PHASE I STUDY OF INTRAVENTRICULAR INFUSIONS OF AUTOLOGOUS EX VIVO EXPANDED NK CELLS IN CHILDREN WITH RECURRENT/REFRACTORY MALIGNANT POSTERIOR FOSSA TUMORS OF THE CENTRAL NERVOUS SYSTEM

Abstract

Prognosis of recurrent/refractory medulloblastoma, ependymoma and atypical teratoid/rhabdoid neoplasms (AT/RT) remains dismal. Preclinical data showing efficacy of activated/propagated NK cells to lyse and kill these tumor cells in culture and in mice have been demonstrated, along with feasibility and safety of infusions of biologic agents into the ventricles. The ongoing phase I trial evaluates for the first time in humans, safety of infusions of autologous NK cells directly into the ventricles of patients with these tumors. Assessment of antitumor activity, and correlative biologic studies are being evaluated to define the immunophenotype and function of expanded NK cells. Patients receive three cycles of NK-cell infusions over 12 weeks through an Ommaya reservoir. To date, 9 patients have been enrolled, 8 patients achieved successful expansion of their NK cells, and 7 patients received up to 27 infusions each of NK cells at doses up to 3x10e7/m2/infusion. Currently this study is enrolling patients in the last cohort. This phase I study has so far demonstrated safety with no dose-limiting toxicity attributable to the infused NK cells. Significant CSF pleocytosis is seen in patients receiving NK cells with the increased dosage, with no evidence of infection. Influx of CD4⁺ and CD8⁺ T cells into the CSF in noted. This could suggest a proinflammatory environment induced by higher doses of NK cell infusion. This study also intends to evaluate the migration and persistence of NK cells with novel neuroimaging techniques to correlate efficacy outcome and the pharmacokinetics of NK cells.