Search

Aims: DNA methylation profiling, recently endorsed by the World Health Organisation (WHO) as a pivotal diagnostic tool for brain tumours, most commonly relies on bead arrays. Despite its widespread use, limited data exist on the technical reproducibility and potential cross-institutional differences. The LOGGIC Core BioClinical Data Bank registry conducted a prospective laboratory comparison trial with 12 international laboratories to enhance diagnostic accuracy for paediatric low-grade gliomas, focusing on technical aspects of DNA methylation data generation and profile interpretation under clinical real-time conditions., Methods: Four representative low-grade gliomas of distinct histologies were centrally selected, and DNA extraction was performed. Participating laboratories received a DNA aliquot and performed the DNA methylation-based classification and result interpretation without knowledge of tumour histology. Additionally, participants were required to interpret the copy number profile derived from DNA methylation data and conduct DNA sequencing of the BRAF hotspot p.V600 due to its relevance for low-grade gliomas. Results had to be returned within 30 days., Results: High technical reproducibility was observed, with a median pairwise correlation of 0.99 (range 0.94-0.99) between coordinating laboratory and participants. DNA methylation-based tumour classification and copy number profile interpretation were consistent across all centres, and BRAF mutation status was accurately reported for all cases. Eleven out of 12 centres successfully reported their analysis within the 30-day timeframe., Conclusion: Our study demonstrates remarkable concordance in DNA methylation profiling and profile interpretation across 12 international centres. These findings underscore the potential contribution of DNA methylation analysis to the harmonisation of brain tumour diagnostics., (© 2024 The Author(s). Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

Background: We applied the strategy for M+ medulloblastoma across all high-risk subgroups, including LC/A histology, TP53 mutations, MYC/MYCN amplification., Methods: Patients over 3-years-old received,after surgery,staging and histo-biological analysis,sequential high-dose-methotrexate(HD-MTX),high-dose-etoposide(HD-VP16),high-dose-cyclophosphamide(HD-Cyclo),high-dose-carboplatin(HD-Carbo).Hyperfractionated-accelerated-radiotherapy-craniospinal(HART-CSI),administered in twice daily 1.3 Gy-fractions reached a total dose tailored to the patients' age and pre-radiation response to chemotherapy(CT): 31.2 Gy if under 10-years-old and complete response(CR) or partial response(PR) obtained or absence of metastatic disease,39 Gy in other/older patients.Boosts to posterior fossa/residual metastatic(M+) deposits were given up to a total dose of 60 Gy/9 Gy,respectively,but avoided if metastatic nodules were very big or patients very young.Two courses of high-dose-thiotepa were delivered in case of not CR/PR after pre-radiotherapy(RT) phase and in all M0 patients either - pre/post HART.Subgrouping was performed where tissue was available., Results: Eighy-nine patients were enrolled,median age 8.8 years,median follow up 136 months.Overall-survival(OS) and event-free-survival(EFS) at 5/15 years were 75.9/66.5% and 68.2/65.3%, respectively;5/28 fatal events were not related to relapse(three developed secondary malignancies).Sex,age less than 10 years,histological subtype,presence of MYC/MYCN amplification,reduction in CSI dose,omission of RT-boosts,implementation of myeloablative therapy,presence/absence of metastases did not impact prognosis.Patients progressing after pre-HART CT(14/89) and stable-disease(SD)+PD after HART(10/89) negatively affected outcome(P<0.001).Subgrouping in 66/89 patients' samples demonstrated a significantly worse EFS for patients with Sonic Hedgehog(SHH)-tumors(#15, 2 with constitutional TP53-mutations) vs. group 3 and 4(15 and 29 patients, respectively, group3/4 in 7).Patients younger than 10 received lower CSI doses if stratified according to CT response., Conclusions: This strategy, partly adopted in the ongoing SIOPE protocol,confirmed improved EFS and OS over previously reported outcomes in all high-risk categories;SHH tumors appeared the most aggressive., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Medulloblastomas (MDBs) are classified into molecular groups showing peculiar immunohistochemical and genetic features and distinct DNA methylation profile. Group 3 and group 4 MDBs have the worst prognosis; the former is treated with high-risk protocols and features MYC amplification, whereas the latter receives standard-risk protocols and harbors MYCN amplification. Herein, we report a unique case of MDB showing histological and immunohistochemical features consistent with non-SHH/non-WNT classic MDB, with both MYCN (30% of tumor cells) and MYC (5-10% tumor cells) amplification in distinct subclones of neoplastic cells at fluorescence in situ hybridization (FISH), characterized by specific patterns. In spite of MYC amplification in only a small percentage of tumor cells, this case had DNA methylation profile consistent with group 3, emphasizing the importance to test both MYC and MYCN amplifications at a single cell level using highly sensitive methods, such as FISH, for diagnostic and therapeutic purposes., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Accurate identification of brain tumor molecular subgroups is increasingly important. We aimed to establish the most accurate and reproducible ependymoma subgroup biomarker detection techniques, across 147 cases from International Society of Pediatric Oncology (SIOP) Ependymoma II trial participants, enrolled in the pan-European "Biomarkers of Ependymoma in Children and Adolescents (BIOMECA)" study., Methods: Across 6 European BIOMECA laboratories, we evaluated epigenetic profiling (DNA methylation array); immunohistochemistry (IHC) for nuclear p65-RELA, H3K27me3, and Tenascin-C; copy number analysis via fluorescent in situ hybridization (FISH) and MLPA (1q, CDKN2A), and MIP and DNA methylation array (genome-wide copy number evaluation); analysis of ZFTA- and YAP1-fusions by RT-PCR and sequencing, Nanostring and break-apart FISH., Results: DNA Methylation profiling classified 65.3% (n = 96/147) of cases as EPN-PFA and 15% (n = 22/147) as ST-ZFTA fusion-positive. Immunohistochemical loss of H3K27me3 was a reproducible and accurate surrogate marker for EPN-PFA (sensitivity 99%-100% across 3 centers). IHC for p65-RELA, FISH, and RNA-based analyses effectively identified ZFTA- and YAP-fused supratentorial ependymomas. Detection of 1q gain using FISH exhibited only 57% inter-center concordance and low sensitivity and specificity while MIP, MLPA, and DNA methylation-based approaches demonstrated greater accuracy., Conclusions: We confirm, in a prospective trial cohort, that H3K27me3 immunohistochemistry is a robust EPN-PFA biomarker. Tenascin-C should be abandoned as a PFA marker. DNA methylation and MIP arrays are effective tools for copy number analysis of 1q gain, 6q, and CDKN2A loss while FISH is inadequate. Fusion detection was successful, but rare novel fusions need more extensive technologies. Finally, we propose test sets to guide future diagnostic approaches., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Background: First-line therapies for medulloblastoma(MBL) are obtaining higher survival-rates while decreasing late-effects, but treatment at relapse is not standardized. We report here the experience with MBL re-irradiation(re-RT), its timing and outcome in different clinical settings and tumor groups., Methods: Patient's staging/treatment at diagnosis, histotypes/molecular subgroups, relapse site/s, re-treatments outcome are reported., Results: 25 patients were included, with a median age of 11.4 years; 8 had metastases. According to 2016-2021 WHO-classification, 14 had SHH subgroup tumors(six TP53 mutated,one + MYC,one + NMYC amplification), 11 non-WNT/non-SHH (two with MYC/MYCN amplification).Thirteen had received HART-CSI, 11 standard-CSI, one HFRT; all post-radiation chemotherapy(CT), 16 also pre-RT. Median time to relapse (local-LR in nine, distant-DR in 14, LR + DR in two) was 26 months. Fourteen patients were re-operated, in five cases excising single DR-sites, thereafter three received CT, two after re-RT; out of 11 patients not re-operated, four had re-RT as first treatment and seven after CT. Re-RT was administered at median 32 months after first RT: focally in 20 cases, craniospinal-CSI in five. Median post-relapse-PFS/after re-RT was 16.7/8.2 months, while overall survival-OS was 35.1/23.9 months, respectively. Metastatic status both at diagnosis/relapse negatively affected outcome and re-surgery was prognostically favorable. PD after re-RT was however significantly more frequent in SHH (with a suggestive association with TP53 mutation, p = 0.050). We did not observe any influence of biological subgroups on PFS from recurrence while SHH showed apparently worse OS compared to non-WNT/non-SHH group., Conclusions: Re-surgery + reRT can prolong survival; a substantial fraction of patients with worse outcome belongs to the SHH-subgroup., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Pediatric high-grade gliomas represent a heterogeneous group of tumors with a wide variety of molecular features. We performed whole exome sequencing and methylation profiling on matched primary and recurrent tumors from four pediatric patients with hemispheric high-grade gliomas. Genetic analysis showed the presence of some variants shared between primary and recurrent tumors, along with other variants exclusive of primary or recurrent tumors. NSD1 variants, all novel and not previously reported, were present at high frequency in our series (100%) and were all shared between the samples, independently of primary or recurrence. For every variant, in silico prediction tools estimated a high probability of altering protein function. The novel NSD1 variant (c.5924T > A; p.Leu1975His) was present in one in four cases at recurrence, and in two in four cases at primary. The novel NSD1 variant (c.5993T > A; p.Met1998Lys) was present in one in four cases both at primary and recurrence, and in one in four cases only at primary. The presence of NSD1 mutations only at recurrence may suggest that they can be sub-clonal, while the presence in both primary and recurrence implies that they can also represent early and stable events. Furthermore, their presence only in primary, but not in recurrent tumors, suggest that NSD1 mutations may also be influenced by treatment.

Neoplastic cells acquire the ability to proliferate endlessly by maintaining telomeres via telomerase, or alternative lengthening of telomeres (ALT). The role of telomere maintenance in pituitary neuroendocrine tumors (PitNETs) has yet to be thoroughly investigated. We analyzed surgical samples of 24 adult recurrent PitNETs (including onset and relapses for 14 of them) and 12 pediatric primary PitNETs. The presence of ALT was assessed using telomere-specific fluorescence in situ hybridization, methylation of telomerase reverse transcriptase promoter (TERTp) by methylation-specific PCR, and ATRX expression by immunohistochemistry. Among the adult recurrent PitNETs, we identified 3/24 (12.5%) ALT-positive cases. ALT was present from the onset and maintained in subsequent relapses, suggesting that this mechanism occurs early in tumorigenesis and is stable during progression. ATRX loss was only seen in one ALT-positive case. Noteworthy, ALT was observed in 3 out of 5 aggressive PitNETs, including two aggressive corticotroph tumors, eventually leading to patient's death. ALT-negative tumors (87.5%) were classified according to their low (29.2%), medium (50%), and high (8.3%) telomere fluorescence intensity, with no significant differences emerging in their molecular, clinical, or pathological characteristics. TERTp methylation was found in 6/24 cases (25%), with a total concordance in methylation status between onset and recurrences, suggesting that this mechanism remains stable throughout disease progression. TERTp methylation did not influence telomere length. In the pediatric cohort of PitNETs, TERTp methylation was also observed in 4/12 cases (33.3%), but no case of ALT activation was observed. In conclusion, ALT is triggered at onset and maintained during tumor progression in a subset of adult PitNETs, suggesting that it could be used for clinical purposes, as a potential predictor of aggressive behavior., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

As a relevant element of novelty, the fifth CNS WHO Classification highlights the distinctive pathobiology underlying gliomas arising primarily in children by recognizing for the first time the families of paediatric-type diffuse gliomas, both high-grade and low-grade. This review will focus on the family of paediatric-type diffuse high-grade gliomas, which includes four tumour types: 1) Diffuse midline glioma H3 K27-altered; 2) Diffuse hemispheric glioma H3 G34-mutant; 3) Diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype; and 4) Infant-type hemispheric glioma. The essential and desirable diagnostic criteria as well as the entities entering in the differential will be discussed for each tumour type. A special focus will be given on the issues encountered in the daily practice, especially regarding the diagnosis of the diffuse paediatric-type high-grade glioma H3-wildtype and IDH-wildtype. The advantages and the limits of the multiple molecular tests which may be utilised to define the entities of this tumour family will be evaluated in each diagnostic context., (Copyright © 2022 Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology.)

The management of patients with Central Nervous System (CNS) malignancies relies on the appropriate classification of these tumors. Recently, the World Health Organization (WHO) has published new criteria underlining the importance of an accurate molecular characterization of CNS malignancies, in order to integrate the information generated by histology. Next generation sequencing (NGS) allows single step sequencing of multiple genes, generating a comprehensive and specific mutational profile of the tumor tissue. We developed a custom NGS-based multi-gene panel (Glio-DNA panel) for the identification of the correct glioma oncotype and the detection of its essential molecular aberrations. Specifically, the Glio-DNA panel targets specific genetic and chromosomal alterations involving ATRX chromatin remodeler (ATRX) , cyclin dependent kinase inhibitor 2A ( CDKN2A) , isocitrate dehydrogenase (NADP+) 1 (IDH1) and the telomerase reverse transcriptase (TERT) promoter while also recognizing the co-deletion of 1p/19q, loss of chromosome 10 and gain of chromosome 7. Furthermore, the Glio-DNA panel also evaluates the methylation level of the O-6-methylguanine-DNA methyltransferase ( MGMT) gene promoter that predicts temozolomide efficacy. As knowledge of the mutational landscape of each glioma is mandatory to define a personalized therapeutic strategy, the Glio-DNA panel also identifies alterations involving "druggable" or "actionable" genes. To test the specificity of our panel, we used two reference mutated DNAs verifying that NGS allele frequency measurement was highly accurate and sensitive. Subsequently, we performed a comparative analysis between conventional techniques - such as immunohistochemistry or fluorescence in situ hybridization - and NGS on 60 diffuse glioma samples that had been previously characterized. The comparison between conventional testing and NGS showed high concordance, suggesting that the Glio-DNA panel may replace multiple time-consuming tests. Finally, the identification of alterations involving different actionable genes matches glioma patients with potential targeted therapies available through clinical trials. In conclusion, our analysis demonstrates NGS efficacy in simultaneously detecting different genetic alterations useful for the diagnosis, prognosis and treatment of adult patients with diffuse glioma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tirrò, Massimino, Broggi, Romano, Minasi, Gianno, Antonelli, Motta, Certo, Altieri, Manzella, Caltabiano, Barbagallo, Buttarelli, Magro, Giangaspero and Vigneri.)

Background: More than 40% of patients with intracranial ependymoma need a salvage treatment within 5 years after diagnosis, and no standard treatment is available as yet. We report the outcome after first relapse of 64 patients treated within the 2nd AIEOP protocol., Methods: We considered relapse sites and treatments, that is, various combinations of complete/incomplete surgery, if followed by standard or hypofractionated radiotherapy (RT) ± chemotherapy (CT). Molecular analyses were available for 38/64 samples obtained at first diagnosis. Of the 64 cases, 55 were suitable for subsequent analyses., Results: The median follow-up was 147 months after diagnosis, 84 months after first relapse, 5-year EFS/OS were 26.2%/30.8% (median EFS/OS 13/32 months) after relapse. For patients with a local relapse (LR), the 5-year cumulative incidence of second LRs was 51.6%, with a 5-year event-specific probability of being LR-free of 40.0%. Tumor site/grade, need for shunting, age above/below 3 years, molecular subgroup at diagnosis, had no influence on outcomes. Due to variation in the RT dose/fractionation used and the subgroup sizes, it was not possible to assess the impact of the different RT modalities. Multivariable analyses identified completion of surgery, the absence of symptoms at relapse, and female sex as prognostically favorable. Tumors with a 1q gain carried a higher cumulative incidence of dissemination after first relapse., Conclusions: Survival after recurrence was significantly influenced by symptoms and completeness of surgery. Only a homogeneous protocol with well-posed, randomized questions could clarify the numerous issues, orient salvage treatment, and ameliorate prognosis for this group of patients., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Urine cytology is a non-invasive test used in combination with cystoscopy for screening and follow-up of urothelial carcinoma (UC). Although cytology can be used to efficiently identify high-grade UC, it has a lower accuracy for the diagnosis of low-grade UC or patients with presence of atypical urothelial cells (AUC). For these reasons, ancillary tests have been added to urine cytology in order to improve the accuracy. However, the poor abundance of neoplastic cells in most samples and the absence of a "tissue-like" structure remains a major challenge. We used a novel synthetic support called CytoMatrix which has the property of capturing and storing cells and micro-macro aggregates within its three-dimensional structure. The urine specimens were obtained from 12 patients: 6 with suspected urothelial neoplasia (low- and high-grade) and 6 with AUC or non-neoplastic samples. The first step is the urine samples preparation, through several centrifugation passages; the second step consists in absorbing cells on the CytoMatrix, and in the subsequent formalin fixation, standard processing and paraffin embedding to prepare FFPE-CytoMatrix block. In the final step, sections are consecutively cut, stained with hematoxylin-eosin (H&E), and analyzed via UroVysion FISH and immunohistochemistry (IHC). Using our simple and reliable protocol, we can improve the quality of urine specimens, allowing a better collection, maintenance, and analysis of cells, with the advantage of using ancillary tests to support cytological diagnosis and the advantage of storing cellular material in a FFPE-CytoMatrix block.

Background: A prospective 2002-2014 study stratified 160 patients by resection extent and histological grade, reporting results in 2016. We re-analyzed the series after a median of 119 months, adding retrospectively patients' molecular features., Methods: Follow-up of all patients was updated. DNA copy number analysis and gene-fusion detection could be completed for 94/160 patients, methylation classification for 68., Results: Progression-free survival (PFS) and overall survival (OS) at 5/10 years were 66/58%, and 80/73%. Ten patients had late relapses (range 66-126 mo), surviving after relapse no longer than those relapsing earlier (0-5 y). On multivariable analysis a better PFS was associated with grade II tumor and complete surgery at diagnosis and/or at radiotherapy; female sex and complete resection showed a positive association with OS. Posterior fossa (PF) tumors scoring ≥0.80 on DNA methylation analysis were classified as PFA (n = 41) and PFB (n = 9). PFB patients had better PFS and OS. Eighteen/32 supratentorial tumors were classified as RELA, and 3 as other molecular entities (anaplastic PXA, LGG MYB, HGNET). RELA had no prognostic impact. Patients with 1q gain or cyclin-dependent kinase inhibitor 2A (CDKN2A) loss had worse outcomes, included significantly more patients >3 years old (P = 0.050) and cases of dissemination at relapse (P = 0.007)., Conclusions: Previously described prognostic factors were confirmed at 10-year follow-up. Late relapses occurred in 6.2% of patients. Specific molecular features may affect outcome: PFB patients had a very good prognosis; 1q gain and CDKN2A loss were associated with dissemination. To draw reliable conclusions, modern ependymoma trials need to combine diagnostics with molecular risk stratification and long-term follow-up., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Mutations of the APC (adenomatous polyposis coli) gene correlate mainly with familial adenomatous polyposis (FAP), but can occasionally be pathogenic for medulloblastoma (MBL) wingless-related integration site (WNT) subtype, the course of which has only recently been described., Methods: We retrieved all patients with documented germline APC mutations and a diagnosis of MBL to examine their outcome, late effects of treatment, and further oncological events., Results: Between 2007 and 2016, we treated six patients, all with a pathogenic APC variant mutation and all with MBL, classic histotype. None had metastatic disease. All patients were in complete remission a median 65 months after treatment with craniospinal irradiation at 23.4 Gy, plus a boost on the posterior fossa/tumor bed up to 54 Gy, followed by cisplatin/carboplatin, lomustine, and vincristine for a maximum of eight courses. Five of six diagnostic revised MRI were suggestive of the WNT molecular subgroup typical aspects. Methylation profile score (in two cases) and copy number variation analysis (chromosome 6 deletion in two cases) performed on four of six retrieved samples confirmed WNT molecular subgroup. Four out of six patients had a positive family history of FAP, while gastrointestinal symptoms prompted its identification in the other two cases. Four patients developed other tumors (desmoid, MELTUMP, melanoma, pancreatoblastoma, thyroid Tir3) from 5 to 7 years after MBL., Discussion: Our data confirm a good prognosis for patients with MBL associated with FAP. Patients' secondary tumors may or may not be related to their syndrome or treatment, but warrant adequate attention when planning shared guidelines for these patients., (© 2021 Wiley Periodicals LLC.)

Purpose: The maintenance of telomere length prevents cancer cell senescence and occurs via two mutually exclusive mechanisms: (a) reactivation of telomerase expression and (b) activation of alternative lengthening of telomeres (ALT). ALT is frequently related to alterations on ATRX, a chromatin-remodelling protein. Recent data have identified different molecular subgroups of paediatric high-grade glioma (pHGG) with mutations of H3F3A, TERTp and ATRX; however, differences in telomere length among these molecular subgroups were not thoroughly examined., Methods: We investigated which genetic alterations trigger the ALT mechanism in 52 IDH-wildtype, 1p/19q-wildtype pHGG. Samples were analysed for telomere length using Tel-FISH. ATRX nuclear loss of expression was assessed by IHC, H3F3A and TERTp mutations by DNA sequencing, and TERTp methylation by MS-PCR., Results: Mutant H3.3 was found in 21 cases (40.3%): 19.2% with K27M mutation and 21.1% with G34R mutation. All H3.3G34R-mutated cases showed the ALT phenotype (100%); on the opposite, only 40% of the H3.3K27M-mutated showed ALT activation. ATRX nuclear loss was seen in 16 cases (30.7%), associated sometimes with the G34R mutation, and never with the K27M mutation. ATRX nuclear loss was always related to telomere elongation. TERTp C250T mutations were rare (5.4%) and were not associated with high intensity Tel-FISH signals, as TERTp hyper-methylation detected in 21% of the cases. H3.3/ATRX/TERTp-wildtype pHGG revealed all basal levels of telomere length., Conclusion: Our results show a strong association between H3.3 mutations and ALT, and highlight the different telomeric profiles in histone-defined subgroups: H3.3-G34R mutants always trigger ALT to maintain telomere length, irrespective of ATRX status, whereas only some H3.3-K27M tumours activate ALT. These findings suggest that acquiring the gly34 mutation on H3.3 might suffice to trigger the ALT mechanism.

Recurrent glycine-to-arginine/valine alterations at codon 34 (G34R/V) within H3F3A gene characterize a subset of hemispheric high-grade gliomas (HGG) affecting children and young adults. These tumors, defined as G34R/V-mutant gliomas, are histologically heterogenous, with microscopic features of either HGG or embryonal tumors (primitve neuroectodermal tumor-like features). To assess the value of immunohistochemistry (IHC) to detect G34R/V-mutated cases, we tested anti-histone G34V (clone 329E5) and anti-histone G34R (clone RM240) antibodies in a series of 28 formalin-fixed and paraffin-embedded samples. A total of 28 cases of hemispheric, IDH-wt HGG mainly affecting children and young adults were evaluated by IHC and by sequencing. The median age of patients at diagnosis was 17 years (0.1 to 26 y). By IHC, 10 of the 28 cases showed nuclear positivity for G34R and 3 of the 28 cases for G34V. Molecular analysis of G34R/V-mutation status was successful in 24 of the 28 cases. Mutation at glycine 34 of the H3F3A gene was identified in 9 of the 24 tumors (37%) by direct sequencing, revealing 7 of 9 positive case by sequencing and 2 of 9 false negative cases by IHC. Two of 15 negative case by sequencing demonstrated a false positivity by IHC. In total, in 4 (16.6%) of 24 cases, IHC and mutational results were discordant: 2 tumors were negative by IHC (false negative) but harbored G34R mutation by sequencing, and 2 cases were positive by IHC (false positive by IHC) but wild type by sequencing. Moreover, most mutated cases showed loss of ATRX expression and/or p53 expression. The positivity by IHC with specific antibody tested is not highly predictive for presence of G34R/V mutation, but confirmation by sequencing is mandatory; G34R/V mutations should be suspected in all hemispheric tumor IDH1/2 wild type, showing loss of OLIG2 and ATRX and/or p53 expression., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Glioblastoma (GBM) is the most common primary malignant brain cancer in adults, with very limited therapeutic options. It is characterized by a severe immunosuppressive milieu mostly triggered by suppressive CD163 + tumor-associated macrophages (TAMs). The efficacy of immune checkpoint inhibitor interventions aimed at rescuing anti-tumor immunity has not been proved to date. Thus, it is critically important to investigate the immunomodulatory mechanisms acting within the GBM microenvironment for the better design of immunotherapeutic strategies., Methods: The immunohistochemical analysis of a panel of immune biomarkers (CD3, FoxP3, CD163, IDO, PDL-1, PD-1 and TIGIT) was performed in paired samples of the tumor core (TC) and peritumoral area (PTA) of nine GBM patients., Results: CD163 + cells were the most common cell type in both the PTA and TC. IDO and PDL-1 were expressed in most of the TC samples, frequently accompanied by TIGIT expression; on the contrary, they were almost absent in the PTA. CD3 + cells were present in both the TC and PTA, to a lesser extent than CD163 + cells; they often were accompanied by PD-1 expression, especially in the TC. FoxP3 was scarcely present., Conclusion: Distinct inhibitory mechanisms can act simultaneously in both the TC and PTA to contribute to the strong immunosuppression observed within the GBM microenvironment. Nevertheless, the PTA shows strongly reduced immunosuppression when compared to the TC, thus representing a potential target for immunotherapies. Moreover, our results support the working hypothesis that immunosuppression and T-cell exhaustion can be simultaneously targeted to rescue anti-tumor immunity in GBM patients.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder caused by mutations in the TP53 gene, predisposing to a wide spectrum of early-onset cancers, including brain tumors. In medulloblastoma patients, the role of TP53 has been extensively investigated, though the prevalence of de novo mutations has not been addressed. We characterized TP53 mutations in a monocentric cohort of consecutive Sonic Hedgehog (SHH)-activated medulloblastoma patients. Germline testing was offered based on tumor p53 immunostaining positivity. Among 24 patients, three (12.5%) showed tumor p53 overexpression, of whom two consented to undergo germline testing and resulted as carriers of TP53 mutations. In the first case, family history was uneventful and the mutation was not found in either of the parents. The second patient, with a family history suggestive of LFS, unexpectedly resulted as a carrier of the mosaic mutation c.742=/C>T p.(Arg248=/Trp). The allele frequency was 26% in normal tissues and 42-77% in tumor specimens. Loss of heterozygosity (LOH) in the tumor was also confirmed. Notably, the mosaic case has been in complete remission for more than one year, while the first patient, as most TP53-mutated medulloblastoma cases from other cohorts, showed a severe and rapidly progressive disease. Our study reported the first TP53 mosaic mutation in medulloblastoma patients and confirmed the importance of germline testing in p53 overexpressed SHH-medulloblastoma, regardless of family history.

Introduction: Medulloblastoma is the most common malignant brain tumor in children, but accounts for only 1% of brain cancers in adults. For standard-risk pediatric medulloblastoma, current therapy includes craniospinal irradiation (CSI) at reduced doses (23.4 Gy) associated with chemotherapy. Whereas most same-stage adult patients are still given CSI at 36 Gy, with or without chemotherapy, we report here on our use of reduced-dose CSI associated with chemotherapy for older patients., Methods: We gathered non-metastatic patients over 18 years old (median age 28 years, range 18-48) with minimal or no residual disease after surgery, no negative histological subtypes, treated between 1996-2018 at the Centre Léon Bérard (Lyon) and the INT (Milano). A series of 54 children with similar tumors treated in Milano was used for comparison., Results: Forty-four adults were considered (median follow-up 101 months): 36 had 23.4 Gy of CSI, and 8 had 30.6 Gy, plus a boost to the posterior fossa/tumor bed; 43 had chemotherapy as all 54 children, who had a median 83-month follow-up. The PFS and OS were 82.2 ± 6.1% and 89 ± 5.2% at 5 years, and 78.5 ± 6.9% and 75.2 ± 7.8% at ten, not significantly different from those of the children. CSI doses higher than 23.4 Gy did not influence PFS. Female adult patients tended to have a better outcome than males., Conclusion: The results obtained in our combined series are comparable with, or even better than those obtained after high CSI doses, underscoring the need to reconsider this treatment in adults.

Medulloblastoma is a highly aggressive brain tumor that typically affects children, while in adults it represents ~1% of all brain tumors. Little is known about microRNA expression profile of the rare adult medulloblastoma. The main aim of this study was to identify peculiar differences in microRNA expression between childhood and adult medulloblastoma. Medulloblastomas were profiled for microRNA expression using the Exiqon Human miRNome panel (I + II) analyzing 752 microRNAs in a training set of six adult and six childhood cases. Then, the most differentially expressed microRNAs were validated in a total of 21 adult and 19 childhood cases. Eight microRNAs (miR-196b-5p, miR-183-5p, miR-200b-3p, miR-196a-5p, miR-193a-3p, miR-29c-3p, miR-33b-5p, and miR-200a-3p) were differentially expressed in medulloblastoma of adults and children. Analysis of the validation set confirmed that miR-196b-5p and miR-200b-3p were significantly overexpressed in medulloblastoma of adults as compared with those of children. We followed an in silico approach to investigate direct targets and the pathways involved for the two microRNAs (miR-196b and miR-200b) differently expressed between adult and childhood medulloblastoma. Adult and childhood medulloblastoma have different miRNA expression profiles. In particular, the differential dysregulation of miR-196b-5p and miR-200b-3p characterizes the miRNA profile of adult medulloblastoma and suggests potential targets for novel diagnostic, prognostic, or therapeutic strategies.

Purpose: The diagnosis of diffuse intrinsic pontine glioma (DIPG) is based largely on a combination of clinical and radiological findings due to the difficulty of obtaining a biopsy. An accurate evaluation of magnetic resonance imaging (MRI) scans is consequently essential. Recent analyses on the genomic landscape of DIPG revealed recurrent mutations in the H3F3A and HIST1H3B histone genes. We reviewed cases with available tumor tissue from institutional DIPG series to ascertain the consistency between their histo-molecular findings and clinical-radiological features., Methods: We conducted a radiological and pathological central review of 22 cases enrolled in institutional DIPG trials. We performed immunohistochemical analyses to detect H3F3A/HIST1H3B K27M mutations, histone trimethylation, and EZH2 expression. Mutational analysis was performed for ACVR1, H3F3A, and HIST1H3B genes., Results: Patients' median age at diagnosis was 8 years, and their median overall survival was 11 months. Nineteen/22 cases (86%) showed evidence of K27M mutation on immunohistochemistry and/or mutation analysis. Histone trimethylation expression was low or lacking in these mutated cases. Sequence analysis revealed 13 cases with H3F3A and 1 case with HIST1H3B K27M mutation. There was no significant difference in EZH2 expression between the K27M mutant and wild-type DIPGs. Upon external, blinded MRI re-evaluation one lesion not consistent with DIPG showed no evidence of K27M mutation and retained histone trimethylation expression., Conclusion: In conclusion, our study demonstrates a high frequency of histone K27M mutations in DIPG when MRI features are carefully assessed, thus confirming the consistency of imaging with biological markers in our institutional series of DIPG.

Purpose: Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB., Methods: We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR)., Results: H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT., Conclusions: Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.

Ependymomas (EPs) are tumors of the brain and spinal cord constituting ∼10% of the childhood central nervous system neoplasms and about 30% in children aged <3 years. Their anatomic distribution varies according to the age, with those arising in the supratentorial (ST) compartment, spinal cord being more common in older children and adults, and those at the infratentorial location are more common and occurring more frequently in infants and children. Recently, molecular classification of EP subgroups has been proposed and a supratentorial ependymoma subgroup characterized by RELA-fusion genes (ST-EP-RELA) has been established. It would be useful to define a standardized, robust method for the diagnosis of these relevant fusion genes. We used real-time polymerase chain reaction, conventional real-time polymerase chain reaction, and Sanger sequencing to characterize RELA fusion status in formalin-fixed paraffin-embedded samples from 42 ST-EPs (12 adults and 30 pediatric). We tested p65/RELA and L1CAM protein immunohistochemistry for their ability to predict RELA-fusion status. We reviewed clinical data to assess significant associations in this anatomic subgroup. Of the 42 patients, we identified RELA-fusion genes in 17 cases. L1CAM immunostaining displayed 94% sensitivity, 76% specificity, 73% positive predictive value (PPV), 95% negative predictive value (NPV). The p65/RELA immunostaining displayed 100% sensitivity, 92% specificity, 89.5% PPV, 100% NPV. Concordant double immunostaining improves PPV to 92.5% and maintains 100% NPV. Immunohistochemistry using both p65/RELA and L1CAM antibodies is valuable for ST-EP-RELA diagnosis: the negativity with both antibodies consistently predicts the absence of RELA fusions, whereas verification of fusion transcripts by molecular analyses is warranted only in single-positive or double-positive staining cases.

Purpose: The aims of patients' radiological surveillance are to: ascertain relapse; apply second-line therapy; accrue patients in phase 1/2 protocols if second-line therapy is not standardized/curative; and assess/treat iatrogenic effects. To lessen the emotional and socioeconomic burdens for patients and families, we ideally need to establish whether scheduled radiological surveillance gives patients a better outcome than waiting for symptoms and signs to appear., Methods: We analyzed a prospective series of 160 newly-diagnosed and treated pediatric/adolescent patients with intracranial ependymoma, comparing patients with recurrent disease identified on scheduled MRI (the RECPT group; 34 cases) with those showing signs/symptoms of recurrent disease (the SYMPPT group; 16 cases). The median follow-up was 67 months., Results: No significant differences emerged between the two groups in terms of gender, age, tumor grade/site, shunting, residual disease, or type of relapse (local, distant, or concomitant). The time to relapse (median 19 months; range 5-104) and the MRI follow-up intervals did not differ between the SYMPPT and RECPT groups. The presence of signs/symptoms was an unfavorable factor for overall survival (OS) after recurrence (5-year OS: 8% vs. 37%, p = 0.001). On multivariable analysis, an adjusted model confirmed a significantly worse OS in the SYMPPT than in the RECPT patients., Conclusions: Symptomatic relapses carried a significantly worse survival for ependymoma patients than recurrences detected by MRI alone. It would therefore be desirable to identify recurrences before symptoms develop. Radiological follow-up should be retained in ependymoma patient surveillance because there is a chance of salvage treatment for relapses found on MRI.

Chordomas arise in the skull base and spine and usually occur in adults and are rare in the pediatric population. Cases of chordoma in pediatric age are often poorly differentiated, showing cytologic atypia, increased cellularity, and mitosis, and their aggressive behavior is associated with a high incidence of metastatic spread and a short patient survival. Recent studies have described loss of SMARCB1/INI1 protein in poorly differentiated chordomas associated not with point mutations but with SMARCB1/INI1 gene deletions instead. In this study, we considered immunohistochemistry and SMARCB1/INI1 mutational status to examine SMARCB1 status in a series of pediatric chordomas (7 classic and 1 poorly differentiated). We performed immunohistochemical tests for INI1, brachyury, S100, and cytokeratins and conducted a genetic analysis on the SMARCB1 coding sequence (NM&#95;003073) using the Sanger method and multiplex ligation-dependent probe amplification to detect abnormal copy numbers of the gene locus. All 8 cases were positive for brachyury, whereas there was no nuclear SMARCB1/INI1 expression in 4 of the 8 cases, including the poorly differentiated chordoma. Genetic analysis identified a missense mutation in 2 cases and a nonsense mutation associated with loss of SMARCB1/INI1 protein and features of poorly differentiated tumor in 1. These mutations were novel variants occurring in heterozygosity, and they were judged to be pathogenic by 3 different bioinformatic tools. In 7 of 8 cases we performed multiplex ligation-dependent probe amplification, and 3 cases showed deletions at the SMARCB1 locus. Our results confirm the pathogenic involvement of SMARCB1/INI1 in childhood chordoma. We also describe 3 novel pathogenic mutations.

Background: This prospective study stratified patients by surgical resection (complete = NED vs incomplete = ED) and centrally reviewed histology (World Health Organization [WHO] grade II vs III)., Methods: WHO grade II/NED patients received focal radiotherapy (RT) up to 59.4 Gy with 1.8 Gy/day. Grade III/NED received 4 courses of VEC (vincristine, etoposide, cyclophosphamide) after RT. ED patients received 1-4 VEC courses, second-look surgery, and 59.4 Gy followed by an 8-Gy boost in 2 fractions on still measurable residue. NED children aged 1-3 years with grade II tumors could receive 6 VEC courses alone., Results: From January 2002 to December 2014, one hundred sixty consecutive children entered the protocol (median age, 4.9 y; males, 100). Follow-up was a median of 67 months. An infratentorial origin was identified in 110 cases. After surgery, 110 patients were NED, and 84 had grade III disease. Multiple resections were performed in 46/160 children (28.8%). A boost was given to 24/40 ED patients achieving progression-free survival (PFS) and overall survival (OS) rates of 58.1% and 68.7%, respectively, in this poor prognosis subgroup. For the whole series, 5-year PFS and OS rates were 65.4% and 81.1%, with no toxic deaths. On multivariable analysis, NED status and grade II were favorable for OS, and for PFS grade II remained favorable., Conclusions: In a multicenter collaboration, this trial accrued the highest number of patients published so far, and results are comparable to the best single-institution series. The RT boost, when feasible, seemed effective in improving prognosis. Even after multiple procedures, complete resection confirmed its prognostic strength, along with tumor grade. Biological parameters emerging in this series will be the object of future correlatives and reports., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Posterior fossa ependymoma comprises two distinct molecular variants termed EPN&#95;PFA and EPN&#95;PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known., Methods: Four independent nonoverlapping retrospective cohorts of posterior fossa ependymomas (n = 820) were profiled using genome-wide methylation arrays. Risk stratification models were designed based on known clinical and newly described molecular biomarkers identified by multivariable Cox proportional hazards analyses., Results: Molecular subgroup is a powerful independent predictor of outcome even when accounting for age or treatment regimen. Incompletely resected EPN&#95;PFA ependymomas have a dismal prognosis, with a 5-year progression-free survival ranging from 26.1% to 56.8% across all four cohorts. Although first-line (adjuvant) radiation is clearly beneficial for completely resected EPN&#95;PFA, a substantial proportion of patients with EPN&#95;PFB can be cured with surgery alone, and patients with relapsed EPN&#95;PFB can often be treated successfully with delayed external-beam irradiation., Conclusion: The most impactful biomarker for posterior fossa ependymoma is molecular subgroup affiliation, independent of other demographic or treatment variables. However, both EPN&#95;PFA and EPN&#95;PFB still benefit from increased extent of resection, with the survival rates being particularly poor for subtotally resected EPN&#95;PFA, even with adjuvant radiation therapy. Patients with EPN&#95;PFB who undergo gross total resection are at lower risk for relapse and should be considered for inclusion in a randomized clinical trial of observation alone with radiation reserved for those who experience recurrence., (© 2016 by American Society of Clinical Oncology.)

Pediatric high-grade gliomas are considered to be different when compared to adult high-grade gliomas in their pathogenesis and biological behavior. Recently, common genetic alterations, including mutations in the H3F3A/ATRX/DAXX pathway, have been described in approximately 30% of the pediatric cases. However, only few cases of infant high-grade gliomas have been analyzed so far. We investigated the molecular features of 35 infants with diffuse high-grade astrocytomas, including 8 anaplastic astrocytomas [World Health Organization (WHO) grade III] and 27 glioblastomas (WHO grade IV) by immunohistochemistry, multiplex ligation probe-dependent amplification (MLPA), pyrosequencing of glioma-associated genes and molecular inversion probe (MIP) assay. MIP and MLPA analyses showed that chromosomal alterations are significantly less frequent in infants compared with high-grade gliomas in older children and adults. We only identified H3F3A K27M in 2 of 34 cases (5.9%), with both tumors located in the posterior fossa. PDGFRA amplifications were absent, and CDKN2A loss could be observed only in two cases. Conversely, 1q gain (22.7%) and 6q loss (18.2%) were identified in a subgroup of tumors. Loss of SNORD located on chromosome 14q32 was observed in 27.3% of the infant tumors, a focal copy number change not previously described in gliomas. Our findings indicate that infant high-grade gliomas appear to represent a distinct genetic entity suggesting a different pathogenesis and biological behavior., (© 2014 International Society of Neuropathology.)

The KIAA1549:BRAF fusion gene is considered a driver genetic event in pilocytic astrocytoma. We investigated a series of 69 pediatric brain neoplasms of diverse histogenesis and grade using the RT-PCR and sequencing. We detected the KIAA1549:BRAF fusion gene in five of 34 non-PA tumors (14.7%), that is, one glioblastoma, one anaplastic astrocytoma, one anaplastic pleomorphic xanthoastrocytoma, 1 ependymoma, and 1 Atypical Teratoid Rhabdoid Tumor. Our study showed that the K-B, although uncommon, it can be detected in non-PA tumors of various histogenesis and grading., (© 2014 Wiley Periodicals, Inc.)

Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human β-catenin cDNA encoding a nondegradable form of β-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of β-catenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UW-B cells was statistically significantly lower than that of all the other control cells (p < 0.01), (C) the mortality of irradiated UW-B cells was statistically significantly higher than that of the controls and their nonirradiated counterparts (p < 0.05), and (D) morphological features of neuronal differentiation were observed in the Wnt-activated cells. In tissue samples, the Ki-67 labeling index (LI) was lower in β-catenin-positive samples compared to non-β-catenin positive ones. The Ki-67 LI median (LI = 40) of the nuclear β-catenin-positive tumor samples was lower than that of non-nuclear β-catenin-positive samples (LI = 50), but the difference was not statistically significant. Overall, our data suggest that activation of the Wnt pathway reduces the proliferation and invasion of MBs and increases the tumor's radiosensitivity.

Radiotherapy is the only treatment definitely indicated for diffuse pontine gliomas (DIPG). Findings on the role of EGFR signaling in the onset of childhood DIPG prompted the use of nimotuzumab, an anti-EGFR monoclonal antibody. Assuming a potential synergy with both radiotherapy and vinorelbine, a pilot phase 2 protocol was launched that combined nimotuzumab with concomitant radiation and vinorelbine. An amendment in July 2011 introduced re-irradiation at relapse. The primary endpoint for first-line treatment was objective response rate (CR + PR + SD) according to the RECIST. This report concerns the outcome of this strategy as a whole. Vinorelbine 20 mg/m(2) was administered weekly, with nimotuzumab 150 mg/m(2) in the first 12 weeks of treatment; radiotherapy was delivered from weeks 3 to 9, for a total dose of 54 Gy. Vinorelbine 25 mg/m(2) and nimotuzumab were given every other week thereafter until the tumor progressed or for up to 2 years. Re-irradiation consisted of 19.8 Gy, fractionated over 11 days. Baseline and latest MRIs were assessed blindly by an outside neuroradiologist. Twenty five children (mean age 7.4 years) were enrolled as of August 2009 (median follow-up 29 months). A response was observed in 24/25 patients (96 %). The nimotuzumab/vinorelbine combination was very well tolerated, with no acute side-effects. Eleven of 16 locally-relapsing patients were re-irradiated. One-year PFS and OS rates were 30 ± 10 % and 76 ± 9 %, respectively; 2-year OS was 27 ± 9 %; the median PFS and OS were 8.5 and 15 months, respectively. This strategy generated interesting results and warrants further investigation.

Background: Extraneural metastases (ENM) rarely occur in medulloblastoma (MBL) patients and only few cases of subcutaneous localizations have been described. ENM indicate an aggressive disease associated with a worse prognosis. The characterization of metastatic tumours might be useful to understand their pathogenesis and to identify the most appropriate therapeutic strategies., Case Presentation: We present the case of a child with Large Cell Anaplastic (LC/A) MBL, who developed multiple subcutaneous metastases in the scalp area after a ventriculo-peritoneal shunting procedure. The disease rapidly progressed and the child died despite chemotherapy and primary tumour surgical debulking.We molecularly classified the tumour as a group 3 MBL; in addition, we derived stem-like cells (SLC) from a metastatic lesion. Primary tumour, metastases and SLC were further analysed, particularly focusing on features linked to the cutaneous dissemination. Indeed, molecules involved in angiogenesis, cell invasion and epidermal growth factor signalling resulted highly expressed., Conclusions: The present report describes a very rare case of subcutaneous metastatic MBL. The tumour, metastases and SLC have been clinically, pathologically and molecularly characterized. Our case is an example of multidisciplinary approach aiming to characterize MBL aggressive behaviour.

Background: High-grade gliomas (HGGs) account for 15% of all pediatric brain tumors and are a leading cause of cancer-related mortality and morbidity. Pediatric HGGs (pHGGs) are histologically indistinguishable from their counterpart in adulthood. However, recent investigations indicate that differences occur at the molecular level, thus suggesting that the molecular path to gliomagenesis in childhood is distinct from that of adults. MicroRNAs (miRNAs) have been identified as key molecules in gene expression regulation, both in development and in cancer. miRNAs have been investigated in adult high-grade gliomas (aHGGs), but scant information is available for pHGGs., Methods: We explored the differences in microRNAs between pHGG and aHGG, in both fresh-frozen and paraffin-embedded tissue, by high-throughput miRNA profiling. We also evaluated the biological effects of miR-17-92 cluster silencing on a pHGG cell line., Results: Comparison of miRNA expression patterns in formalin versus frozen specimens resulted in high correlation between both types of samples. The analysis of miRNA profiling revealed a specific microRNA pattern in pHGG with an overexpression and a proliferative role of the miR-17-92 cluster. Moreover, we highlighted a possible quenching function of miR-17-92 cluster on its target gene PTEN, together with an activation of tumorigenic signaling such as sonic hedgehog in pHGG., Conclusions: Our results suggest that microRNA profiling represents a tool to distinguishing pediatric from adult HGG and that miR-17-92 cluster sustains pHGG.

Background: Medulloblastoma histological classification has gained in importance and newer treatment protocols will include histology stratification. We centrally reviewed medulloblastoma cases from past 10 years reassessing their histology to ascertain its prognostic significance., Methods: Samples from 125 consecutive patients (99 males; 10 under age 3 years) were reviewed according to the two WHO classifications of 2000/2007., Results: Eighty-two patients did not have metastases, the primary tumor was completely resected in 101. The median follow-up was 96 months. Treatment was: our institutional protocol, that is, hyperfractionated accelerated radiotherapy (HART), for 39 non-metastatic cases up to 2003; according to the European PNET IV protocol in 31 cases; a HART-based strategy in 39 metastatic cases; tailored to the age below 3 years and based on high-dose chemotherapy in 10; and tailored to the patients conditions in 7. The 5-year PFS/OS rates were 76% and 81%, respectively. Histology was classic in 93 cases, nodular/desmoplastic in 20, anaplastic/large-cell in 9, and with extensive nodularity (MBEN) in 3. Stratification by residual disease after resection, metastases, age, or protocols was not prognostic. Histology suggested 5-year PFS rates of 82% for the desmoplastic and MBEN variants, 78% for classic medulloblastoma, 44% for the anaplastic/large-cell variants (P = 0.01). Multivariable analysis demonstrated statistically significant difference in PFS by histology (P = 0.02), due to the poor prognosis of anaplastic/large-cell medulloblastoma., Conclusions: Tailoring treatments to known risk factors cancelled all prognostic differences, except for anaplasia (not considered as such within previous trials) which proved the most powerful prognostic factor, warranting appropriate treatment intensification., (Copyright © 2012 Wiley Periodicals, Inc.)

KIAA1549-BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of the KIAA1549-BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAF(v600E) mutation was also screened. The KIAA1549-BRAF fusion gene was evaluated by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549-BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAF(v600E) in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549-BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co-presence of 1p/19q loss, IDH mutations and KIAA1549-BRAF fusion. Among the 17 cases with KIAA1549-BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAF(v600E) mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co-deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549-BRAF fusion gene and BRAF(v600E) mutation may be responsible for deregulation of the Ras-RAF-ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co-present with IDH mutations and 1p/19q loss., (© 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.)

Several molecular biomarkers have been suggested as predictors of outcome for pediatric ependymomas but deserve further validation in independent case series. We analyzed intracranial ependymomas belonging to a series of 60 patients prospectively treated according to the protocol sponsored by the Italian Association of Pediatric Hematology-Oncology. We used a tissue microarray to analyze nucleolin (NCL), cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein 53 (TP53), and epidermal growth factor receptor (EGFR) by immunohistochemistry and by 1q gain by fluorescent in situ hybridization. The mRNA expression levels of EGFR, human telomerase reverse-transcriptase (HTERT), and Prominin 1 (PROM 1)/CD133 were evaluated by quantitative real-time PCR from cases with fresh-frozen tumor material available. Univariate and multivariate analyses of updated clinical data confirmed the prognostic significance of surgery (P < .01) and tumor grading (P < .05) for both relapse-free survival (RFS) and overall survival (OS). Among biomolecular markers, HTERT mRNA expression emerged with the strongest association with OS at multivariate analysis (hazard ratio [HR] = 9.9; P = .011); the 5-year OS was 84% versus 48% in the subgroups with HTERT median value <6 versus ≥ 6, respectively (P = .005). Five-year RFS was 46% versus 20% in the subgroups with low versus high NCL protein expression, respectively (P = .004), while multivariate Cox analyses gave suggestively high HRs for high versus low NCL (HR = 1.9; P = .090). The other genes tested were not significant at multivariate analyses, and genetic alterations of CDKN2A, TP53, EGFR, and HTERT loci were rare. The PROM1/CD133 cancer stem cell marker was strongly expressed at both RNA and protein levels in a substantial fraction of cases and was suggestively associated with a more indolent form of the disease. We conclude that NCL and HTERT represent the strongest prognostic biomarkers of RFS and OS, respectively, in our ependymoma case series.

The Ras signaling pathway, consisting of mitogen-activated protein kinase (MAPK) and PI3K/AKT signaling, is a prominent oncogenic pathways in adult diffuse gliomas, but few studies have evaluated such pathways in pediatric malignant gliomas. We investigated by immunohistochemistry MAPK and AKT signaling in a series of 28 pediatric high-grade gliomas (WHO grade III and IV). We sought a possible association of phospho-ERK (p-ERK) and phospho-AKT (p-AKT) with expression of other proteins involved in the Ras pathway, that is, YKL40, epidermal growth factor receptor (EGFR), EGFR vIII and c-Met. Moreover we correlated the expression of p-ERK and p-AKT with prognosis. No cases showed expression for c-Met and EGFR, and only one case was positive for EGFR vIII. YKL-40 protein was expressed in 43% of cases. We detected expression of p-ERK and p-AKT in 61% and 57%, respectively, of pediatric high grade gliomas. Statistical analysis comparing the two groups in term of high and low p-ERK and p-AKT expression showed a trend toward worse overall survival in patients with high expression of p-AKT. The activation of ERK and AKT suggest a possible role of this protein in inducing activation of the Ras signaling pathway in pediatric high-grade gliomas. Moreover high levels of p-AKT are associated with worse overall survival., (© 2011 Japanese Society of Neuropathology.)

Besides its action on the nervous system, dopamine (DA) plays a role on neural-immune interactions. Here we review the current evidence on the dopaminergic system in human peripheral blood lymphocytes (PBL). PBL synthesize DA through the tyrosine-hydroxylase/DOPA-decarboxylase pathway, and express DA receptors and DA transporter (DAT) on their plasma membrane. Stimulation of DA receptors on PBL membrane contributes to modulate the development and initiation of immune responses under physiological conditions and in immune system pathologies such as autoimmunity or immunodeficiency.The characterization of DA system in PBL gave rise to a further line of research investigating the feasibility of PBL as a cellular model for studying DA derangement in neuropsychiatric disorders. Several reports showed changes of the expression of DAT and/or DA receptors in PBL from patients suffering from several neuropsychiatric disorders, in particular parkinsonian syndromes, schizophrenia and drug- or alcohol-abuse. Despite some methodological and theoretical limitations, these findings suggest that PBL may prove a cellular tool with which to identify the derangement of DA transmission in neuropsychiatric diseases, as well as to monitor the effects of pharmacological treatments.

The modulation of expression of the dopamine transporter by dopaminergic drugs was investigated by flow cytometry in peripheral blood lymphocytes from patients suffering Parkinson's disease. An 8-week in vivo exposure to pramipexole (0.7 mg free base, 3 times a day) or ropinirole (12 mg, once daily), but not levodopa/carbidopa (100/25 mg, 3 times a day), significantly reduced the mean fluorescence intensity of the dopamine transporter in peripheral blood lymphocytes. These results demonstrate that levodopa differs from dopamine agonists in its regulation of dopamine transporter expression in peripheral blood lymphocytes.

The objective of this study was to evaluate, in a series of 43 pediatric high-grade gliomas (21 anaplastic astrocytoma WHO grade III and 22 glioblastoma WHO grade IV), the prognostic value of histological grading and expression of p53 and YKL-40. Moreover, mutational screening for TP53 and IDH1 was performed in 27 of 43 cases. The prognostic stratification for histological grading showed no difference in overall (OS) and progression-free survival (PFS) between glioblastomas and anaplastic astrocytomas. Overexpression of YKL40 was detected in 25 of 43 (58%) cases, but YKL-40 expression was not prognostic in terms of OS and PFS. p53 protein expression was observed in 13 of 43 (31%) cases but was not prognostic. TP53 mutations were detected in five of 27 (18%) cases (four glioblastomas and one anaplastic astrocytoma). Patients with TP53 mutation had a shorter median OS (9 months) and PFS (8 months) than those without mutations (OS, 17 months; PFS, 16 months), although this trend did not reach statistical significance (p = 0.07). IDH1 mutations were not detected in any of the cases analyzed. Our results suggest that in pediatric high-grade gliomas: (i) histological grading does not have strong prognostic significance, (ii) YKL-40 overexpression is less frequent than adult high-grade gliomas and does not correlate with a more aggressive behavior, (iii) TP53 mutations but not p53 expression may correlate with a more aggressive behavior, and (iv) IDH1 mutations are absent. These observations support the concept that, despite identical histological features, the biology of high-grade gliomas in children differs from that in adults, and therefore different prognostic factors are needed.