Optimizing reirradiation for relapsed medulloblastoma: identifying the ideal patient and tumor profiles.

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.

Case report of a pediatric medulloblastoma with concurrent MYC and MYCN subclonal amplification in distinct populations of neoplastic cells.

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.

Optimizing biomarkers for accurate ependymoma diagnosis, prognostication, and stratification within International Clinical Trials: A BIOMECA study.

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.

Paediatric-type diffuse high-grade gliomas in the 5th CNS WHO Classification.

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.

A Custom DNA-Based NGS Panel for the Molecular Characterization of Patients With Diffuse Gliomas: Diagnostic and Therapeutic Applications.

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.

Alternative Lengthening of Telomeres (ALT) and Telomerase Reverse Transcriptase Promoter Methylation in Recurrent Adult and Primary Pediatric Pituitary Neuroendocrine Tumors.

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.

Treatment and outcome of intracranial ependymoma after first relapse in the 2nd AIEOP protocol.

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.

NSD1 Mutations and Pediatric High-Grade Gliomas: A Comparative Genomic Study in Primary and Recurrent Tumors.

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.

Correction: Visani et al. miR-196B-5P and miR-200B-3P Are Differentially Expressed in Medulloblastomas of Adults and Children. Diagnostics 2020, 10, 265.

The authors wish to make the following corrections to this paper [...].

Improvement of the Collection, Maintenance, and Analysis of Neoplastic Cells from Urine Specimens with the Use of CytoMatrix.

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.

Medulloblastoma and familial adenomatous polyposis: Good prognosis and good quality of life in the long-term?

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.

Correlation Between Immunohistochemistry and Sequencing in H3G34-Mutant Gliomas.

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.

Second series by the Italian Association of Pediatric Hematology and Oncology of children and adolescents with intracranial ependymoma: an integrated molecular and clinical characterization with a long-term follow-up.

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.

Alternative lengthening of telomeres in molecular subgroups of paediatric high-grade glioma.

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.

Immunohistochemical Characterization of Immune Infiltrate in Tumor Microenvironment of Glioblastoma.

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.

Pre- and Post-Zygotic TP53 De Novo Mutations in SHH-Medulloblastoma.

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.

Reduced-dose craniospinal irradiation is feasible for standard-risk adult medulloblastoma patients.

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.

miR-196B-5P and miR-200B-3P Are Differentially Expressed in Medulloblastomas of Adults and Children.

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.

Retrospective analysis on the consistency of MRI features with histological and molecular markers in diffuse intrinsic pontine glioma (DIPG).

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.

Telomere elongation via alternative lengthening of telomeres (ALT) and telomerase activation in primary metastatic medulloblastoma of childhood.

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.