Biomolecular characterization of metastatic medulloblastoma and study of telomere lengthening control

Medulloblastoma (MDB) is a malignant embryonic brain tumor and occurs typically in pediatric patients. Medulloblastoma cells can disseminate through the cerebrospinal fluid in the leptomeningeal space; approximately 30% of children present metastasis at the onset and no gold standard treatment has been defined for these patients. Genetic, epigenetic and molecular analyses identified four molecular subgroups (WNT, SHH, group 3 and group 4), associated with prognostic stratification of patients; however, previous works in literature included only small amount of metastatic cases, not analyzed independently from the non-metastatic counterpart. Furthermore, recent studies evidenced that mechanisms of telomeres elongation can be activated in pediatric brain tumours and telomeres maintenance was enriched in SHH and group 3 non-metastatic MDB; however, elongation of telomeres was not previously investigated in metastatic medulloblastomas. Therefore, our aim is to characterize a series of 39 pediatric MDB, selected from a cohort of 60, with leptomeningeal dissemination at the onset, studying molecular features involved in malignancy, metastasis, telomeres elongation and senescence escape. We analyzed several biomarkers and we correlated results to outcome of patients, evaluating the prognostic relevance of molecular biomarkers and subgroups. Furthermore, we analyzed the activation of mechanisms involved in control of telomeres lengthening, in order to figure out if telomeres elongation could have a role in metastatic medulloblastomas. We show that distribution of metastatic MDB into four molecular subgroups is highly similar to the distribution of non-metastatic cases, reflecting a high molecular heterogeneity; interestingly, our molecular subgrouping system defines high-risk (group SHH, 3 and 4) and standard-risk (group WNT and Not Classifiable) patients. Furthermore, we evidence that FSTL5 over-expression is associated exclusively with groups 3/4 and with poor outcome of patients, highlighting that FSTL5 can be used to better define molecular subgroups, prognosis and risk stratification of metastatic medulloblastomas. In addition, we analyzed H3.3 and ATRX mutations, involved in activation of the Alternative Lengthening of Telomeres (ALT) pathway, and the mutation and methylation status of TERT promoter, involved in telomerase reactivation. We evidence that metastatic MDB activate elongation of telomeres both via telomerase (14%) and via ALT mechanism (27%), triggered by ATRX mutations; interestingly, ALT pathway is highly activated in our cohort compared to MDB previously analysed in literature (<5%), highlighting the differences between metastatic and non-metastatic tumors in control of telomeres elongation and senescence escape. Furthermore, metastatic MDB show a higher reactivation of telomerase compared to pHGG (0%), triggered by TERT promoter mutations in combination with hyper-methylation. Our findings suggest that immortalization of tumor cells in metastatic MDB is a common process to escape from senescence and characterizes all molecular subgroups. In conclusion, our results contribute to improve the current characterization of pediatric patients with metastatic medulloblastoma; however, further studies will be necessary to increase the number of cases and to analyze, with statistical significance, the molecular subgroups, FSTL5 expression, and telomeres elongation, which could be used to “personalize” treatments or develop targeted therapies, reducing the side effects of the current therapeutic protocols

Biomolecular characterization of metastatic medulloblastoma and study of telomere lengthening control

Medulloblastoma (MDB) is a malignant embryonic brain tumor and occurs typically in pediatric patients. Medulloblastoma cells can disseminate through the cerebrospinal fluid in the leptomeningeal space; approximately 30% of children present metastasis at the onset and no gold standard treatment has been defined for these patients. Genetic, epigenetic and molecular analyses identified four molecular subgroups (WNT, SHH, group 3 and group 4), associated with prognostic stratification of patients; however, previous works in literature included only small amount of metastatic cases, not analyzed independently from the non-metastatic counterpart. Furthermore, recent studies evidenced that mechanisms of telomeres elongation can be activated in pediatric brain tumours and telomeres maintenance was enriched in SHH and group 3 non-metastatic MDB; however, elongation of telomeres was not previously investigated in metastatic medulloblastomas. Therefore, our aim is to characterize a series of 39 pediatric MDB, selected from a cohort of 60, with leptomeningeal dissemination at the onset, studying molecular features involved in malignancy, metastasis, telomeres elongation and senescence escape. We analyzed several biomarkers and we correlated results to outcome of patients, evaluating the prognostic relevance of molecular biomarkers and subgroups. Furthermore, we analyzed the activation of mechanisms involved in control of telomeres lengthening, in order to figure out if telomeres elongation could have a role in metastatic medulloblastomas. We show that distribution of metastatic MDB into four molecular subgroups is highly similar to the distribution of non-metastatic cases, reflecting a high molecular heterogeneity; interestingly, our molecular subgrouping system defines high-risk (group SHH, 3 and 4) and standard-risk (group WNT and Not Classifiable) patients. Furthermore, we evidence that FSTL5 over-expression is associated exclusively with groups 3/4 and with poor outcome of patients, highlighting that FSTL5 can be used to better define molecular subgroups, prognosis and risk stratification of metastatic medulloblastomas. In addition, we analyzed H3.3 and ATRX mutations, involved in activation of the Alternative Lengthening of Telomeres (ALT) pathway, and the mutation and methylation status of TERT promoter, involved in telomerase reactivation. We evidence that metastatic MDB activate elongation of telomeres both via telomerase (14%) and via ALT mechanism (27%), triggered by ATRX mutations; interestingly, ALT pathway is highly activated in our cohort compared to MDB previously analysed in literature (<5%), highlighting the differences between metastatic and non-metastatic tumors in control of telomeres elongation and senescence escape. Furthermore, metastatic MDB show a higher reactivation of telomerase compared to pHGG (0%), triggered by TERT promoter mutations in combination with hyper-methylation. Our findings suggest that immortalization of tumor cells in metastatic MDB is a common process to escape from senescence and characterizes all molecular subgroups. In conclusion, our results contribute to improve the current characterization of pediatric patients with metastatic medulloblastoma; however, further studies will be necessary to increase the number of cases and to analyze, with statistical significance, the molecular subgroups, FSTL5 expression, and telomeres elongation, which could be used to “personalize” treatments or develop targeted therapies, reducing the side effects of the current therapeutic protocols

FSTL5 expression is a marker of Group C metastatic medulloblastomas

INTRODUCTION: Medulloblastoma (MB) is the most commonmalignant brain tumor in children. Four different molecular subgroups are recognized, which differ in gene expression, genomic aberrations, histology, demographics and survival:WNT and SHH groups, having specific mutations in the homonymous pathway, and groups C and D having several genetic alternations not specific to a single pathway. The gene for follistatin-like protein 5, FSTL5, is overexpressed in nonSHH/nonWNT MBs poorly characterized. Highexpression of FSTL5 is significantly associated with reduced event-free and overall survival in non-WNT/non-SHHMBs. The major aim of this project is to study the FSTL5 expression level in pediatric MBs with metastasis at the onset. METHOD: We investigated the protein expression of biomarkers involved in metastatic pathways by IHC and FSTL5 expression level by RT-PCR in 26 metastatic MBs samples and correlated these data with the outcomes by Kaplan-Meier statistic analysis. RESULTS: 83% of Group C MBs showed high level of FSTL5 while none of these presented down-expression. Low-expression level of FSTL5 was find in 60% of SHH MBs and none showed over-expression. Kaplan-Meier test revealed that, in our cohort, highexpression ofFSTL5didnot correlatewithworse outcomewhile lowexpression of FSTL5 was associated with good prognosis and the co-presence of FSTL5 with other biomarkers correlated with poorer prognosis. CONCLUSION: FSTL5 is a marker of Group C in medulloblastomas with metastasis at the onset and the results highlighted decreased FSTL5 expression as a marker of good prognosis. Group C MBs have characteristic molecular features that confirm the poorest outcome also inMBs with metastasis at the onset

Brafv600e and Ctbn1 Mutational Study in Rathke's Cleft Cysts

Aim: Rathke's cleft cysts and craniopharyngiomas tipically involve sellar region and their histogenetic relationship is still matter of debate. Clinical and histopathologic differentiation of cystic lesions from the sellar region, that is, craniopharyngiomas (CPs) and Rathke cleft cysts (RCCs), is challenging and has great importance with respect to variable clinical manifestation and adapted surgical treatment strategies in both entities. The recent acquisition that adamantinomatous and papillary craniopharyngiomas bear distinct molecular alterations i.e., β-catenin (CTNNB1) and BRAFv600 mutations respectively, has suggest to screen for such alteration a series of Rathke cyst to seek a possible relation with one of the two craniopharyngioma type. Methods: Seven Rathke's cleft cysts were analyzed for BRAF and CTNNB1 mutational status by sequencing and immunohistochemistry. Radiological, clinical and histological features were performed. Results: None of the 7 Rathke's cleft cysts harbor BRAFV600E mutation. No CTNNB1 mutation was found. Radiological, clinical and histological re-evaluation of the cases confirmed the diagnosis of Rathke's cleft cysts. Conclusion: BRAFV600E and CTNNB1 mutations appeared, as most reliable factor for the differentiation between purely cystic CPs and RCCs, whereas tumor location, tumor size, and radiological parameter of the tumor were less consistent parameters. This study again confirms that craniopharyngiomas (CPs) and Rathke cleft cysts (RCCs), are associated with distinct pathogenic pathways

Role of 1p/19q Codeletion in Diffuse Low-grade Glioma Tumour Prognosis

Background/Aim: In the latest 2021 WHO classification of central nervous system tumours (CNS), gliomas that present isocitrate dehydrogenase (IDH) mutations are defined as diffuse low-grade gliomas (DLGGs). IDH mutations are commonly observed in this tumour type. The Extent of Resection (EOR) positively influence survival; however, it is still debated whether the predictive value of EOR is independent of the 1p/19q co-deletion. We carried out a retrospective analysis on patients operated on for DLGG at the Sant’Andrea University Hospital Sapienza University of Rome, correlating the outcome with the presence of 1p/19q co-deletion and EOR. Patients and Methods: The study examined 66 patients with DLGG who had undergone surgery for tumour resection between 2008 and 2018. Patients with DLGG were divided into two groups; diffuse astrocytoma (DA) in which 1p/19q codeletion is absent and oligodendroglioma (OG) in which 1p/19q codeletion is present. According to EOR, both groups were divided into two subgroups: subtotal resection (STR) and gross total resection (GTR). Three end-point variables were considered: overall survival (OS), progression-free survival (PFS) and time to malignant transformation (TMT). Results: In the DA group, the GTR subgroup had an average OS of 81.6 months, an average PFS of 45.9 months and an average TMT of 63.6 months. After surgery, these patients had an average Karnofsky Performance Score (KPS) of 83.4. The STR subgroup had an average OS of 60.4 months, PFS was 38.7 months, and TMT was 46.4 months, post-operative KPS was 83.4. In contrast, in the OG group, the GTR averagely had 101.7 months of OS, 64.9 months of PFS, 80.3 months of TMT and an average post-operative KPS of 84.2, and the STR subgroup had an average of OS of 73.3 months, PFS of 48.2 months, TMT of 57.3 and an average postoperative KPS of 96.2. Conclusion: In patients affected by DLGGs, 1p/19q codeletion is significantly associated with prolonged survival and longer time-to-malignant transformation (TMT) compared to the absence of 1p/19q codeletion. Also, the extent of surgical resection (EOR) in DLGG patients has been confirmed as one of the main prognostic factors. However, its predictive value is substantially influenced by the presence of the 1p/19q codeletion

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

Mechanisms of telomere maintenance in pediatric brain tumors: promising targets for therapy – a narrative review

Recent advances in genetic and molecular characterization of telomere maintenance mechanisms (TMMs) highlighted their strong relationship with cancer pathogenesis; neoplastic cells rely on two mechanisms to maintain telomere length and escape from replicative senescence: (a) reactivation of telomerase expression and (b) activation of alternative lengthening of telomere (ALT). Our aims are to describe the role of telomere maintenance in the context of recently published literature regarding pediatric brain cancers and to discuss the emerging therapeutic strategies to target telomerase-positive and ALT-positive tumors. In this review, we illustrate the incidence of TMM via telomerase or ALT and discuss the importance of analyzing telomere length and ALT-associated genetic alterations in certain histological/molecular subtypes of pediatric brain tumors, as potential therapeutic biomarkers. Telomerase-dependent TMM is a common mechanism in SHH-medulloblastomas and ependymomas, which could potentially benefit from antitelomerase therapies, while ALT-dependent TMM is more frequently activated in α-thalassemia/mental retardation syndrome X-linked/H3.3-mutated pediatric high-grade gliomas, metastatic medulloblastomas, and choroid plexus tumors, which could potentially be treated with ALT-targeted drugs. Conversely, pediatric low-grade gliomas lack both mechanisms of telomere maintenance, and anti-TMM therapies do not appear to be a promising strategy for these tumors

Letter to the editors: pathogenetic analysis of sinonasal teratocarcinosarcomas reveal actionable β-catenin overexpression and a β-catenin mutation

We read with great interest the article “Pathogenetic Analysis of Sinonasal Teratocarcinosarcomas Reveal Actionable β- Catenin Overexpression and a β-Catenin Mutation” by Birkeland et al.1 In the article, the authors performed targeted exome sequencing on an index Sinonasal Teratocarcinosarcoma (SNTCS) specimen and identified an activating mutation in the β-catenin gene (CTNNB1, c.134C &gt; T, p.S45F). In addition, they confirmed β-catenin overexpression and nuclear localization via immunohistochemistry in the index tumor and in a subsequent case. Based on their findings, the authors suggested “a role for the Wnt/β-catenin pathway in SNTCS tumorigenesis,” postulated that “this mutation is a potential genetic driver mutation and an alluring prospect for treatment using inhibitors of the Wnt/β-catenin pathway” and underlined the importance “to screen for p.S45F mutations and other mutations/aberrations in β-catenin in other teratocarcinosarcoma specimens to identify if this is a common driver mutation in these tumors.

Distribution and prognostic impact of molecular subgroups in a homogeneously treated series of metastatic medulloblastoma

Medulloblastoma (MDB) is the most common malignant paediatric brain tumor. Prognostic system based on clinical parameters and histopathological variants is commonly used in clinical practice. Four different molecular subgroups are recognized: WNT and SHH, having specific homonymous pathwayalterations;Cand D, havingseveral genetic alterationsand associated to a worse outcome, but the system has not been prospectively validated in metastatic cohorts. Purpose of this studywasto evaluate distribution andprognostic impact of the four molecular subgroups in 47 MDB metastatic at the onset, homogenously treated in a single institution (Gandola et al, JCO 2009). Subgroup biomarkers were investigated by IHC, RT-PCR, mRNA sequencing, FISH; results were correlated with patient outcomes by Kaplan-Meier. We identify 11% WNT with nuclear b-catenin, 19% SHH, 26%groupCand15%group D;29%were unclassifiable (NC) having heterogeneous biomarkers. MYC amplification was more frequent (32.5%) compared to MYCN (2.7%). WNT and NC groups showed longer (not significant) OSand PFS compared to SHH,Cand D. Furthermore, low expression of FSTL5 was associated with good prognosis (OS rate 90%, PFS rate 100%), while FSTL5 higher expression correlate with worse outcome (OS and PFS rate 66%); difference was statistically significant (p ¼ 0,05). We have previously shown that histological variants maintain prognostic value in metastatic MDB; on the opposite, molecular sub-grouping is inefficient to allow a better risk stratification in our metastatic MDB cohort; furthermore, FSTL5 gene expression might be used in metastatic MDB as prognostic factor to better define patient outcome