WNT-activated Medulloblastoma Research Articles

MDB-02. RECURRNET SPINAL DESMOID FIBROMATOSIS IN A CHILD WITH APC GERMLINE MUTATION-RELATED MEDULLOBLASTOMA

Abstract BACKGROUND Familial adenomatous polyposis (FAP) is an autosomal-dominant dominant Familial Cancer Syndrome FCs Caused by Germline inactivation of adenomatous polyposis coli (APC) tumor suppressor gene, results in up-regulation of the WNT signaling pathway which increases the risk of colorectal cancer CRC, colon polyposis and extracolonic tumors like gastric and duodenal carcinomas WNT-activated Medulloblastoma MBL and Desmoid fibromatosis (DF) DF are locally aggressive soft tissue tumors with high local recurrence rates after Surgical excision role of adjuvant chemotherapy or Radiotherapy is still unclear. METHODS A 6-year-old boy product of non-consanguineous marriage with positive family HX brain tumor Diagnosed with Non-Metastatic Average Risk MBL WHO grade IV classic histology, he had craniotomy and Gross tumor resection followed by craniospinal irradiation at 23.4 Gy, plus a boost to posterior fossa/tumor bed up to 54 Gy, followed by 8 maintenance chemotherapy cycles as HIT-MED protocol Cisplatin/VCR/CCNU total. 3 years post end of therapy the pateint was in complete remission from MBL when routing MRI surveillance showed a new left paravertebral muscle lesion within the irradiation field from T1 to T4 levels which progressed gradually over a short follow-up time he had surgical resection of the tumor with Pathological diagnosis of Desmoid fibromatosis. after six months he had a second recurrence of the spinal tumor that needed a second surgical resection. Result: positive Genetic testing for Germline pathogenic mutation of APC gene exons 7 to 16 confirmed the diagnosis of autosomal familial adenomatous polyposis type 1(FAP1). Up to date, he is stable on endoscopic and MRI surveillance with radiological evidence of slow progression of the paraspinal tumor CONCLUSION Medulloblastomas associated with APC germline mutation have favorable outcomes that may need de-escalating therapeutic protocol with reduced intensity craniospinal irradiation aiming to reduce the incidence of secondary tumors after adjuvant treatment for MBL

Cribriform-morular Thyroid Carcinoma Arising in a Medulloblastoma Survivor: Two Metachronous Tumors Shared with the Activation of the Wnt Signaling Pathway.

Wnt signaling pathway activation is involved in the pathogenesis of a series of malignant tumors and is characterized by the nuclear accumulation of β-catenin protein. The occurrence of two or more Wnt pathway-associated tumors in a single individual is uncommon and generally attributed to inherited cancer syndrome, especially familial adenomatous polyposis (FAP). Herein, we presented a rare case of a child who suffered from the occurrence of Wnt-activated medulloblastoma and cribriform-morular thyroid carcinoma (CMTC) within a 9-year interval. She had no history of FAP and harbored an unexpected somatic mutation of the APC gene in the CMTC tumor. The potential agents involved in the pathogenesis of the two molecular-linked tumors other than FAP were discussed in this report.

WNT-Activated Medulloblastoma in A 6-Year-Old Boy

<p><strong>Background:</strong> Medulloblastoma is the most common malignant brain tumor of childhood. Prognosis is mostly favorable, but may be affected by histological and molecular subtypes. Long-term therapy-related morbidity also remains a significant concern.</p><p><strong>Case Presentation</strong>: A 6-year-old boy with brainstem/midline cerebellum tumor. Histopathology found area of necrosis, sheets of malignant undifferentiated round cells with hyperchromatic nuclei and scanty cytoplasm, no nodule and no anaplasia. Immunohistochemistry found p53 wild-type staining pattern along with b-catenin diffuse cytoplasmic and focal nuclear staining. This indicated a diagnosis of WNT-activated medulloblastoma, World Health Organization (WHO) grade IV, with classic histological features.</p><p><strong>Discussion: </strong>WNT-activated medulloblastomas with classic histological features and no anaplasia were reported to have the most favorable prognosis. The current patient showed negative staining for GFAP, Olig2, EMA, H3K27M, EZHIP, and LIN28A, with retained staining for INI1 and BRG1, thus excluding several differential diagnosis such as atypical teratoid/rhabdoid tumor, embryonal tumor with multilayered rosettes, small cell glioblastoma, Ewing sarcoma, high-grade neuroepithelial tumor with BCOR alteration or diffuse midline glioma. Histopathology in combination with immunohistochemical and molecular subtyping of medulloblastoma can help to refine diagnosis, exclude differential diagnosis, and improve counseling in regards to overall prognosis.</p>

High frequency of WNT-activated medulloblastomas with CTNNB1 wild type suggests a higher proportion of hereditary cases in a Latin-Iberian population.

Medulloblastomas are the most common primary malignant brain tumors in children. They are divided into molecular subgroups: WNT-activated, SHH-Activated, TP53 mutant or wild type, and non-WNT/non-SHH (Groups 3 and 4). WNT-activated medulloblastomas are usually caused by mutations in the CTNNB1 gene (85%-90%), and most remaining cases of CTNNB1 wild type are thought to be caused by germline mutations in APC. So far, the frequencies of CTNNB1 have been reported mainly in North American and European populations. The aim of this study was to report the frequency of CTNNB1 mutations in WNT-activated medulloblastomas in a Latin-Iberian population and correlate with their clinicopathological characteristics. A total of 266 medulloblastomas from seven different institutions from Brazil (n=211), Portugal (n=38), and Argentina (n=17) were evaluated. Following RNA and DNA isolation from formalin-fixed, paraffin-embedded (FFPE) tumor tissues, the molecular classification and CTNNB1 mutation analysis were performed by nCounter and Sanger sequencing, respectively. WNT-activated medulloblastomas accounted for 15% (40/266) of the series. We observed that 73% of WNT-activated medulloblastomas harbored CTNNB1 mutations. CTNNB1 wild-type cases (27%) were more prevalent in female individuals and suggested to be associated with a worse outcome. Among the CTNNB1 wild-type cases, the available analysis of family history revealed two cases with familiar adenomatous polyposis, harboring APC germline variants. We observed a lower incidence of CTNNB1 mutations in WNT-activated medulloblastomas in our Latin-Iberian cohort compared to frequencies previously described in other populations. Considering that CTNNB1 wild-type cases may exhibit APC germline mutations, our study suggests a higher incidence (~30%) of hereditary WNT-activated medulloblastomas in the Latin-Iberian population.

A Pilot Study Omitting Radiation in the Treatment of Children with Newly Diagnosed Wnt-Activated Medulloblastoma.

Treatment of wingless (WNT)-activated medulloblastoma (WNT+MB) with surgery, irradiation (XRT), and chemotherapy results in excellent outcomes. We studied the efficacy of therapy de-intensification by omitting XRT entirely in children with WNT+MB. Tumors were molecularly screened to confirm the diagnosis of WNT+MB. Eligible children were treated within 31 days following surgery with nine cycles of adjuvant chemotherapy per ACNS0331. No XRT was planned. The primary endpoint was the occurrence of relapse, progression, or death in the absence of XRT within the first two years after study enrollment. Four events in the first 10 evaluable patients would result in early study closure. Fourteen children were prescreened, and nine met the protocol definition of WNT+MB. Six of the nine eligible patients consented to protocol therapy, and five completed planned protocol therapy. The first two children enrolled relapsed shortly after therapy completion with local and leptomeningeal recurrences. The study was closed early due to safety concerns. Both children are surviving after XRT and additional chemotherapy. A third child relapsed at completion of therapy but died of progressive disease 35 months from diagnosis. Two children finished treatment but immediately received post-treatment XRT to guard against early relapse. The final child's treatment was aborted in favor of a high-dose therapy/stem cell rescue approach. Although OS at 5 years is 83%, no child received only planned protocol therapy, with all receiving eventual XRT and/or alternative therapy. Radiotherapy is required to effectively treat children with WNT-altered medulloblastoma. See related commentary by Gottardo and Gajjar, p. 4996.

Genetic alterations of TP53 and OTX2 indicate increased risk of relapse in WNT medulloblastomas

This study aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in WNT-activated medulloblastoma (WNT-MB). In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by DNA sequencing. Chromosomal copy-number aberrations were assessed by molecular inversion probe technology (MIP), SNP6, or 850k methylation array hybridization. Prognostic impact was evaluated in 120 patients with follow-up data from the HIT2000 medulloblastoma trial or HIT registries. CTNNB1 mutations were present in 92.2%, and APC mutations in 6.8% of samples. One CTNNB1 wild-type tumor gained WNT activation due to homozygous FBXW7 deletion. Monosomy 6 was present in 78.6%, and more frequent in children than adults. 16.1% of tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. Loss of heterozygosity at the TP53 locus (chromosome 17p13.1) was found in 40.7% (11/27) of TP53 mutant tumor samples and in 12.6% of TP53 wild-type cases (13/103). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; 5-year-PFS 68% versus 93%, p = 0.001), and were enriched for chromosomes 17p (p = 0.001), 10, and 13 losses. Gains of OTX2 (14q22.3) occurred in 38.9% of samples and were associated with poor PFS and OS (5-year-PFS 72% versus 93%, p = 0.017 resp. 5-year-OS 83% versus 97%, p = 0.006). Multivariable Cox regression analysis for PFS/OS identified both genetic alterations as independent prognostic markers. Our data suggest that patients with WNT-MB carrying TP53 mutations or OTX2 gains (58.1%) are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

Immunohistochemical staining of LEF-1 is a useful marker for distinguishing WNT-activated medulloblastomas

ObjectivesTo investigate lymphoid enhancer factor 1 (LEF-1) protein expression in medulloblastomas (MBs) and its correlation with molecular grouping of MBs.MethodsExpressions of LEF-1 and β-catenin were detected by immunohistochemistry, and molecular grouping was performed based on the NanoString and sequencing techniques for 30 MBs.ResultsBy genetic defining, 3 MBs were WNT-activated, 11 were SHH-activated, 3 were in Group 3 and 13 in Group 4 respectively. Nuclear LEF-1 staining was found in 8 MBs using immunohistochemical method. Three out of 8 showed diffuse and strong nuclear LEF-1 staining which were proved to be WNT-activated genetically, while the other 5 MBs with focal staining were SHH-activated genetically. The expression of LEF-1 protein was significantly correlated with genetically defined WNT-activated MBs (P < 0.0001). We also found focal nuclear β-catenin expression ( less than 1% of tumor cells) in 5 MBs. LEF-1 positivity was significantly correlated nuclear β-catenin expression (p < 0.001).ConclusionsImmunohistochemical staining of LEF-1 can be used as a supplement for β-catenin to diagnosis WNT-activated Medulloblastomas, when β-catenin is difficult to recognize for its cytoplasm/membrane staining background. Diffuse nuclear staining of LEF-1 indicates WNT-activated MB.

JS04.4.A Beyond β-catenin: Genetic alterations ofTP53 andOTX2 and older age indicate increased risk of relapse in WNT medulloblastomas

Abstract Background This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of age, TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers. Material and Methods In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Association with prognosis was evaluated in 133 patients with follow-up data from the HIT2000 medulloblastoma trial, HIT registries, and the NOA-07 trial. Results CTNNB1 mutations were present in 92.2% of the samples. APC mutations were found in 6.8% (13 samples). One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, and more frequent in children compared to adolescents/adults (≥16 years). Adolescents/adults showed worse overall survival (OS; p=0.009) compared to children, but not worse progression-free survival (PFS; p=0.106). With an age cut-off at 18 years, no survival difference was found. Also adolescents alone (16-20 years) had worse OS (p=0.003) compared to children, whereas in patients ≥21 (n=12 adults with PFS/OS data) no tumor progression/relapse occurred. Only one adult died due to therapy-related complications. WNT-MB patients with tumors harboring TP53 mutations (24/133, 18.1%) showed significant worse PFS (p=0.001), which was also found in children and adolescents individually (p=0.004, resp. p=0.017). Gains of the OTX2 locus on chromosome 14q found in 40.2% (35/87) of samples were independent of TP53 mutations and also associated with poor PFS and OS (p=0.034, resp. p=0.016). Individual analyses of OTX2 gains within age groups showed only worse OS in children (p=0.012). Multivariate Cox regression analysis for PFS identified both genetic alterations, but not age, as independent prognostic markers. For OS, multivariate analysis found OTX2 gains and older age as independent prognostic markers. Conclusion Our data suggest that adolescent patients with WNT-MB and those patients carrying TP53 mutations or OTX2 gains - independent of age - are at higher risk of relapse. Eligibility of these patients for therapy de-escalation trials needs to be debated.

MEDB-61. Genetic alterations ofTP53 andOTX2 indicate increased risk of relapse in WNT medulloblastomas

PURPOSE: This genetic analysis of WNT-activated medulloblastomas (WNT-MBs) aimed to re-evaluate the prognostic impact of TP53 mutations and to identify specific chromosomal aberrations as possible prognostic markers in a retrospective cohort of patients treated according to the protocols of the HIT medulloblastoma studies. PATIENTS AND METHODS: In a cohort of 191 patients with WNT-MBs, mutations in CTNNB1, APC, and TP53 were analyzed by Sanger and/or NGS panel sequencing. Chromosomal copy number aberrations (CNAs) were assessed by high-resolution, genome-wide molecular inversion probe technology (MIP), SNP6 array, and/or 850k methylation bead-array hybridization. Complete clinical data were available from 120 patients. RESULTS: Patients with WNT-MBs had a female predominance (1.4:1) and a median age of 13 years (range 3-69 years). CTNNB1 mutations were present in 92.2% of the samples, APC mutations in 6.8%. One CTNNB1 wildtype tumor gained WNT-activation due to a homozygous deletion of FBXW7. Monosomy 6 was present in 78.6%, but more frequent in children compared to adults. 16.1% of the tumor samples showed TP53 mutations, of those 60% with nuclear positivity for the p53 protein. A loss of heterozygosity at the TP53 locus on chromosome 17p13.1 was found in 40.7% (11/27) of TP53 mutant tumor samples and in 18.5% of the whole cohort (24/130 cases). Patients with tumors harboring TP53 mutations showed significant worse progression-free survival (PFS; p=0.001), but not overall survival (OS) and were enriched for chromosomes 17p (p=0.001), 10, and 13 losses. Gains of the OTX2 locus on chromosome 14q were also associated with poor PFS and OS (p=0.017 resp. p=0.006). Multivariate Cox regression analysis identified both genetic alterations as independent prognostic markers for PFS and OS. CONCLUSION: For ongoing and future de-escalation trials for patients with WNT medulloblastomas, we recommend to exclude patients with tumors carrying TP53 mutations or OTX2 gains.

MEDB-32. Reducing treatment-related toxicity for children with WNT-activated medulloblastoma

WNT-medulloblastoma has an excellent prognosis, with an overall survival rate of 90% among children receiving standard-of-care (SOC) surgical resection, radiotherapy, and chemotherapy. Unfortunately, while curative, this treatment is associated with major, long-term, debilitating motor, developmental, and neuroendocrine side effects. Therefore, it is crucial we develop effective, less toxic therapies for these children. Similarities have been demonstrated between cancer cell lysosomes and those of patients with Niemann-Pick, a lysosomal storage disease characterised by lysosomal fragility and sphingomyelin accumulation. A class of drugs known as Functional Inhibitors of Acid Sphingomyelinase (FIASMAs), increase lysosomal sphingomyelin and destabilise the cancer cell’s more fragile lysosomal membrane which leads to the induction of cell-death pathways via lysosomal membrane permeabilisation. Loratadine, an antihistamine with high FIASMA activity, consistently induced lysosomal membrane permeabilisation, leading to increased cell-death, in our panel of mouse and human WNT-medulloblastoma lines. Loratadine exhibited no detrimental effect on normal mouse embryonic stem cells from the lower rhombic lip – the putative cell of origin in WNT-medulloblastoma. Luciferase-expressing mouse WNT-medulloblastoma cells were orthotopically implanted into CD1-nude mice and monitored for tumour development via bioluminescent imaging. Upon tumour engraftment, mice were subjected to reduced SOC (radiotherapy and adjuvant vincristine) plus a clinically relevant dose of loratadine. Response and survival were compared to mice treated with full SOC (radiotherapy, vincristine, cisplatin, and etoposide). Mice treated with 2mg/kg/day of loratadine following reduced SOC demonstrated increased survival when compared to those treated with full SOC (p=0.02) along with a significant reduction in weight loss during treatment (p=<0.0001). This work suggests that loratadine, or other FIASMA compounds, may be good alternative adjuvant therapies for WNT-medulloblastoma. Using less toxic adjuvants could improve long-term outcomes through reducing therapeutic related toxicities for children with this devastating disease.

MEDB-65. Molecular subclassification of a national cohort of pediatric medulloblastoma based on methylation profile

INTRODUCTION: Pediatric Medulloblastoma (MB) accounts for approximately 20% of all childhood brain tumors. Molecular subgroups namely WNT, SHH, Group 3 and Group 4, exhibit divergent biology, and clinical outcomes. DNA methylation analysis is a robust option to classify pediatric MB into molecular subgroups, which allows the optimization of diagnosis and stratification of the treatment. We review the first experience of molecular subclassification carried out at the national level in our country. METHODS: Multi-center centralized prospective and retrospective study of frozen tumor samples at diagnosis from pediatric MB patients diagnosed in Spanish hospitals, from April 2021 to December 2021. A registry was created with histology review, immunohistochemical (IHC) subgrouping, and a molecular subgrouping based on the Minimal Methylation Classifier (MIMIC) from Schwalbe et al., 2017. The time from the sample centralized reception to the study result was also collected. RESULTS: 25 frozen MB tumor samples from patients at diagnosis were included. 6 were retrospective and 19 prospective. IHC classified 19 cases (76%) as non-WNT/non-SHH MBs, 3 (12%) as WNT-activated and 3 (12%) as SHH-activated. MIMIC classified, in the non-WNT/non-SHH, 6 tumors (24%) as Group 3 and 13 (52%) as Group 4. 2 cases (8%) were WNT-activated MBs and 3 (12%) were SHH-activated MBs. Only 1 case (4%) was unclassified by MIMIC (WNT using IHC). Comparing both methods (IHC and MIMIC), diagnosis agreed in 96% of cases. The response time ranged from 5 to 10 days. CONCLUSIONS: DNA methylation profiling has proven to be a robust and quick option to classify MB into subgroups and it correlates with the IHC diagnosis. This tool was successfully implemented in our national routine diagnosis, enabling a reliable and rapid molecular subgrouping classification.

A CTNNB1-altered medulloblastoma shows the immunophenotypic, DNA methylation and transcriptomic profiles of SHH-activated, and not WNT-activated, medulloblastoma.

Recent advancements in molecular characterisation have identified four principal molecular groups of medulloblastoma: WNT, SHH, group 3 and group 4. Each has its characteristic clinical features, signature genetic alterations and distinct DNA methylome profiles. Thus far, CTNNB1 mutations have been considered pathognomonic of WNT-activated medulloblastoma. Furthermore, it has been shown that CTNNB1 mutations dominantly drive the WNT-activated phenotype in medulloblastoma, even in the presence of alterations in the SHH pathway. We herein report an illustrative case that challenges this belief-a medulloblastoma with a pathogenic CTNNB1 mutation that otherwise showed the histopathology, immunophenotype and methylation and transcriptomic profiles of an SHH-activated medulloblastoma. Detailed molecular analyses, including whole exome sequencing, transcriptome analysis and DNA methylation profiling with DKFZ brain tumour classifier and St. Jude MLPnet neural network classifier analyses, have been performed on the tumour. Our example emphasises the diagnostic value of the immunohistochemistry panel with YAP1, GAB1 and β-catenin and DNA methylation profiling, combined with exome sequencing, in the characterisation of medulloblastoma. CTNNB1 mutations are not specific for WNT-activated medulloblastoma, and different CTNNB1 mutations have diverse oncogenic potential.

SIOP PNET5 MB Trial: History and Concept of a Molecularly Stratified Clinical Trial of Risk-Adapted Therapies for Standard-Risk Medulloblastoma.

Simple SummaryThe European trial SIOP PNET5 MB was initiated in 2014 and will remain open to recruitment until 2022. It is the first European trial using clinical, histological, and molecular parameters to stratify treatments for childhood medulloblastoma, based on disease risk. In the standard-risk stratum, a randomized intensification of carboplatin concomitant to radiotherapy is investigated. In the favourable-risk stratum, defined by localized WNT subgroup disease, reduction of craniospinal radiotherapy intensity (from 24 to 18 Gy) and reduced maintenance chemotherapy is investigated for children <16 years old at diagnosis. Two additional exploratory strata (WNT-HR and SHH-TP53) have been implemented during the trial. The use of biological parameters for stratification has proven feasible in a prospective multicentre setting, and may improve future risk-adapted treatment. The primary endpoint is 3-year event-free survival. Late effects on hearing, endocrine- and neurologic function, alongside health-related quality of life (e.g., health status, behavioural outcomes), are secondary endpoints.Background. SIOP PNET5 MB was initiated in 2014 as the first European trial using clinical, histological, and molecular parameters to stratify treatments for children and adolescents with standard-risk medulloblastoma. Methods. Stratification by upfront assessment of molecular parameters requires the timely submission of adequate tumour tissue. In the standard-risk phase-III cohort, defined by the absence of high-risk criteria (M0, R0), pathological (non-LCA), and molecular biomarkers (MYCN amplification in SHH–MB or MYC amplification), a randomized intensification by carboplatin concomitant with radiotherapy is investigated. In the LR stratum for localized WNT-activated medulloblastoma and age <16 years, a reduction of craniospinal radiotherapy dose to 18 Gy and a reduced maintenance chemotherapy are investigated. Two additional strata (WNT-HR, SHH-TP53) were implemented during the trial. Results. SIOP PNET5 MB is actively recruiting. The availability of adequate tumour tissue for upfront real-time biological assessments to assess inclusion criteria has proven feasible. Conclusion. SIOP PNET5 MB has demonstrated that implementation of biological parameters for stratification is feasible in a prospective multicentre setting, and may improve risk-adapted treatment. Comprehensive research studies may allow assessment of additional parameters, e.g., novel medulloblastoma subtypes, and identification and validation of biomarkers for the further refinement of risk-adapted treatment in the future.

OMIC-07. FEASIBILITY AND UTILITY OF EPIGENOMIC PROFILING FOR CHILDHOOD CNS TUMORS IN HONG KONG

Genome-wide DNA methylation profiling has emerged as an important diagnostic tool that complements histopathology for CNS tumors in children and adults. Literature describing its application in Asian countries is nonetheless limited. Herein, we report the feasibility and utility of adopting such platform for children diagnosed with CNS tumors in Hong Kong. A multi-institutional cohort (n=94, 97% of Chinese ethnicity) with CNS embryonal or high grade neuroepithelial tumors (HGNET) diagnosed in Hong Kong from 1996–2020 was assembled based on tissue availability. DNA was extracted from FFPE tumor material (median 301ng, range 13-1000ng), bisulfite converted and profiled with the Infinium Methylation EPIC BeadChip kit. Raw data were analyzed on the German Cancer Research Center MNP 2.0 classifier and through unsupervised dimensionality-reduction analysis (t-SNE) referencing a published CNS tumor reference dataset (GSE90496). The radio-histologic diagnosis included medulloblastoma (n=65), ATRT (n=9), pineal parenchymal tumors (n=7), ETMR (n=5), CNS-PNET (n=4) and other embryonal tumors/HGNETs (n=4). Methylation class could be assigned based on results from MNP 2.0 (calibrated score ≥ 0.9) in 62 patients (66%, including 2 clustering with control) and t-SNE in 22 (23%), while no-match was encountered in 10 (11%). Methylation-based analysis allowed confirmation of diagnosis and assignment of molecular subgroup in 64 patients (68%), confirmation of histologic diagnosis alone in 5 (5%) and resulted in revision/reassignment of diagnosis in 13 (14%). Among medulloblastoma samples that were assigned molecular tumor classes (n=57), 8 clustered with WNT-activated medulloblastoma, 13 with SHH-activated medulloblastoma, 10 with Group 3 medulloblastoma, 21 with Group 4 medulloblastoma, and 5 with non-medulloblastoma entities (high-grade gliomas=3, ETMR=1, ATRT=1). In conclusion, epigenomic profiling allowed refinement of disease classification for pediatric CNS tumors. Availability of such methodology in Asia sets the stage for international collaborations in molecularly-driven trials.

GLI3Is Associated With Neuronal Differentiation in SHH-Activated and WNT-Activated Medulloblastoma.

Glioma-associated oncogene homolog 3 (GLI3), whose main function is to inhibit GLI1, has been associated with neuronal differentiation in medulloblastoma. However, it is not clear what molecular subtype(s) show increased GLI3 expression. GLI3 levels were assessed by immunohistochemistry in 2 independent cohorts, including a total of 88 cases, and found to be high in both WNT- and SHH-activated medulloblastoma. Analysis of bulk mRNA expression data and single cell RNA sequencing studies confirmed that GLI1 and GLI3 are highly expressed in SHH-activated medulloblastoma, whereas GLI3 but not GLI1 is highly expressed in WNT-activated medulloblastoma. Immunohistochemical analysis has shown that GLI3 is expressed inside the neuronal differentiated nodules of SHH-activated medulloblastoma, whereas GLI1/2 are expressed in desmoplastic areas. In contrast, GLI3 is diffusely expressed in WNT-activated medulloblastoma, whereas GLI1 is suppressed. Our data suggest that GLI3 may be a master regulator of neuronal differentiation and morphology in these subgroups.

Pattern of Relapse and Treatment Response in WNT-Activated Medulloblastoma

SummaryOver the past decade, wingless-activated (WNT) medulloblastoma has been identified as a candidate for therapy de-escalation based on excellent survival; however, a paucity of relapses has precluded additional analyses of markers of relapse. To address this gap in knowledge, an international cohort of 93 molecularly confirmed WNT MB was assembled, where 5-year progression-free survival is 0.84 (95%, 0.763–0.925) with 15 relapsed individuals identified. Maintenance chemotherapy is identified as a strong predictor of relapse, with individuals receiving high doses of cyclophosphamide or ifosphamide having only one very late molecularly confirmed relapse (p = 0.032). The anatomical location of recurrence is metastatic in 12 of 15 relapses, with 8 of 12 metastatic relapses in the lateral ventricles. Maintenance chemotherapy, specifically cumulative cyclophosphamide doses, is a significant predictor of relapse across WNT MB. Future efforts to de-escalate therapy need to carefully consider not only the radiation dose but also the chemotherapy regimen and the propensity for metastatic relapses.

Pattern of Relapse and Treatment Response in WNT-Activated Medulloblastoma

Wingless-activated (WNT) medulloblastoma have been identified over the past decade as being a candidate for therapy de-escalation based on their excellent survival, however a paucity of relapses in any single cohort have precluded any additional analysis of markers of relapse. To address this gap in knowledge, an international cohort of 91 WNT-MB was assembled where we identified 14 relapsed cases where five-year progression free survival was 0.84 (95% 0.763-0.925). Maintenance chemotherapy was identified as a strong predictor of relapse, where no relapses were observed in cases receiving high doses of cyclophosphamide or ifosphamide (p=0.0026). The location of relapse was metastatic in 10/14 of relapses, with 7/10 metastatic relapses presenting along the ependymal lining in the lateral ventricles. Maintenance chemotherapy, specifically cumulative cyclophosphamide dose is a significant predictor of relapse across WNT-MB. Future efforts to de-escalate therapy need to carefully consider not only the radiation dose but also the chemotherapy regimen, and the propensity to metastatic relapses.

Immunohistochemical detection of ALK protein identifies APC mutated medulloblastoma and differentiates the WNT-activated medulloblastoma from other types of posterior fossa childhood tumors

Expression of the ALK gene strongly correlates with the WNT-activated medulloblastomas, which are routinely identified by detection of CTNNB1 mutation. However, some tumors have mutations in other than CTNNB1 genes. Therefore, we investigated if ALK expression may identify WNT-activated tumors without CTNNB1 mutation. In addition, we examined if ALK expression may differentiate WNT-activated medulloblastoma from other malignant posterior fossa tumors. ALK expression was analyzed using immunohistochemistry (clone D5F3) in 70 patients with posterior fossa tumours. Among 55 medulloblastomas, 6 tumors showed ALK expression in > 50% of tumor cells. In one tumor, with ALK positive reaction, negative nuclear reaction against β-catenin and the lack of CTNNB1 mutation, next generation sequencing revealed a presence of pathogenic variant c.3366_3369del in the APC gene, with homozygous deletion leading to inactivation of both copies in tumor cells. MLPA analysis displayed the presence of chromosome 6 monosomy, therefore, confirming the WNT type of this tumor. All analyzed 19 anaplastic ependymomas, 4 choroid plexus carcinomas and 2 atypical teratoid rhabdoid tumors were immunonegative for ALK expression. Therefore, we propose, that immunohistochemical detection of ALK protein should be highly recommended in routine investigation, in parallel to already established methods for identification and differentiation of WNT-activated medulloblastoma.

DNA methylation profiling is a method of choice for molecular verification of pediatric WNT-activated medulloblastomas.

Wingless-activated medulloblastoma (WNT MB) represents a well-characterized molecular variant accounting for 10-15% of all MB and is associated with a favorable clinical outcome. Patients with localized WNT MBs could benefit from de-intensification of combined treatment, which would require an accurate diagnosis of these tumors. However, despite the presence of molecular features related with a WNT MB signature (nuclear ß-catenin immunoexpression, CTNNB1 mutation, and monosomy 6), a prompt and reliable diagnostic verification of these tumors is not yet feasible. In the current study, we analyzed 78 samples of WNT MB treated in a single institute through genome-wide DNA methylation and targeted next generation sequencing to elaborate an optimal method for WNT MB molecular verification. We found that DNA methylation profiling discloses significant advantages for molecular diagnostic of WNT MB. All other "routine" methods applied, such as ß-catenin immunohistochemistry, CTNNB1 mutation analysis, and detection of monosomy 6, failed to identify all WNT MB cases. Survival analysis revealed that application of a reduced radiotherapy protocol for WNT MB treatment had no influence on patients' survival. Only one patient died due to local relapse but recurrent tumor was pathologically and molecularly diagnosed as a secondary glioblastoma. DNA methylation analysis should be considered as a method of choice for further clinically relevant stratification of WNT MB and for correct diagnosis of the recurrent tumors. WNT MB patients with localized disease could benefit from treatment de-intensification.

Medulloblastoma, WNT-activated/SHH-activated: clinical impact of molecular analysis and histogenetic evaluation.

Medulloblastoma (MDB) is a small cell poorly differentiated embryonal tumor of the cerebellum, which more frequently compromises children. Overall prognosis is favorable, but dependent of stage, histopathological pattern and molecular group. Approximately 30% of the affected patients will die from the disease. WHO 2016 Classification of Tumors of the Central Nervous System (CNS) has been classified MDB into four principal groups: WNT-activated MDB, SHH-activated MDB, group 3 MDB, and group 4 MDB. WNT-activated MDB is associated to monosomy 6, CTNNB1, DDX3X and TP53 mutations, beta-catenin nuclear immunoexpression, and a better prognosis than SHH-activated MDB. WNT-activated tumors account approximately for 10% of cases of MDBs, and are thought to arise from cells in the dorsal brain stem/lower rhombic lip progenitor cells. SHH-activated MDB more frequently arises in the lateral hemispheres of the cerebellum, and clinical outcome in this group is variable. TP53-mutant SHHactivated MDB usually shows the large cell/anaplastic pattern, and can be related to MYCN amplification, GLI2 amplification and 17p loss. TP53-wildtype SHH-activated MDB is more commonly of desmoplastic/nodular morphology, and can be related to PTCH1 deletion and 10q loss. Gene expression and methylation profiling is the gold standard for defining molecular groups of MDB. In immunohistochemistry assays, anti-GAB1 antibody expression is positive in tumors showing SHH pathway activation or PTCH mutation, while positive immunoexpression for YAP1 antibody can be only found in WNT-activated and SHH-activated MDB.