Transcription factors are frequent cancer driver genes, exhibiting noted specificity based on the precise cell of origin. We demonstrate that ZIC1 exhibits loss-of-function (LOF) somatic events in group 4 (G4) medulloblastoma through recurrent point mutations, subchromosomal deletions and mono-allelic epigenetic repression (60% of G4 medulloblastoma). In contrast, highly similar SHH medulloblastoma exhibits distinct and diametrically opposed gain-of-function mutations and copy number gains (20% of SHH medulloblastoma). Overexpression of ZIC1 suppresses the growth of group 3 medulloblastoma models, whereas it promotes the proliferation of SHH medulloblastoma precursor cells. SHH medulloblastoma ZIC1 mutants show increased activity versus wild-type ZIC1, whereas G4 medulloblastoma ZIC1 mutants exhibit LOF phenotypes. Distinct ZIC1 mutations affect cells of the rhombic lip in diametrically opposed ways, suggesting that ZIC1 is a critical developmental transcriptional regulator in both the normal and transformed rhombic lip and identifying ZIC1 as an exquisitely context-dependent driver gene in medulloblastoma.

2002 Background: The 1st and 2 nd interim analyses of the CATNON trial on anaplastic glioma (NCT00626990) showed benefit from adjuvant (adj) temozolomide (TMZ) on overall survival (OS) in patients with IDH mutant (mt) tumors, but no benefit of concurrent (conc) TMZ regardless of Isocitrate dehydrogenase 1 and 2 (IDH) mutation (mt) status. We now present the final analysis and the exploratory molecular marker analysis of the study. Methods: The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed non-codeleted anaplastic glioma to either 59.4 Gy radiotherapy (RT) alone; the same RT with concTMZ; the same RT and 12 cycles of adjTMZ or the same RT with both concTMZ and adjTMZ. Methylation status including MGMT promoter methylation status were assessed with the Infinium MethylationEPIC Beadchip. IDH mutation (mt) status and glioma specific alterations were assessed with a glioma targeted panel using Agilent SureSelect baits. Results: After a median follow-up of 10.9 years and with 499 events observed, in the intent-to-treat population the hazard ratio (HR) for OS adjusted for stratification factors after concTMZ was 0.906 (95%CI 0.760, 1.082; p=0.28) and after adjTMZ 0.647 (95%CI 0.541, 0.773; p <0.0001). In 660 patients IDH status could be determined: IDH was mt in 444 tumors and wild type (wt) in 216 tumors. Median OS was 1.7 yrs in patients with IDHwt tumors and 8.5 years in patients with IDHmt tumors. Benefit to TMZ was limited to patients with anaplastic glioma IDHmt of which 199 were still alive (45%). For patients with IDHmt tumors the HR for concTMZ was 0.81 (95% CI 0.63-1.04; p=0.09) and for adjTMZ 0.54 (95% CI 0.42-0.69,p < 0.0001). No benefit was observed of concTMZ in IDHmt glioma patients that also received adjTMZ (HR 0.92 95% CI 0.63-1.36; p=0.69). In patients with IDHmt tumors that had received any TMZ median OS was 10.3 years, the median OS in patients treated with adjTMZ was 12.5 years (95% CI 9.4-15.0; p<0.0001). In exploratory analysis, high-copy number Amplification of PDGFR and CDK4 ; Homozygous deletion of the CDKN2A/B locus, total copy number alterations, methylation subtype (A_IDH vs A_IDH_HG, G-CIMP high versus low, MGMT -promoter methylation as determined by methylation arrays) were all associated with outcome but none was predictive for benefit to TMZ. Conclusions: Despite more follow-up, concTMZ did not improve OS regardless of IDH status. AdjTMZ increased OS in patients with IDHmt tumors but not in patients with IDHwt tumors. Molecular factors of known prognostic significance for IDHmt 1p/19q intact anaplastic glioma did not predict benefit to TMZ. Median OS in patients with IDHmt glioma having received adjTMZ after RT was 12.5 years. Standard of post-operative care in patients with high grade IDHmt astrocytoma should be RT followed by 12 cycles adjTMZ. Funding Source: MSD. Clinical trial information: NCT00626990 .

2000 Background: The 1st interim analysis of the CATNON trial showed benefit from adjuvant (adj) temozolomide (TMZ) on overall survival (OS) but remained inconclusive about concurrent (conc) TMZ. A 2 nd interim analysis was planned after 356 events. Methods: The 2x2 factorial design phase III CATNON trial randomized 751 adult patients with newly diagnosed non-codeleted anaplastic glioma to either 59.4 Gy radiotherapy (RT) alone; the same RT with concTMZ; the same RT and 12 cycles of adjTMZ or the same RT with both concTMZ and adjTMZ ( doi: 10.1016/S0140-6736(17)31442-3). MGMT promoter methylation ( MGMTmeth) status was re-assessed with the Infinium Methylation EPIC Beadchip using the MGMT_STP27 model. Isocitrate dehydrogenase 1 and 2 ( IDH) mutation (mt) status was assessed with glioma targeted Agilent SureSelect baits sequence using an Illumina HiSeq2500 Rapid PE100. Results: With a median follow-up of 56 months and 356 events, the hazard ratio (HR) for OS adjusted for stratification factors after concTMZ was 0.968 (99.1% CI 0.73, 1.28). 5-year OS was 50.2% with and 52.7% without concTMZ (95% CI [44.4, 55.7] and [46.9, 58.1]). An IDHmt was found in 335 of 480 assessed cases (70%). Median OS was 19 mo (95% CI 16.3, 22.3) in IDHwt tumors and 116 mo (95% CI 82.0, 116.6) in IDHmt tumors. HR for OS after concTMZ in patients with known IDH status. Clinical trial information: NCT00626990. IDHmt was predictive of benefit from adjTMZ ( IDHmt HR: 0.41, 95% CI 0.27, 0.64; IDHwt: HR 1.05, 95% CI 0.73, 1.52; interaction test p = 0.001). In IDHmt patients that received adjTMZ, the HR for OS after concTMZ was 0.71 (95% CI 0.35, 1.42, p=0.32). MGMTmeth was found in 288 of 410 assessed cases (70%), interaction test for concTMZ (p = 0.092) and adjTMZ (p = 0.166) did not reach statistical significance. Conclusions: In the entire study cohort, concTMZ did not increase OS. However, in IDHmt tumors a trend towards benefit of concTMZ is present. AdjTMZ increased OS in IDHmt but not in IDHwt tumors. The ongoing molecular analyses and further follow-up will allow full assessment of efficacy in the molecular subgroups.[Table: see text]

The evolutionary processes that drive malignant progression of IDH-mutant astrocytomas remain unclear. Here, we performed multiomics on matched initial and recurrent tumor samples from a cohort of 105 patients and overlaid the data with detailed clinical annotation. We identified overlapping features associated with malignant progression that are derived from three molecular mechanisms: cell cycling, tumor cell (de)differentiation and remodeling of the extracellular matrix. Together, they provide a rationale of the underlying biology of tumor malignancy. DNA methylation levels decreased over time, predominantly in tumors with malignant transformation, and co-occurred with poor prognostic genetic events. We identified a DNA methylation-based signature strongly associated with survival, which allows objective, molecular-based grading of IDH-mutant astrocytomas to aid clinical decision making. Our findings were validated on large, independent cohorts of IDH-mutant astrocytoma samples. Lastly, in this retrospective study, we found little effect of radiotherapy or chemotherapy on the molecular features associated with malignant progression. Vallentgoed et al. integrate clinical and multiomic data from persons with matched initial and recurrent IDH-mutant astrocytomas to identify progression-associated mechanisms and report a DNA methylation-based signature associated with survival.

Background:To discover immune response-associated gene expressions related to brain metastases of skin melanomas, we analyzed a group of fifteen primary skin melanomas consisting of five tumors from patients who did not develop systemic metastases (NM); ten from patients with systemic metastasis of which five with (BM), and five without brain involvement (OOM). Regions of interest (ROIs) harboring tumor cells only; immune cells only; or immune cell infiltration in tumor cell regions, were separately analyzed. Methods:we profiled the tumor immune microenvironment (TIME) of primary skin melanomas by using a panel of 77 immune response-related oligo-nucleotide targets in the GeoMx Digital Spatial profiler (Nanostring Technologies). Results:Melanomas from patients who developed brain metastases contained significantly higher levels of (CD45+) immune cells and overexpressed fibronectin and CTLA-4 in all ROIs compared to the melanomas that had metastasized to other sites than the brain (p<0.05). Distant metastases were accompanied by lower expression of CD137 (4-1BB). Downregulation of VISTA, IDO1 and ICOS was associated with melanomas that gave rise to distant metastases including brain. Conclusion:Lower expression of CD137 (4-1BB) is linked with the formation of distant metastases and the expressions of fibronectin and CTLA-4 herald the formation of brain metastasis in particular.

Supplementary Figure S1. Assessment of the linearity of the effect of age and tumor volume.

The appearance of brain metastasis is the most serious complication of breast cancer with mostly fatal outcomes. To reach the brain, tumor cells need to pass the blood-brain barrier (BBB). The molecular mechanisms underlying penetration of the BBB are largely unknown. Previously we found that tumor-infiltrating T lymphocytes enhance the development of brain metastasis of estrogen receptor-negative (ER-) breast cancer. In the current study, we investigate the contribution of T lymphocytes and the IFN-γ pathway in enabling breast cancer cells to pass the in vitro BBB. CD8+ cells display the strongest stimulatory effect on breast cancer cell passage. We show that inhibition of the IFN-γ receptor in MDA-MB-231 breast cancer cells, or neutralization of soluble IFN-γ, impairs the in vitro trespassing of breast cancer cells. Importantly, we validated our findings using gene expression data of breast cancer patients. The CXCL-9,-10,-11/CXCR3 axis, dependent on IFN-γ signaling activity, was overexpressed in primary breast cancer samples of patients who developed brain metastasis. The data support a role for T-lymphocytes and the IFN-γ pathway in the formation of brain metastasis of ER- breast cancer, and offer targets to design future therapies for preventing breast cancer cells to cross the BBB.

Background: In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored how T lymphocytes promote breast cancer cell penetration through the blood brain barrier (BBB). Methods: An in vitro BBB model was employed to study the effects of T lymphocytes on BBB trespassing capacity of three different breast carcinoma cell lines. Differential protein expression was explored by comparing the proteomes of the breast cancer cells before and after co-culture with activated T lymphocytes using liquid chromatography-mass spectrometry (LC-MS). siRNA was used to silence protein expression in the breast cancer cells to study contribution to in vitro BBB passage. Furthermore, protein expression in primary breast cancer tissues was explored and related to brain-metastatic potential. Results: Co-culturing with activated T lymphocytes or their conditioned medium (CM) resulted in increased passage through the in vitro BBB. The effects were less for cell line MDA-MB-231-B2M2 (brain affinity) as compared to MDA-MB-231 and SK-BR-7. Mass spectrometry-based proteomics revealed significant alterations in the expression of 35 proteins by the breast cancer cell lines upon T cell contact. Among the proteins is coronin-1A, a protein related to cell motility. Knockdown of CORO1A in the breast cancer cells reduced their ability to cross the artificial BBB to 60%. The effects were significantly less for the cell line derived from breast cancer with affinity for brain. The expression of coronin-1A was confirmed by immunohistochemistry and RT-PCR of 52 breast cancer samples of patients with metastasized breast cancers, with and without brain locations. Lastly, CORO1A upregulation was validated in a publicly available mRNA expression database from 204 primary breast cancers with known metastatic sites. Conclusions: We conclude that T lymphocytes trigger cancer cells to express proteins including coronin-1A that enable the cancer cells to cross an in vitro BBB. In addition, a prominent role of coronin-1A in the formation of cerebral metastases in breast cancer patients is strongly suggestive by its upregulation in tissue samples of breast cancer patients with brain metastases.

We describe a 46-year-old patient with an IDH-wildtype diffusely infiltrating atypical teratoid/rhabdoid tumour (AT/RT), SHH-1B molecular subtype. The unusual histology and subsequent diagnosis in an adult patient will be discussed. In this case report, we describe a 46-year-old patient with a brain lesion identified with imaging studies following a minor head injury. Initially suspected to be diffuse glioma, detailed histological, immunohistochemical and molecular investigations revealed characteristics consistent with an atypical teratoid/rhabdoid tumour (AT/RT), SHH-1B molecular subtype. image

Abstract The immune response–gut microbiota interaction is implicated in various human diseases, including cancer. Identifying the link between the gut microbiota and systemic inflammatory markers and their association with cancer will be important for our understanding of cancer etiology. The current study was performed on 8090 participants from the population‐based Rotterdam study. We found a significant association (false discovery rate [FDR] ≤0.05) between lymphocytes and three gut microbial taxa, namely the family Streptococcaceae, genus Streptococcus, and order Lactobacillales. In addition, we identified 95 gut microbial taxa that were associated with inflammatory markers (p < 0.05). Analyzing the cancer data, we observed a significant association between higher systemic immune‐inflammation index (SII) levels at baseline (hazard ratio (HR): 1.65 [95% confidence interval (CI); 1.10–2.46, p ≤ 0.05]) and a higher count of lymphocytes (HR: 1.38 [95% CI: 1.15–1.65, p ≤ 0.05]) and granulocytes (HR: 1.69 [95% CI: 1.40–2.03, p ≤ 0.05]) with increased risk of lung cancer after adjusting for age, sex, body mass index (BMI), and study cohort. This association was lost for SII and lymphocytes after additional adjustment for smoking (SII = HR:1.46 [95% CI: 0.96–2.22, p = 0.07] and lymphocytes = HR: 1.19 [95% CI: 0.97–1.46, p = 0.08]). In the stratified analysis, higher count of lymphocyte and granulocytes at baseline were associated with an increased risk of lung cancer in smokers after adjusting for age, sex, BMI, and study cohort (HR: 1.33 [95% CI: 1.09–1.62, p ≤0.05] and HR: 1.57 [95% CI: 1.28–1.92, p ≤0.05], respectively). Our study revealed a positive association between gut microbiota, higher SII levels, and higher lymphocyte and granulocyte counts, with an increased risk of developing lung cancer.

In previous work we discovered that T lymphocytes play a prominent role in the rise of brain metastases of ER-negative breast cancers. In the present study we explored how T lymphocytes promote breast cancer cell penetration through the blood brain barrier (BBB). An in vitro BBB model was employed to study the effects of T lymphocytes on BBB trespassing capacity of three different breast carcinoma cell lines. Differential protein expression was explored by comparing the proteomes of the breast cancer cells before and after co-culture with activated T lymphocytes using liquid chromatography-mass spectrometry (LC-MS). siRNA was used to silence protein expression in the breast cancer cells to study contribution to in vitro BBB passage. Furthermore, protein expression in primary breast cancer tissues was explored and related to brain-metastatic potential. Co-culturing with activated T lymphocytes or their conditioned medium (CM) resulted in increased passage through the in vitro BBB. The effects were less for cell line MDA-MB-231-B2M2 (brain affinity) as compared to MDA-MB-231 and SK-BR-7. Mass spectrometry-based proteomics revealed significant alterations in the expression of 35 proteins by the breast cancer cell lines upon T cell contact. Among the proteins is coronin-1 A, a protein related to cell motility. Knockdown of CORO1A in the breast cancer cells reduced their ability to cross the artificial BBB to 60%. The effects were significantly less for the cell line derived from breast cancer with affinity for brain. The expression of coronin-1A was confirmed by immunohistochemistry and RT-PCR of 52 breast cancer samples of patients with metastasized breast cancers, with and without brain locations. Lastly, CORO1A upregulation was validated in a publicly available mRNA expression database from 204 primary breast cancers with known metastatic sites. We conclude that T lymphocytes trigger cancer cells to express proteins including coronin-1A that enable the cancer cells to cross an in vitro BBB. In addition, a prominent role of coronin-1A in the formation of cerebral metastases in breast cancer patients is strongly suggestive by its upregulation in tissue samples of breast cancer patients with brain metastases.

Cerebral hypoxia significantly impacts the progression of brain tumors and their resistance to radiotherapy. This study employed streamlined quantitative blood-oxygen-level-dependent (sqBOLD) MRI to assess the oxygen extraction fraction (OEF)—a measure of how much oxygen is being extracted from vessels, with higher OEF values indicating hypoxia. Simultaneously, we utilized vessel size imaging (VSI) to evaluate microvascular dimensions and blood volume. A cohort of ten patients, divided between those with glioma and those with brain metastases, underwent a 3 Tesla MRI scan. We generated OEF, cerebral blood volume (CBV), and vessel size maps, which guided 3–4 targeted biopsies per patient. Subsequent histological analyses of these biopsies used hypoxia-inducible factor 1-alpha (HIF-1α) for hypoxia and CD31 for microvasculature assessment, followed by a correlation analysis between MRI and histological data. The results showed that while the sqBOLD model was generally applicable to brain tumors, it demonstrated discrepancies in some metastatic tumors, highlighting the need for model adjustments in these cases. The OEF, CBV, and vessel size maps provided insights into the tumor’s hypoxic condition, showing intertumoral and intratumoral heterogeneity. A significant relationship between MRI-derived measurements and histological data was only evident in the vessel size measurements (r = 0.68, p < 0.001).

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is however difficult and, apart from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. Experimental Design: RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n=138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. We used the DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) that is based on classification scores derived from a CNS-tumor classifier. We found that the CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with i) an upregulation of cell cycling genes; ii) a downregulation of glial differentiation genes; iii) an upregulation of embryonic development genes (e.g. HOX, PAX and TBX) and iv) an upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes.

Abstract BACKGROUND We previously reported on progression-free survival of our randomized phase 3 trial [Lancet Oncol. 2016;17:1521-1532]. Here we report results of long-term follow-up (FU) including overall survival (OS) and molecular subgroup analyses. METHODS 487 patients were randomized between 2005-2012 to either standard radiotherapy (RT, 50.4 Gy/ 28 fractions) or primary dose-dense temozolomide [TMZ] chemotherapy (75 mg/m² daily x 21/28 days, up to 12 cycles). Treatment at progression was at investigators discretion and commonly included cross-over to the alternative treatment modality. RESULTS At a median follow-up of 13 years, 68% of patients had died. Median age at inclusion was 45 years (range 18-75), 96% had a performance status of 0-1. There was no significant difference in PFS nor OS: PFS was 3.6 years (95% CI: 3.0-4.1) and 3.1 years (95% CI: 2.7-3.6), HR 1.12 (95% CI: 0.92-1.36); OS 6.6 years (95% CI: 5.8-7.5) and 8.0 years (95% CI: 6.9-9.1), HR 0.87 (95% CI: 0.69-1.09), for RT or TMZ, respectively. Subgroup analyses were performed for patients when available tissue allowed for molecular analyses (n=351/487,73%). In astrocytoma, IDHmt/1p/19q non-codeleted (n = 178), median OS with RT was 6.6 years (95% CI: 5.3-7.6) and with TMZ was 6.7 years (95% CI: 5.7-8.0); HR 0.98 (95% CI: 0.67-1.44). For oligodendroglioma, IDHmt/codeleted (n = 109) median OS with RT was 12.9 years (95% CI: 9.4 - NE) and with TMZ 14.9 years (95% CI: 10.1 - NE); HR 1.01 (95% CI: 0.56-1.81). For a heterogenous group of IDHwt tumors (n = 64), median OS with RT was 2.5 years (95% CI: 1.8-3.3) and with TMZ 4.7 years (95% CI: 2.2-7.2); HR 0.37 (95% CI: 0.18-0.77). CONCLUSIONS Primary treatment with RT or TMZ provided comparable PFS and OS. Choice of primary treatment can be tailored to individual tumor characteristics and patient preference.

AbstractPurpose: IDH-mutant glioma is classified as oligodendroglioma or astrocytoma based on 1p19q-codeletion. Whether prognostic factors are similar between these tumor types is not well understood. Experimental Design: Retrospective cohort study. Molecular characterization was performed with targeted next-generation sequencing. Tumor volumes were calculated using semiautomatic 3D segmentation on all pre- and post-operative MRI scans. Overall survival was assessed with the Cox-proportional hazards model. Results: A total of 383 patients with newly diagnosed IDH-mutant glioma were followed up for a median of 7.2 years. Grades 3 and 4 patients had significantly lower Karnofsky performance, with tumors having more contrast enhancement. Patients also received more aggressive postsurgery treatment. Postoperative tumor volume is significantly and independently associated with survival (HR, per cm3 1.19; 95% CI, 1.03–1.39) in IDH-mutant glioma. A separate analysis of oligodendroglioma and astrocytoma showed a significant association of postoperative tumor volume in astrocytoma but not in oligodendroglioma. Higher age and histologic tumor grade were associated with worse survival in patients with oligodendroglioma but not with astrocytoma. Conclusions: Our data support an initial strategy of extensive resection in patients with oligodendroglioma and astrocytoma. Other important prognostic factors differ between these tumor types, urging researchers and clinicians to keep treating these tumors as separate entities.

Abstract BACKGROUND Metastatic infant medulloblastoma (MB) is a major challenge given the devastating long-term effects of craniospinal irradiation to the developing brain. However large cohorts of metastatic infant medulloblastoma have not been analysed to date. METHODS A multicentre molecularly informed international cohort of children <6 years old with metastatic medulloblastoma was assembled and analysed. RESULTS Eighty-four patients with a median age of 2.95 (0.66-6.58) years were identified. 48.8% were Group3-MB, 23.8% Group4-MB and 27.4% SHH-MB. 78% of Group3-MB were subtype II (40.4%) and IV (38.1%), 50% of Group 4 were subtype VII, and within SHH, SHHß and SHHγ were 52.2% and 43.5% respectively. 28.5% of all Group3-MB harboured MYC amplification. A multivariable analysis incorporating subgroup, MYC amplification and upfront craniospinal irradiation revealed that survival was superior for SHH patients compared to Group 3 irrespective of MYC status (Group3-veMYCamp: HR 3.05 95%-CI 1.05-8.87 p=0.04, Group3+veMYCamp HR 7.87 95%-CI 2.49-24.87 p=0.00044). Group 4 had a trend to a poor outcome (HR 2.63 95%-CI 0.82-8.39 p=0.1). SHH subjects had a superior outcome with 5-year PFS of 0.657 (95%-CI 0.47-0.92), without a significant difference between SHHß and SHHγ. High-dose chemotherapy regimens portended to a superior outcome for both subtypes of SHH-MB. 5-year PFS for non-radiated SHH-MB was 61% (95%-CI 40.8-91.4), compared to non-radiated Group3-MB (18.5%; 95%-CI 5.4-62.8) and Group4-MB (20%; 95%-CI 3.4-100). SHH-MB patients had more local than metastatic relapses compared to non-SHH-MB (p=0.01). CONCLUSIONS In the largest study dedicated to metastatic infant medulloblastoma assembled to date, outcomes for infants with Group 3- and 4-MB with metastatic disease treated with radiation sparing approaches is dismal despite intensified regimens. MYC amplification portends to a near uniformly fatal prognosis with current treatment irrespective of upfront radiotherapy. Novel radiation sparing approaches are urgently needed for this group. Infants with both metastatic SHHß and SHHγ benefit from intensified therapy.

Performance of DASL and RNA-seq on RNA isolated from FFPE tissues, correlation with snap frozen samples.

Example of genomic breakpoints identified by RNA sequencing.

Supplementary Figure 3 - PDF file 186K, SAM analysis of survival within CIMP+ or CIMP- tumors