Influence of Acoustic Parameters and Sonication Schemes on Transcranial Blood-Brain Barrier Disruption Induced by Pulsed Weakly Focused Ultrasound.

Pulsed ultrasound combined with microbubbles use can disrupt the blood-brain barrier (BBB) temporarily; this technique opens a temporal window to deliver large therapeutic molecules into brain tissue. There are published studies to discuss the efficacy and safety of the different ultrasound parameters, microbubble dosages and sizes, and sonication schemes on BBB disruption, but optimal the paradigm is still under investigation. Our study is aimed to investigate how different sonication parameters, time, and microbubble dose can affect BBB disruption, the dynamics of BBB disruption, and the efficacy of different sonication schemes on BBB disruption. Method: We used pulsed weakly focused ultrasound to open the BBB of C57/B6 mice. Evans blue dye (EBD) was used to determine the degree of BBB disruption. With a given acoustic pressure of 0.56 MPa and pulse repetitive frequency of 1 Hz, burst lengths of 10 ms to 50 ms, microbubbles of 100 µL/kg to 300 µL/kg, and sonication times of 60 s to 150 s were used to open the BBB for parameter study. Brain EBD accumulation was measured at 1, 4, and 24 h after sonication for the time-response relationship study; EBD of 100 mg/kg to 200 mg/kg was administered for the dose-response relationship study; EBD injection 0 to 6 h after sonication was performed for the BBB disruption dynamic study; brain EBD accumulation induced by one sonication and two sonications was investigated to study the effectiveness on BBB disruption; and a histology study was performed for brain tissue damage evaluation. Results: Pulsed weakly focused ultrasound opens the BBB extensively. Longer burst lengths and a larger microbubble dose result in a higher degree of BBB disruption; a sonication time longer than 60 s did not increase BBB disruption; brain EBD accumulation peaks 1 h after sonication and remains 81% of the peak level 24 h after sonication; the EBD dose administered correlates with brain EBD accumulation; BBB disruption decreases as time goes on after sonication and lasts for 6 h at least; and brain EBD accumulation induced by two sonication increases 74.8% of that induced by one sonication. There was limited adverse effects associated with sonication, including petechial hemorrhages and mild neuronal degeneration. Conclusions: BBB can be opened extensively and reversibly by pulsed weakly focused ultrasound with limited brain tissue damage. Since EBD combines with albumin in plasma to form a conjugate of 83 kDa, these results may simulate ultrasound-induced brain delivery of therapeutic molecules of this size scale. The result of our study may contribute to finding the optimal paradigm of focused ultrasound-induced BBB disruption.

Notch signaling and natural killer cell infiltration in tumor tissues underlie medulloblastoma prognosis.

Medulloblastoma is the most common embryonic brain tumor in children. We investigated a cohort of 52 Asian medulloblastoma patients aged between 0 and 19 years old, who received surgical resections and post-resection treatments in the Taipei Medical University Hospital and the Taipei Veterans General Hospital. Genome-wide RNA sequencing was performed on fresh-frozen surgical tissues. These data were analyzed using the CIBERSORTx immune deconvolution software. Two external clinical and molecular datasets from United States (n = 62) and Canada (n = 763) were used to evaluate the transferability of the gene-signature scores across ethnic populations. The abundance of 13 genes, including DLL1, are significantly associated with overall survival (All Cox regression P < 0.001). A gene-signature score was derived from the deep transcriptome, capable of indicating patients' subsequent tumor recurrence (Hazard Ratio [HR] 1.645, confidence interval [CI] 1.337-2.025, P < 0.001) and mortality (HR 2.720, CI 1.798-4.112, P < 0.001). After the adjustment of baseline clinical factors, the score remains indicative of recurrence-free survival (HR 1.604, CI 1.292-1.992, P < 0.001) and overall survival (HR 2.781, CI 1.762-4.390, P < 0.001). Patients stratified by this score manifest not only distinct prognosis but also different molecular characteristics: Notch signaling ligands and receptors are comparatively overexpressed in patients with poorer prognosis, while tumor infiltrating natural killer cells are more abundant in patients with better prognosis. Additionally, immunohistochemical staining showed the DLL1 protein, a major ligand in the Notch signaling pathway, and the NCAM1 protein, a representative biomarker of natural killer cells, are present in the surgical tissues of patients of four molecular subgroups, WNT, SHH, Group 3 and Group 4. NCAM1 RNA level is also positively associated with the mutation burden in tumor (P = 0.023). The gene-signature score is validated successfully in the Canadian cohort (P = 0.009) as well as its three molecular subgroups (SHH, Group 3 and Group 4; P = 0.047, 0.018 and 0.040 respectively). In conclusion, pediatric medullablastoma patients can be stratified by gene-signature scores with distinct prognosis and molecular characteristics. Ligands and receptors of the Notch signaling pathway are overexpressed in the patient stratum with poorer prognosis. Tumor infiltrating natural killer cells are more abundant in the patient stratum with better prognosis.

Upregulation of Protein Synthesis and Proteasome Degradation Confers Sensitivity to Proteasome Inhibitor Bortezomib in Myc-Atypical Teratoid/Rhabdoid Tumors.

Atypical teratoid rhabdoid tumors (ATRTs) are among the most malignant brain tumors in early childhood and remain incurable. Myc-ATRT is driven by the Myc oncogene, which directly controls the intracellular protein synthesis rate. Proteasome inhibitor bortezomib (BTZ) was approved by the Food and Drug Administration as a primary treatment for multiple myeloma. This study aimed to determine whether the upregulation of protein synthesis and proteasome degradation in Myc-ATRTs increases tumor cell sensitivity to BTZ. We performed differential gene expression and gene set enrichment analysis on matched primary and recurrent patient-derived xenograft (PDX) samples from an infant with ATRT. Concomitant upregulation of the Myc pathway, protein synthesis and proteasome degradation were identified in recurrent ATRTs. Additionally, we found the proteasome-encoding genes were highly expressed in ATRTs compared with in normal brain tissues, correlated with the malignancy of tumor cells and were essential for tumor cell survival. BTZ inhibited proliferation and induced apoptosis through the accumulation of p53 in three human Myc-ATRT cell lines (PDX-derived tumor cell line Re1-P6, BT-12 and CHLA-266). Furthermore, BTZ inhibited tumor growth and prolonged survival in Myc-ATRT orthotopic xenograft mice. Our findings suggest that BTZ may be a promising targeted therapy for Myc-ATRTs.

Molecular-Clinical Correlation in Pediatric Medulloblastoma: A Cohort Series Study of 52 Cases in Taiwan.

In 2016, a project was initiated in Taiwan to adopt molecular diagnosis of childhood medulloblastoma (MB). In this study, we aimed to identify a molecular-clinical correlation and somatic mutation for exploring risk-adapted treatment, drug targets, and potential genetic predisposition. In total, 52 frozen tumor tissues of childhood MBs were collected. RNA sequencing (RNA-Seq) and DNA methylation array data were generated. Molecular subgrouping and clinical correlation analysis were performed. An adjusted Heidelberg risk stratification scheme was defined for updated clinical risk stratification. We selected 51 genes for somatic variant calling using RNA-Seq data. Relevant clinical findings were defined. Potential drug targets and genetic predispositions were explored. Four core molecular subgroups (WNT, SHH, Group 3, and Group 4) were identified. Genetic backgrounds of metastasis at diagnosis and extent of tumor resection were observed. The adjusted Heidelberg scheme showed its applicability. Potential drug targets were detected in the pathways of DNA damage response. Among the 10 patients with SHH MBs analyzed using whole exome sequencing studies, five patients exhibited potential genetic predispositions and four patients had relevant germline mutations. The findings of this study provide valuable information for updated risk adapted treatment and personalized care of childhood MBs in our cohort series and in Taiwan.

Downregulation of miR-137 and miR-6500-3p promotes cell proliferation in pediatric high-grade gliomas.

Pediatric high-grade gliomas (pHGGs) are aggressive brain tumors affecting children, and outcomes have remained dismal, even with access to new multimodal therapies. In this study, we compared the miRNomes and transcriptomes of pediatric low- (pLGGs) and high-grade gliomas (pHGGs) using small RNA sequencing (smRNA-Seq) and gene expression microarray, respectively. Through integrated bioinformatics analyses and experimental validation, we identified miR-137 and miR-6500-3p as significantly downregulated in pHGGs. miR-137 or miR-6500-3p overexpression reduced cell proliferation in two pHGG cell lines, SF188 and UW479. CENPE, KIF14 and NCAPG levels were significantly higher in pHGGs than pLGGs, and were direct targets of miR-137 or miR-6500-3p. Furthermore, knockdown of CENPE, KIF14 or NCAPG combined with temozolomide treatment resulted in a combined suppressive effect on pHGG cell proliferation. In summary, our results identify novel mRNA/miRNA interactions that contribute to pediatric glioma malignancy and represent potential targets for the development of new therapeutic strategies.

Integrated genomics has identified a new AT/RT-like yet INI1-positive brain tumor subtype among primary pediatric embryonal tumors.

BACKGROUND: Pediatric embryonal brain tumors (PEBTs), which encompass medulloblastoma (MB), primitive neuroectodermal tumor (PNET) and atypical teratoid/rhabdoid tumor (AT/RT), are the second most prevalent pediatric brain tumor type. AT/RT is highly malignant and is often misdiagnosed as MB or PNET. The distinction of AT/RT from PNET/MB is of clinical significance because the survival rate of patients with AT/RT is substantially lower. The diagnosis of AT/RT relies primarily on morphologic assessment and immunohistochemical (IHC) staining for a few known markers such as the lack of INI1 protein expression. However, in our clinical practice we have observed several AT/RT-like tumors, that fulfilled histopathological and all other biomarker criteria for a diagnosis of AT/RT, yet retained INI1 immunoreactivity. Recent studies have also reported preserved INI1 immunoreactivity among certain diagnosed AT/RTs. It is therefore necessary to re-evaluate INI1(+), AT/RT-like cases. METHOD: Sanger sequencing, array CGH and mRNA microarray analyses were performed on PEBT samples to investigate their genomic landscapes. RESULTS: Patients with AT/RT and those with INI(+) AT/RT-like tumors showed a similar survival rate, and global array CGH analysis and INI1 gene sequencing showed no differential chromosomal aberration markers between INI1(-) AT/RT and INI(+) AT/RT-like cases. We did not misdiagnose MBs or PNETs as AT/RT-like tumors because transcriptome profiling revealed that not only did AT/RT and INI(+) AT/RT-like cases express distinct mRNA and microRNA profiles, their gene expression patterns were different from those of MBs and PNETs. The most similar transcriptome profile to that of AT/RTs was the profile of embryonic stem cells. However; the transcriptome profile of INI1(+) AT/RT-like tumors was more similar to that of somatic neural stem cells, while the profile of MBs was closer to that of fetal brain tissue. Novel biomarkers were identified that can be used to distinguish INI1(-) AT/RTs, INI1(+) AT/RT-like cases and MBs. CONCLUSION: Our studies revealed a novel INI1(+) ATRT-like subtype among Taiwanese pediatric patients. New diagnostic biomarkers, as well as new therapeutic tactics, can be developed according to the transcriptome data that were unveiled in this work.

Downregulation of SUN2, a novel tumor suppressor, mediates miR-221/222-induced malignancy in central nervous system embryonal tumors.

Embryonal tumors of the central nervous system represent a highly malignant tumor group of medulloblastoma (MB), atypical teratoid/rhabdoid tumor (AT/RT) and primitive neuroectodermal tumor that frequently afflict children. AT/RT is often misdiagnosed as MB/primitive neuroectodermal tumor but with higher recurrence and lower survival rates. Pathogenesis of AT/RT is largely unknown. In this study, we report both the miRNome and transcriptome traits in AT/RT and MB by using small RNA sequencing and gene expression microarray analyses. Our findings demonstrate that the miR-221/222-encoded micro RNAs are abundantly expressed in AT/RT but not in MB, which contribute substantially to the malignancy of embryonal tumors. miR-221/222 targeted SUN2, a newly discovered tumor suppressor, directly to increase cell proliferation and tumor malignancy in vitro and in vivo. Immunohistochemistry against SUN2 in a tissue microarray of 33 AT/RT and 154 MB tumor specimens also detected less SUN2 protein in AT/RT. Collectively, this study uncovers a novel tumor suppressor, SUN2, plays a critical role in miR-221/222-mediated AT/RT malignancy as well as supports miR-221/222 and SUN2 represent new promising targets for more active therapies in AT/RT. In addition, our miRNome and transcriptome data also provide a roadmap for further embryonal tumor research.

Intact INI1 gene region with paradoxical loss of protein expression in AT/RT: implications for a possible novel mechanism associated with absence of INI1 protein immunoreactivity.

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant central nervous system tumor often misdiagnosed as some other type of pediatric embryonal tumor, such as medulloblastoma (MB). Distinguishing AT/RT from primitive neuroectodermal tumor/MB is of clinical significance, as the reported survival rate of patients with AT/RT is much lower than that of patients with average-risk primitive neuroectodermal tumor/MB. The diagnosis of AT/RT currently relies primarily on the morphologic assessment and immunostaining of a few known markers, such as the lack of INI1 protein expression. Immunohistochemical staining of INI1 is considered very sensitive and is highly specific for the detection of INI1 genetic defects. Genetic studies have shown that deletion or mutation of the INI1 gene, which is located on 22q11.2, occurs in AT/RT lesions. During our gene expression microarray analysis, we unexpectedly found a subgroup of AT/RT patients still expressing INI1 mRNA, even though INI1 proteins were negative by immunohistochemistry in those cases. Direct DNA sequencing showed no INI1 sequence alternation in 3 of 4 AT/RTs. Point mutation was found in only 1 allele of the fourth case, which would result in a frameshift mutation and generate a new INI1 protein with an extra 100-aa tail. Global array comparative genomic hybridization analysis confirmed no aberration around the INI1 gene at 22q11.2. It also extended our knowledge on the chromosomal aberration situations in our series. This study reveals that a novel yet unidentified posttranscriptional regulatory mechanism(s) for INI1 protein synthesis exists in AT/RT tumor cells.

Pediatric primary central nervous system germ cell tumors of different prognosis groups show characteristic miRNome traits and chromosome copy number variations.

BACKGROUND: Intracranial pediatric germ cell tumors (GCTs) are rare and heterogeneous neoplasms and vary in histological differentiation, prognosis and clinical behavior. Germinoma and mature teratoma are GCTs that have a good prognosis, while other types of GCTs, termed nongerminomatous malignant germ cell tumors (NGMGCTs), are tumors with an intermediate or poor prognosis. The second group of tumors requires more extensive drug and irradiation treatment regimens. The mechanisms underlying the differences in incidence and prognosis of the various GCT subgroups are unclear. RESULTS: We identified a distinct mRNA profile correlating with GCT histological differentiation and prognosis, and also present in this study the first miRNA profile of pediatric primary intracranial GCTs. Most of the differentially expressed miRNAs were downregulated in germinomas, but miR-142-5p and miR-146a were upregulated. Genes responsible for self-renewal (such as POU5F1 (OCT4), NANOG and KLF4) and the immune response were abundant in germinomas, while genes associated with neuron differentiation, Wnt/beta-catenin pathway, invasiveness and epithelial-mesenchymal transition (including SNAI2 (SLUG) and TWIST2) were abundant in NGMGCTs. Clear transcriptome segregation based on patient survival was observed, with malignant NGMGCTs being closest to embryonic stem cells. Chromosome copy number variations (CNVs) at cytobands 4q13.3-4q28.3 and 9p11.2-9q13 correlated with GCT malignancy and clinical risk. Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5) within 4q13.3-4q28.3 were more abundant in germinomas. CONCLUSIONS: Our results integrate molecular profiles with clinical observations and provide insights into the underlying mechanisms causing GCT malignancy. The genes, pathways and microRNAs identified have the potential to be novel therapeutic targets.