Identifying predictors of glioma evolution from longitudinal sequencing.

Clonal evolution drives cancer progression and therapeutic resistance. Recent studies have revealed divergent longitudinal trajectories in gliomas, but early molecular features steering posttreatment cancer evolution remain unclear. Here, we collected sequencing and clinical data of initial-recurrent tumor pairs from 544 adult diffuse gliomas and performed multivariate analysis to identify early molecular predictors of tumor evolution in three diffuse glioma subtypes. We found that CDKN2A deletion at initial diagnosis preceded tumor necrosis and microvascular proliferation that occur at later stages of IDH-mutant glioma. Ki67 expression at diagnosis was positively correlated with acquiring hypermutation at recurrence in the IDH-wild-type glioma. In all glioma subtypes, MYC gain or MYC-target activation at diagnosis was associated with treatment-induced hypermutation at recurrence. To predict glioma evolution, we constructed CELLO2 (Cancer EvoLution for LOngitudinal data version 2), a machine learning model integrating features at diagnosis to forecast hypermutation and progression after treatment. CELLO2 successfully stratified patients into subgroups with distinct prognoses and identified a high-risk patient group featured by MYC gain with worse post-progression survival, from the low-grade IDH-mutant-noncodel subtype. We then performed chronic temozolomide-induction experiments in glioma cell lines and isogenic patient-derived gliomaspheres and demonstrated that MYC drives temozolomide resistance by promoting hypermutation. Mechanistically, we demonstrated that, by binding to open chromatin and transcriptionally active genomic regions, c-MYC increases the vulnerability of key mismatch repair genes to treatment-induced mutagenesis, thus triggering hypermutation. This study reveals early predictors of cancer evolution under therapy and provides a resource for precision oncology targeting cancer dynamics in diffuse gliomas.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

scONE-seq: A single-cell multi-omics method enables simultaneous dissection of phenotype and genotype heterogeneity from frozen tumors.

Single-cell multi-omics can provide a unique perspective on tumor cellular heterogeneity. Most previous single-cell whole-genome RNA sequencing (scWGS-RNA-seq) methods demonstrate utility with intact cells from fresh samples. Among them, many are not applicable to frozen samples that cannot produce intact single-cell suspensions. We have developed scONE-seq, a versatile scWGS-RNA-seq method that amplifies single-cell DNA and RNA without separating them from each other and hence is compatible with frozen biobanked samples. We benchmarked scONE-seq against existing methods using fresh and frozen samples to demonstrate its performance in various aspects. We identified a unique transcriptionally normal-like tumor clone by analyzing a 2-year frozen astrocytoma sample, demonstrating that performing single-cell multi-omics interrogation on biobanked tissue by scONE-seq could enable previously unidentified discoveries in tumor biology.

Cancer-associated fibroblasts potentiate colorectal cancer progression by crosstalk of the IGF2-IGF1R and Hippo-YAP1 signaling pathways.

Colorectal cancer (CRC) is one of the most common cancers worldwide. The tumor microenvironment exerts crucial effects in driving CRC progression. Cancer-associated fibroblasts (CAFs) serve as one of the most important tumor microenvironment components promoting CRC progression. This study aimed to elucidate the novel molecular mechanisms of CAF-secreted insulin-like growth factor (IGF) 2 in colorectal carcinogenesis. Our results indicated that IGF2 was a prominent factor upregulated in CAFs compared with normal fibroblasts. CAF-derived conditioned media (CM) promoted tumor growth, migration, and invasion of HCT 116 and DLD-1 cells. IGF1R expression is significantly increased in CRC, serving as a potent receptor in response to IGF2 stimulation and predicting unfavorable outcomes for CRC patients. Apart from the PI3K-AKT pathway, RNA-seq analysis revealed that the YAP1-target signature serves as a prominent downstream effector to mediate the oncogenic signaling of IGF2-IGF1R. By single-cell RNA sequencing (scRNA-seq) and immunohistochemical validation, IGF2 was found to be predominantly secreted by CAFs, whereas IGF1R was expressed mainly by cancer cells. IGF2 triggers the nuclear accumulation of YAP1 and upregulates YAP1 target signatures; however, these effects were abolished by either IGF1R knockdown or inhibition with picropodophyllin (PPP), an IGF1R inhibitor. Using CRC organoid and in vivo studies, we found that cotargeting IGF1R and YAP1 with PPP and verteporfin (VP), a YAP1 inhibitor, enhanced antitumor effects compared with PPP treatment alone. In conclusion, this study revealed a novel molecular mechanism by which CAFs promote CRC progression. The findings highlight the translational potential of the IGF2-IGF1R-YAP1 axis as a prognostic biomarker and therapeutic target for CRC. © 2022 The Pathological Society of Great Britain and Ireland.

Combinations of Single-Gene Biomarkers Can Precisely Stratify 1,028 Adult Gliomas for Prognostication.

Advanced genomic techniques have now been incorporated into diagnostic practice in neuro-oncology in the literature. However, these assays are expensive and time-consuming and demand bioinformatics expertise for data interpretation. In contrast, single-gene tests can be run much more cheaply, with a short turnaround time, and are available in general pathology laboratories. The objective of this study was to establish a molecular grading scheme for adult gliomas using combinations of commonly available single-gene tests. We retrospectively evaluated molecular diagnostic data of 1,275 cases of adult diffuse gliomas from three institutions where we were testing for IDH1/2 mutation, TERTp mutation, 1p19q codeletion, EGFR amplification, 10q deletion, BRAF V600E, and H3 mutations liberally in our regular diagnostic workup. We found that a molecular grading scheme of Group 1 (1p19q codeleted, IDH mutant), Group 2 (IDH mutant, 1p19q non-deleted, TERT mutant), Group 3 (IDH mutant, 1p19q non-deleted, TERT wild type), Group 4 (IDH wild type, BRAF mutant), Group 5 (IDH wild type, BRAF wild type and not possessing the criteria of Group 6), and Group 6 (IDH wild type, and any one of TERT mutant, EGFR amplification, 10q deletion, or H3 mutant) could significantly stratify this large cohort of gliomas for risk. A total of 1,028 (80.6%) cases were thus classifiable with sufficient molecular data. There were 270 cases of molecular Group 1, 59 cases of molecular Group 2, 248 cases of molecular Group 3, 27 cases of molecular Group 4, 117 cases of molecular Group 5, and 307 cases of molecular Group 6. The molecular groups were independent prognosticators by multivariate analyses and in specific instances, superseded conventional histological grades. We were also able to validate the usefulness of the Groups with a cohort retrieved from The Cancer Genome Atlas (TCGA) where similar molecular tests were liberally available. We conclude that a single-gene molecular stratification system, useful for fine prognostication, is feasible and can be adopted by a general pathology laboratory.

Establishment and characterization of meningioma patient-derived organoid.

Meningioma is a central nervous system tumor originated from arachnoid cells. 2D cell culture is widely used as a platform for tumor research as it enables us to culture cells in in vitro and a controlled environment. However, in 2D culture condition, 3D architecture of in vivo tumor mass is lost and phenotypic change may occur. Due to the drawbacks of 2D cell culture, organoid culture is seen as an alternative platform for disease modeling, drug testing and personalized medicine. The objective of this study was to establishing protocol for culturing cells from patient meningioma tissue in in vitro 3D environment. Eight meningiomas were collected for the 3D organoid culture. Cells of 5 meningioma tissues survived and proliferated. Under 3D culture condition, cell aggregates were formed and cytoplasmic processes linking the cell aggregates could be observed. In H&E staining, ovaloid cells and spindle cells were observed. Resembling cultured organoids observed under the light microscope, cell aggregates were also observed in the H&E staining. Epithelial Membrane Antigen (EMA) staining was positive. In 4 (80%) cultured organoids, low Ki67 index (≤6%) were measured. In one cultured organoid, a high Ki67 index (12.8%) was seen. The result of this study revealed the feasibility of culturing meningioma cells in in vitro 3D culture condition. Organoid technology showed its potential as an alternative platform for meningioma research.

Mismatch repair proteins PMS2 and MLH1 can further refine molecular stratification of IDH-mutant lower grade astrocytomas.

The diagnostic role of Isocitrate Dehydrogenase (IDH) mutation status in adult lower grade astrocytomas was first formally presented within the WHO Classification of Tumours of the Central Nervous System (2016). IDH-mutant astrocytomas are not as common as IDH-wildtype astrocytomas but are of better prognosis. Our previous study provided an evident that IDH-mutant lower grade astrocytomas is not a homogeneous group and could be further stratified by PDGFRA amplification, CDK4 amplification and CDKN2A deletion. In this study, we detected the expressions of DNA mismatch repair (MMR) proteins (PMS2, MLH1, MSH2, MSH6) and PD-L1 by immunohistochemistry in 147 IDH-mutant lower grade astrocytomas and explored their clinical relevance. The loss of was identified in 28.6%, 1.4%, 8.8% and 13.6%, respectively. PD-L1 expression was detected in 1.4% of this cohort. Survival analysis revealed that loss of PMS2 was correlated with shorter OS (p < 0.001) and PFS (p = 0.005). Loss of PMS2 or MLH1 was associated with shorter OS (p < 0.001) and PFS (p = 0.008). In IDH-mutant lower grade astrocytomas without CDKN2A deletion, loss of PMS2 was associated with poorer OS (p < 0.001) and PFS (p = 0.001). Furthermore, among IDH-mutant lower grade astrocytomas lacking the three biomarkers (PDGFRA, CDK4 and CDKN2A), loss of PMS2 was also associated with a poorer OS (p < 0.001) and PFS (p = 0.003). Our data illustrated the potential application of MMR genes in stratification of IDH-mutant lower grade astrocytomas without PDGFRA, CDK4 and CDKN2A copy number alterations.

Molecular landscape of IDH-mutant primary astrocytoma Grade IV/glioblastomas.

WHO 2016 classified glioblastomas into IDH-mutant and IDH-wildtype with the former having a better prognosis but there was no study on IDH-mutant primary glioblastomas only, as previous series included secondary glioblastomas. We recruited a series of 67 IDH-mutant primary glioblastomas/astrocytoma IV without a prior low-grade astrocytoma and examined them using DNA-methylation profiling, targeted sequencing, RNA sequencing and TERT promoter sequencing, and correlated the molecular findings with clinical parameters. The median OS of 39.4 months of 64 cases and PFS of 25.9 months of 57 cases were better than the survival data of IDH-wildtype glioblastomas and IDH-mutant secondary glioblastomas retrieved from datasets. The molecular features often seen in glioblastomas, such as EGFR amplification, combined +7/-10, and TERT promoter mutations were only observed in 6/53 (11.3%), 4/53 (7.5%), and 2/67 (3.0%) cases, respectively, and gene fusions were found only in two cases. The main mechanism for telomere maintenance appeared to be alternative lengthening of telomeres as ATRX mutation was found in 34/53 (64.2%) cases. In t-SNE analyses of DNA-methylation profiles, with an exceptional of one case, a majority of our cases clustered to IDH-mutant high-grade astrocytoma subclass (40/53; 75.5%) and the rest to IDH-mutant astrocytoma subclass (12/53; 22.6%). The latter was also enriched with G-CIMP high cases (12/12; 100%). G-CIMP-high status and MGMT promoter methylation were independent good prognosticators for OS (p = 0.022 and p = 0.002, respectively) and TP53 mutation was an independent poor prognosticator (p = 0.013) when correlated with other clinical parameters. Homozygous deletion of CDKN2A/B was not correlated with OS (p = 0.197) and PFS (p = 0.278). PDGFRA amplification or mutation was found in 16/59 (27.1%) of cases and was correlated with G-CIMP-low status (p = 0.010). Aside from the three well-known pathways of pathogenesis in glioblastomas, chromatin modifying and mismatch repair pathways were common aberrations (88.7% and 20.8%, respectively), the former due to high frequency of ATRX involvement. We conclude that IDH-mutant primary glioblastomas have better prognosis than secondary glioblastomas and have major molecular differences from other commoner glioblastomas. G-CIMP subgroups, MGMT promoter methylation, and TP53 mutation are useful prognostic adjuncts.

Clinical and mutational profiles of adult medulloblastoma groups.

Adult medulloblastomas are clinically and molecularly understudied due to their rarity. We performed molecular grouping, targeted sequencing, and TERT promoter Sanger sequencing on a cohort of 99 adult medulloblastomas. SHH made up 50% of the cohort, whereas Group 3 (13%) was present in comparable proportion to WNT (19%) and Group 4 (18%). In contrast to paediatric medulloblastomas, molecular groups had no prognostic impact in our adult cohort (p = 0.877). Most frequently mutated genes were TERT (including promoter mutations, mutated in 36% cases), chromatin modifiers KMT2D (31%) and KMT2C (30%), TCF4 (31%), PTCH1 (27%) and DDX3X (24%). Adult WNT patients showed enrichment of TP53 mutations (6/15 WNT cases), and 3/6 TP53-mutant WNT tumours were of large cell/anaplastic histology. Adult SHH medulloblastomas had frequent upstream pathway alterations (PTCH1 and SMO mutations) and few downstream alterations (SUFU mutations, MYCN amplifications). TERT promoter mutations were found in 72% of adult SHH patients, and were restricted to this group. Adult Group 3 tumours lacked hallmark MYC amplifications, but had recurrent mutations in KBTBD4 and NOTCH1. Adult Group 4 tumours harboured recurrent mutations in TCF4 and chromatin modifier genes. Overall, amplifications of MYC and MYCN were rare (3%). Since molecular groups were not prognostic, alternative prognostic markers are needed for adult medulloblastoma. KMT2C mutations were frequently found across molecular groups and were associated with poor survival (p = 0.002). Multivariate analysis identified histological type (p = 0.026), metastasis (p = 0.031) and KMT2C mutational status (p = 0.046) as independent prognosticators in our cohort. In summary, we identified distinct clinical and mutational characteristics of adult medulloblastomas that will inform their risk stratification and treatment.

Patient-derived tumor organoid predicts drugs response in glioblastoma: A step forward in personalized cancer therapy?

Despite significant medical advances, glioblastoma multiforme (GBM) remains a formidable therapeutic challenge. Advent of targeted capture sequencing and patients-derived organoid cultures may hold the key to scientifically sound individualized treatment approaches. We report on our initial experience of using the combination of these two technologies to create a tailored approach of systemic therapies for a patient with GBM, which challenges the conventional treatment paradigm, as well as specifically highlighting the complexities of such an approach and the potential for a more favorable treatment outcome.

IDH mutant lower grade (WHO Grades II/III) astrocytomas can be stratified for risk by CDKN2A, CDK4 and PDGFRA copy number alterations.

In the 2016, WHO classification of tumors of the central nervous system, isocitrate dehydrogenase (IDH) mutation is a main classifier for lower grade astrocytomas and IDH-mutated astrocytomas is now regarded as a single group with longer survival. However, the molecular and clinical heterogeneity among IDH mutant lower grade (WHO Grades II/III) astrocytomas have only rarely been investigated. In this study, we recruited 160 IDH mutant lower grade (WHO Grades II/III) astrocytomas, and examined PDGFRA amplification, CDKN2A deletion and CDK4 amplification by FISH analysis, TERT promoter mutation by Sanger sequencing and ATRX loss and p53 expression by immunohistochemistry. We identified PDGFRA amplification, CDKN2A homozygous deletion and CDK4 amplification in 18.8%, 15.0% and 18.1% of our cohort respectively, and these alterations occurred in a mutually exclusive fashion. PDGFRA amplification was associated with shorter PFS (P = 0.0003) and OS (P < 0.0001). In tumors without PDGFRA amplification, CDKN2A homozygous deletion or CDK4 amplification was associated with a shorter OS (P = 0.035). Tumors were divided into three risk groups based on the presence of molecular alterations: high risk (PDGFRA amplification), intermediate risk (CDKN2A deletion or CDK4 amplification) and low risk (neither CDKN2A deletion and CDK4 amplification nor PDGFRA amplification). These three risk groups were significantly different in overall survival with mean survivals of 40.5, 62.9 and 71.5 months. The high-risk group also demonstrated a shorter PFS compared to intermediate- (P = 0.036) and low-risk (P < 0.0001) groups. One limitation of this study is the relatively short follow-up period, a common confounding factor for studies on low-grade tumors. Our data illustrate that IDH mutant lower grade astrocytomas is not a homogeneous group and should be molecularly stratified for risk.

Identification of subsets of IDH-mutant glioblastomas with distinct epigenetic and copy number alterations and stratified clinical risks.

BACKGROUND: IDH-mutant glioblastoma is classified by the 2016 CNS WHO as a group with good prognosis. However, the actual number of cases examined in the literature is relatively small. We hypothesize that IDH-mutant glioblastoma is not a uniform group and should be further stratified. METHODS: We conducted methylation profiles and estimated copy number variations of 57 IDH-mutant glioblastomas. RESULTS: Our results showed that 59.6% and 40.4% of tumors belonged to glioma-CpG island methylator phenotype (G-CIMP)-high and G-CIMP-low methylation subgroups, respectively. G-CIMP-low subgroup was associated with significantly worse overall survival (OS) as compared to G-CIMP-high (P = .005). CDKN2A deletion (42.1%) was the most common gene copy number variation, and was significantly associated with G-CIMP-low subgroup (P = .004). Other frequent copy number changes included mesenchymal-epithelial transition (MET) (5.3%), CCND2 (19.3%), PDGFRA (14.0%), CDK4 (12.3%), and EGFR (12.3%) amplification. Both CDKN2A deletion (P = .036) and MET amplification (P < .001) were associated with poor OS in IDH-mutant glioblastomas. Combined epigenetic signature and gene copy number variations separated IDH-mutant glioblastomas into Group 1 (G-CIMP-high), Group 2 (G-CIMP-low without CDKN2A nor MET alteration), and Group 3 (G-CIMP-low with CDKN2A and/or MET alteration). Survival analysis revealed Groups 1 and 2 exhibited a favorable OS (median survival: 619 d [20.6 mo] and 655 d [21.8 mo], respectively). Group 3 exhibited a significant shorter OS (median survival: 252 d [8.4 mo]). Multivariable analysis confirmed the independent prognostic significance of our Groups. CONCLUSIONS: IDH-mutant glioblastomas should be stratified for risk with combined epigenetic signature and CDKN2A/MET status and some cases have poor outcome.

FGF18, a prominent player in FGF signaling, promotes gastric tumorigenesis through autocrine manner and is negatively regulated by miR-590-5p.

Fibroblast growth factors (FGFs) and their receptors are significant components during fundamental cellular processes. FGF18 plays a distinctive role in modulating the activity of both tumor cells and tumor microenvironment. This study aims to comprehensively investigate the expression and functional role of FGF18 in gastric cancer (GC) and elucidate its regulatory mechanisms. The upregulation of FGF18 was detected in seven out of eleven (63.6%) GC cell lines. In primary GC samples, FGF18 was overexpressed in genomically stable and chromosomal instability subtypes of GC and its overexpression was associated with poor survival. Knocking down FGF18 inhibited tumor formation abilities, induced G1 phase cell cycle arrest and enhanced anti-cancer drug sensitivity. Expression microarray profiling revealed that silencing of FGF18 activated ATM pathway but quenched TGF-ß pathway. The key factors that altered in the related signaling were validated by western blot and immunofluorescence. Meanwhile, treating GC cells with human recombinant FGF18 or FGF18-conditioned medium accelerated tumor growth through activation of ERK-MAPK signaling. FGF18 was further confirmed to be a direct target of tumor suppressor, miR-590-5p. Their expressions showed a negative correlation in primary GC samples and more importantly, re-overexpression of FGF18 partly abolished the tumor-suppressive effect of miR-590-5p. Our study not only identified that FGF18 serves as a novel prognostic marker and a therapeutic target in GC but also enriched the knowledge of FGF-FGFR signaling during gastric tumorigenesis.

Pediatric low-grade gliomas can be molecularly stratified for risk.

Pediatric low-grade gliomas (PLGGs) consist of a number of entities with overlapping histological features. PLGGs have much better prognosis than the adult counterparts, but a significant proportion of PLGGs suffers from tumor progression and recurrence. It has been shown that pediatric and adult low-grade gliomas are molecularly distinct. Yet the clinical significance of some of newer biomarkers discovered by genomic studies has not been fully investigated. In this study, we evaluated in a large cohort of 289 PLGGs a list of biomarkers and examined their clinical relevance. TERT promoter (TERTp), H3F3A and BRAF V600E mutations were detected by direct sequencing. ATRX nuclear loss was examined by immunohistochemistry. CDKN2A deletion, KIAA1549-BRAF fusion, and MYB amplification were determined by fluorescence in situ hybridization (FISH). TERTp, H3F3A, and BRAF V600E mutations were identified in 2.5, 6.4, and 7.4% of PLGGs, respectively. ATRX loss was found in 4.9% of PLGGs. CDKN2A deletion, KIAA1549-BRAF fusion and MYB amplification were detected in 8.8, 32.0 and 10.6% of PLGGs, respectively. Survival analysis revealed that TERTp mutation, H3F3A mutation, and ATRX loss were significantly associated with poor PFS (p < 0.0001, p < 0.0001, and p = 0.0002) and OS (p < 0.0001, p < 0.0001, and p < 0.0001). BRAF V600E was associated with shorter PFS (p = 0.011) and OS (p = 0.032) in a subset of PLGGs. KIAA1549-BRAF fusion was a good prognostic marker for longer PFS (p = 0.0017) and OS (p = 0.0029). MYB amplification was also a favorable marker for a longer PFS (p = 0.040). Importantly, we showed that these molecular biomarkers can be used to stratify PLGGs into low- (KIAA1549-BRAF fusion or MYB amplification), intermediate-I (BRAF V600E and/or CDKN2A deletion), intermediate-II (no biomarker), and high-risk (TERTp or H3F3A mutation or ATRX loss) groups with distinct PFS (p < 0.0001) and OS (p < 0.0001). This scheme should aid in clinical decision-making.

Oligodendrogliomas in pediatric and teenage patients only rarely exhibit molecular markers and patients have excellent survivals.

Although oligodendrogliomas appear histologically similar in adult and pediatric patients, the latter have only been rarely studied and most of those studies did not have long follow-up. We examined 55 oligodendroglial tumors from pediatric and teenage patients for their biomarkers with formalin-fixed paraffin-embedded tissues and studied their survival status. None of the tumors harbored 1p/19q codeletion or IDH mutation. Mutations in TERTp (4%), BRAF (11%), FGFR1 (3%) and H3F3A (5%), fusions of BRAF (8%) and FGFR1 (8%) were found sparingly and almost all in a mutually exclusive manner. Molecular events were exclusively found in tumors with classic oligodendroglial histology. Survival analysis showed remarkably excellent prognosis compared to the adult counterparts. 5-year overall survival was 95% in our cohort with median follow-up of 8.1 years and in nine patients with follow-up more than 10 years, the 10-year overall survival was 100%. The 5-year and 10-year progression-free survivals of our cohort were 89 and 77%, respectively. FGFR1 fusion seemed to confer a poor prognosis in pediatric oligodendrogliomas. Patients receiving adjuvant chemotherapy (p = 0.046) or harboring Grade II histology (p < 0.001) had longer interval to recurrence. Our study demonstrated the distinct indolent clinical course of pediatric and teenage oligodendrogliomas compared to the adult tumors. Molecular markers commonly seen in adult oligodendrogliomas and other pediatric low-grade gliomas were only rarely seen. Since there is no clinical or molecular evidence suggesting that pediatric "oligodendrogliomas" are the same as adult oligodendrogliomas albeit histologic similarity, a case can be made for their separation from adult oligodendrogliomas in the next WHO classification.

Specific targeting of point mutations in EGFR L858R-positive lung cancer by CRISPR/Cas9.

Cancer cells are defined genetically by the mutations they harbor, commonly single nucleotide substitutions. Therapeutic approaches which specifically target cancer cells by recognizing these defining genetic aberrations are expected to exhibit minimal side-effects. However, current protein-based targeted therapy is greatly limited by the range of genes that can be targeted, as well as by acquired resistance. We hypothesized that a therapeutic oligonucleotide-based strategy may address this need of specific cancer targeting. We used CRISPR/Cas9 system to target a commonly occurring EGFR point mutation, L858R, with an oligonucleotide guide that recognizes L858R as the suitable protospacer-adjacent motif (PAM) sequence for DNA cleavage. We found that this strategy, which utilized PAM to differentiate cancer mutation from normal, afforded high specificity to the extent of a single nucleotide substitution. The anti-L858R vehicle resulted in selective genome cleavage only in L858R mutant cells, as detected by Sanger sequencing and T7 Endonuclease I assay. Wild-type cells were unaffected by the same treatment. Digital PCR revealed 37.9 ± 8.57% of L858R gene copies were targeted in mutant. Only treated mutant cells, but not wild-type cells, showed reduction in EGFR expression and decreased cell proliferation. Treated mutant cells also formed smaller tumor load in vivo. This targeting approach is expected to be able to target a significant subset of the 15-35% cancer mutations with C > G, A > G, and T > G point mutations. Thus, this strategy may serve as a useful approach to target cancer-defining mutations with specificity, to the extent of differentiating the change of a single nucleotide.

SRGAP1, a crucial target of miR-340 and miR-124, functions as a potential oncogene in gastric tumorigenesis.

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-124 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of 12 (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited Wnt/ß-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-124. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-124. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating Wnt/ß-catenin pathway.