Sequencing of cerebrospinal fluid cell-free DNA facilitated early differential diagnosis of intramedullary spinal cord tumors.

Pre-surgery differential diagnosis is valuable for personalized treatment planning in intramedullary spinal cord tumors. This study assessed the performance of sequencing cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) for differential diagnosis of these tumors. Prospectively enrolling 45 patients with intramedullary spinal cord lesions, including diffuse midline glioma (DMG), H3K27-altered (14/45), glioblastoma (1/45), H3-wildtype-astrocytoma (10/45), ependymoma (11/45), and other lesions (9/45), CSF samples were collected via lumbar puncture (41/45), intraoperative extraction (3/45), and Ommaya reservoir (1/45). Then, these samples underwent targeted sequencing along with paired tissue DNA. DMG, H3K27-altered patients exhibited a higher ctDNA positivity (85.7%, 12/14) compared to patients with H3-wildtype-astrocytoma (0/8, P = 0.0003), ependymoma (2/10, P = 0.003), and glioneuronal tumor (0/3, P = 0.009). The histological-grade-IV (P = 0.0027), Ki-67 index ≥10% (P = 0.014), and tumor reaching spinal cord surface (P = 0.012) are also associated with higher ctDNA positivity. Interestingly, for patients with TERT promoter mutant tumors, TERT mutation was detectable in the CSF cfDNA of one DMG case, but not other five cases with histological-grade-II tumors. Shared copy number variants were exclusively observed in DMG, H3K27-altered, and showed a strong correlation (Correlation = 0.95) between CSF and tissue. Finally, H3K27M mutations in CSF exhibited high diagnostic efficiency for DMG, H3K27-altered (Sensitivity = 85.7%, Specificity = 100.0%, AUC = 0.929). Notably, H3K27M was detectable in CSF from patients with recurrent tumors, making it easily applicable for postoperative monitoring. In conclusion, the molecular profile from ctDNA released into CSF of malignant tumors was more frequently detected compared to relatively benign ones. Sequencing of ctDNA in CSF exhibited high efficiency for the differential diagnosis of DMG, H3K27-altered.

MET fusions and splicing variants is a strong adverse prognostic factor in astrocytoma, isocitrate dehydrogenase mutant.

Liu et al. describe the adverse prognostic role of MET fusions and splicing variants in astrocytoma, isocitrate dehydrogenase mutant. On this basis, MET fusions and splicing variants was suggested to be a biomarker for the diagnosis of high-grade astrocytoma, isocitrate dehydrogenase mutant.

A Multiethnic Germline-Somatic Association Database Deciphers Multilayered and Interconnected Genetic Mutations in Cancer.

Inherited germline and acquired somatic alterations can both promote human tumor development. Elucidating the cooperation between somatic and germline genetic alterations that drive tumorigenesis could help inform precision cancer prevention and treatment strategies. Here, leveraging genomic genotyping and sequencing data from 9,029 patients with cancer with European, East Asian, and African ancestry, we performed a pan-cancer analysis to evaluate the associations between germline SNPs and somatic alterations, including single-nucleotide variant and small insertion/deletion mutations, copy-number variation, tumor mutational burden, and mutational signatures. Genome-wide significant germline-somatic pairs were abundant, and most of the associations were observed in one cancer type and one ancestry group. A user-friendly interactive Multiethnic Germline-Somatic Association (MGSA) database (http://wanglab-hkust.cn:3838/MGSA/) was developed, which can be used to query, browse, and download the results of the association analyses. Moreover, the MGSA database offers additional survival analysis and functional annotation. Together, this work provides a resource for uncovering the clinical and biological roles of associations between germline variants and somatic alterations in human cancer. SIGNIFICANCE: Comprehensive analysis of connections between germline variants and somatic events in cancer offers a resource for investigating the functional significance of genetic mutations and exploring genetic factors contributing to racial disparities.

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.

Tumor-associated monocytes promote mesenchymal transformation through EGFR signaling in glioma.

The role of brain immune compartments in glioma evolution remains elusive. We profile immune cells in glioma microenvironment and the matched peripheral blood from 11 patients. Glioblastoma exhibits specific infiltration of blood-originated monocytes expressing epidermal growth factor receptor (EGFR) ligands EREG and AREG, coined as tumor-associated monocytes (TAMo). TAMo infiltration is mutually exclusive with EGFR alterations (p = 0.019), while co-occurring with mesenchymal subtype (p = 4.7 × 10-7) and marking worse prognosis (p = 0.004 and 0.032 in two cohorts). Evolutionary analysis of initial-recurrent glioma pairs and single-cell study of a multi-centric glioblastoma reveal association between elevated TAMo and glioma mesenchymal transformation. Further analyses identify FOSL2 as a TAMo master regulator and demonstrates that FOSL2-EREG/AREG-EGFR signaling axis promotes glioma invasion in vitro. Collectively, we identify TAMo in tumor microenvironment and reveal its driving role in activating EGFR signaling to shape glioma evolution.

Genomic profiling and prognostic factors of H3 K27M-mutant spinal cord diffuse glioma.

H3 K27-altered diffuse midline glioma is a highly lethal pediatric-type tumor without efficacious treatments. Recent findings have highlighted the heterogeneity among diffuse midline gliomas with different locations and ages. Compared to tumors located in the brain stem and thalamus, the molecular and clinicopathological features of H3 K27-altered spinal cord glioma are still largely elusive, thus hindering the accurate management of patients. Here we aimed to characterize the clinicopathological and molecular features of H3 K27M-mutant spinal cord glioma in 77 consecutive cases. We found that the H3 K27M-mutant spinal cord glioma, with a median age of 35 years old, had a significantly better prognosis than H3 K27M-mutant brain tumors. We noticed a molecular heterogeneity of H3 K27M-mutant spinal cord astrocytoma via targeted sequencing with 34 cases. TP53 mutation which occurred in 58.8% of cases is mutually exclusive with PPM1D (26%) and NF1 (44%) mutations. The TP53-mutant cases had a significantly higher number of copy number variants (CNV) and a marginally higher proportion of pediatric patients (age at diagnosis <18 years old, p = 0.056). Cox regression and Kaplan-Meier curve analysis showed that the higher number of CNV events (≥3), chromosome (Chr) 9p deletion, Chr 10p deletion, ATRX mutation, CDK6 amplification, and retinoblastoma protein (RB) pathway alteration are associated with worse survival. Cox regression analysis with clinicopathological features showed that glioblastoma histological type and a high Ki-67 index (>10%) are associated with a worse prognosis. Interestingly, the histological type, an independent prognostic factor in multivariate Cox regression, can also stratify molecular features of H3 K27M-mutant spinal cord glioma, including the RB pathway, KRAS/PI3K pathway, and chromosome arms CNV. In conclusion, although all H3 K27M-mutant spinal cord diffuse glioma were diagnosed as WHO Grade 4, the histological type, molecular features representing chromatin instability, and molecular alterations associated with accelerated cell proliferative activity should not be ignored in clinical management.

Epidemiological characteristics and genetic alterations in adult diffuse glioma in East Asian populations.

Understanding the racial specificities of diseases-such as adult diffuse glioma, the most common primary malignant tumor of the central nervous system-is a critical step toward precision medicine. Here, we comprehensively review studies of gliomas in East Asian populations and other ancestry groups to clarify the racial differences in terms of epidemiology and genomic characteristics. Overall, we observed a lower glioma incidence in East Asians than in Whites; notably, patients with glioblastoma had significantly younger ages of onset and longer overall survival than the Whites. Multiple genome-wide association studies of various cohorts have revealed single nucleotide polymorphisms associated with overall and subtype-specific glioma susceptibility. Notably, only 3 risk loci-5p15.33, 11q23.3, and 20q13.33-were shared between patients with East Asian and White ancestry, whereas other loci predominated only in particular populations. For instance, risk loci 12p11.23, 15q15-21.1, and 19p13.12 were reported in East Asians, whereas risk loci 8q24.21, 1p31.3, and 1q32.1 were reported in studies in White patients. Although the somatic mutational profiles of gliomas between East Asians and non-East Asians were broadly consistent, a lower incidence of EGFR amplification in glioblastoma and a higher incidence of 1p19q-IDH-TERT triple-negative low-grade glioma were observed in East Asian cohorts. By summarizing large-scale disease surveillance, germline, and somatic genomic studies, this review reveals the unique characteristics of adult diffuse glioma among East Asians, to guide clinical management and policy design focused on patients with East Asian ancestry.

Clinical management and survival outcomes of patients with different molecular subtypes of diffuse gliomas in China (2011-2017): a multicenter retrospective study from CGGA.

OBJECTIVE: We aimed to summarize the clinicopathological characteristics and prognostic features of various molecular subtypes of diffuse gliomas (DGs) in the Chinese population. METHODS: In total, 1,418 patients diagnosed with DG between 2011 and 2017 were classified into 5 molecular subtypes according to the 2016 WHO classification of central nervous system tumors. The IDH mutation status was determined by immunohistochemistry and/or DNA sequencing, and 1p/19q codeletion was detected with fluorescence in situ hybridization. The median clinical follow-up time was 1,076 days. T-tests and chi-square tests were used to compare clinicopathological characteristics. Kaplan-Meier and Cox regression methods were used to evaluate prognostic factors. RESULTS: Our cohort included 15.5% lower-grade gliomas, IDH-mutant and 1p/19q-codeleted (LGG-IDHm-1p/19q); 18.1% lower-grade gliomas, IDH-mutant (LGG-IDHm); 13.1% lower-grade gliomas, IDH-wildtype (LGG-IDHwt); 36.1% glioblastoma, IDH-wildtype (GBM-IDHwt); and 17.2% glioblastoma, IDH-mutant (GBM-IDHm). Approximately 63.3% of the enrolled primary gliomas, and the median overall survival times for LGG-IDHm, LGG-IDHwt, GBM-IDHwt, and GBM-IDHm subtypes were 75.97, 34.47, 11.57, and 15.17 months, respectively. The 5-year survival rate of LGG-IDHm-1p/19q was 76.54%. We observed a significant association between high resection rate and favorable survival outcomes across all subtypes of primary tumors. We also observed a significant role of chemotherapy in prolonging overall survival for GBM-IDHwt and GBM-IDHm, and in prolonging post-relapse survival for the 2 recurrent GBM subtypes. CONCLUSIONS: By controlling for molecular subtypes, we found that resection rate and chemotherapy were 2 prognostic factors associated with survival outcomes in a Chinese cohort with DG.

WES data from 286 diffuse gliomas under the 2021 WHO Classification of Tumors of the Central Nervous System.

Diffuse gliomas (DGs) are the most common and lethal primary neoplasms in the central nervous system. The latest 2021 World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) was published in 2021, immensely changing the approach to diagnosis and decision making. As a part of the Chinese Glioma Genome Atlas (CGGA) project, our aim was to provide genomic profiling of gliomas in a Chinese cohort. Two hundred eighty six gliomas with different grades were collected over the last decade. Using the Illumina HiSeq platform, over 75.8 million high-quality 150 bp paired-end reads were generated per sample, yielding a total of 43.4 billion reads. We also collected each patient's clinical and pathological information and used it to annotate their genetic data. All patients were diagnosed and classified by neuro-pathologist under the 2021 WHO classification. This dataset provides an important reference for researchers and will significantly advance our understanding of gliomas.

Natural Coevolution of Tumor and Immunoenvironment in Glioblastoma.

Isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM) has a dismal prognosis. A better understanding of tumor evolution holds the key to developing more effective treatment. Here we study GBM's natural evolutionary trajectory by using rare multifocal samples. We sequenced 61,062 single cells from eight multifocal IDH wild-type primary GBMs and defined a natural evolution signature (NES) of the tumor. We show that the NES significantly associates with the activation of transcription factors that regulate brain development, including MYBL2 and FOSL2. Hypoxia is involved in inducing NES transition potentially via activation of the HIF1A-FOSL2 axis. High-NES tumor cells could recruit and polarize bone marrow-derived macrophages through activation of the FOSL2-ANXA1-FPR1/3 axis. These polarized macrophages can efficiently suppress T-cell activity and accelerate NES transition in tumor cells. Moreover, the polarized macrophages could upregulate CCL2 to induce tumor cell migration. SIGNIFICANCE: GBM progression could be induced by hypoxia via the HIF1A-FOSL2 axis. Tumor-derived ANXA1 is associated with recruitment and polarization of bone marrow-derived macrophages to suppress the immunoenvironment. The polarized macrophages promote tumor cell NES transition and migration. This article is highlighted in the In This Issue feature, p. 2711.

The Water Transport System in Astrocytes-Aquaporins.

Astrocytes have distinctive morphological and functional characteristics, and are found throughout the central nervous system. Astrocytes are now known to be far more than just housekeeping cells in the brain. Their functions include contributing to the formation of the blood-brain barrier, physically and metabolically supporting and communicating with neurons, regulating the formation and functions of synapses, and maintaining water homeostasis and the microenvironment in the brain. Aquaporins (AQPs) are transmembrane proteins responsible for fast water movement across cell membranes. Various subtypes of AQPs (AQP1, AQP3, AQP4, AQP5, AQP8 and AQP9) have been reported to be expressed in astrocytes, and the expressions and subcellular localizations of AQPs in astrocytes are highly correlated with both their physiological and pathophysiological functions. This review describes and summarizes the recent advances in our understanding of astrocytes and AQPs in regard to controlling water homeostasis in the brain. Findings regarding the features of different AQP subtypes, such as their expression, subcellular localization, physiological functions, and the pathophysiological roles of astrocytes are presented, with brain edema and glioma serving as two representative AQP-associated pathological conditions. The aim is to provide a better insight into the elaborate "water distribution" system in cells, exemplified by astrocytes, under normal and pathological conditions.

Recurrent PTPRZ1-MET fusion and a high occurrence rate of MET exon 14 skipping in brain metastases.

Identifying molecular features is an essential component of the management and targeted therapy of brain metastases (BMs). The molecular features are different between primary lung cancers and BMs of lung cancer. Here we report the DNA and RNA mutational profiles of 43 pathological samples of BMs. In addition to previously reported mutational events associated with targeted therapy, PTPRZ1-MET, which was previously exclusively identified in glioma, was present in two cases of BMs of lung cancer. Furthermore, MET exon 14 skipping may be more common (6/37 cases) in BMs of lung cancer than the frequency previously reported in lung cancer. These findings highlight the clinical significance of targeted DNA plus RNA sequencing for BMs and suggest PTPRZ1-MET and MET exon 14 skipping as critical molecular events that may serve as targets of targeted therapy in BMs.

Molecular pathology and clinical implications of diffuse glioma.

ABSTRACT: The prognosis for diffusely infiltrating gliomas at World Health Organization (WHO) grade 2-4 remains dismal due to their heterogeneity. The rapid development of genome-wide molecular-profiling-associated studies has greatly promoted the accuracy of glioma classification. Thus, the latest version of the WHO classification of the central nervous system tumors published in 2021 has incorporated more molecular biomarkers together with histological features for the diagnosis of gliomas. Advanced usage of molecular pathology in clinical diagnostic practice provides also new opportunities for the therapy of patients with glioma, including surgery, radiotherapy and chemotherapy, targeted therapy, immunotherapy, and more precision clinical trials. Herein, we highlight the updates in the classification of gliomas according to the latest WHO guidelines and summarize the clinically relevant molecular markers by focusing on their applications in clinical practice. We also review the advances in molecular features of gliomas, which can facilitate the development of glioma therapies, thereby discussing the challenges and future directions of molecular pathology toward precision medicine for patients with glioma.

Molecular Characterization and Clinical Relevance of ANXA1 in Gliomas via 1,018 Chinese Cohort Patients.

Annexin A1 (ANXA1) is a calcium-dependent phospholipid-binding protein and has been implicated in multiple functions essential in cancer, including cell proliferation, apoptosis, chemosensitivity, metastasis, and invasion. However, the biological role and clinical behavior of ANXA1 in glioma remain unclear. In this study, RNA-seq (n = 1018 cases) and whole-exome sequencing (WES) (n = 286 cases) data on a Chinese cohort, RNA-seq data with different histological regions of glioblastoma blocks (n = 270 cases), and scRNA-seq data (n = 7630 cells) were used. We used the R software to perform statistical calculations and graph rendering. We found that ANXA1 is closely related to the malignant progression in gliomas. Meanwhile, ANXA1 is significantly associated with clinical behavior. Furthermore, the mutational profile revealed that glioma subtypes classified by ANXA1 expression showed distinct genetic features. Functional analyses suggest that ANXA1 correlates with the immune-related function and cancer hallmark. At a single-cell level, we found that ANXA1 is highly expressed in M2 macrophages and tumor cells of the mesenchymal subtype. Importantly, our result suggested that ANXA1 expression is significant with the patient's survival outcome. Our study revealed that ANXA1 was closely related to immune response. ANXA1 plays a key factor in M2 macrophages and MES tumor cells. Patients with lower ANXA1 expression levels tended to experience improved survival. ANXA1 may become a valuable factor for the diagnosis and treatment of gliomas in clinical practice.

YTHDF2 facilitates UBXN1 mRNA decay by recognizing METTL3-mediated m6A modification to activate NF-κB and promote the malignant progression of glioma.

BACKGROUND: The prognosis for diffuse gliomas is very poor and the mechanism underlying their malignant progression remains unclear. Here, we aimed to elucidate the role and mechanism of the RNA N6,2'-O-dimethyladenosine (m6A) reader, YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), in regulating the malignant progression of gliomas. METHODS: YTHDF2 mRNA levels and functions were assessed using several independent datasets. Western blotting, quantitative polymerase chain reaction, and immunohistochemistry were used to evaluate the expression levels of YTHDF2 and other molecules in human and mouse tumor tissues and cells. Knockdown and overexpression were used to evaluate the effects of YTHDF2, methyltransferase-like 3 (METTL3), and UBX domain protein 1 (UBXN1) on glioma malignancy in cell and orthotopic xenograft models. RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were performed to study the mechanisms underlying the oncogenic role of YTHDF2. RESULTS: YTHDF2 expression was positively associated with a higher malignant grade and molecular subtype of glioma and poorer prognosis. YTHDF2 promoted the malignant progression of gliomas in both in vitro and in vivo models. Mechanistically, YTHDF2 accelerated UBXN1 mRNA degradation via METTL3-mediated m6A, which, in turn, promoted NF-κB activation. We further revealed that UBXN1 overexpression attenuated the oncogenic effect of YTHDF2 overexpression and was associated with better survival in patients with elevated YTHDF2 expression. CONCLUSIONS: Our findings confirmed that YTHDF2 promotes the malignant progression of gliomas and revealed important insight into the upstream regulatory mechanism of NF-κB activation via UBXN1 with a primary focus on m6A modification.

Spinal Cord Diffuse Midline Gliomas With H3 K27m-Mutant: Clinicopathological Features and Prognosis.

BACKGROUND: "Diffuse midline glioma, H3 K27M-mutant" (DMG) mainly arises within the pontine, thalamic, and spinal cord regions. Because of the rarity of spinal cord gliomas, the general knowledge surrounding DMGs is mainly based on pontine and thalamic gliomas, whereas tumor location tends to influence the clinicopathological features and prognosis. OBJECTIVE: To determine the clinicopathological characteristics and molecular profiles of DMGs located in the spinal cord. METHODS: The clinical and molecular pathologic features and prognosis were comprehensively analyzed in a series of 44 patients with spinal cord DMGs. RESULTS: The median age was 36 yr, and 88.7% of patients (39/44) were adults (≥18 yr). Histopathologically, malignant grades included grade II (16 cases), grade III (20 cases), and grade IV (8 cases). Compared with patients with histological grade IV, patients with lower histological grade (grade II/III) were older (37 vs 24 yr, P = .020) and were associated with longer overall survival (24.1 vs 8.6 mo, P = .007). All 30 tested tumors were isocitrate dehydrogenase (IDH) wild type, and 96% of cases (22/23) presented with unmethylated O6-methylguanine-DNA methyltransferase. Univariate and multivariate analyses showed that histological grade and presurgery McCormick Scale scores were independent prognostic factors for overall survival, whereas extensive surgical resection and chemoradiotherapy were not significantly associated with improved survival. The most frequent anatomic locations were the cervical enlargement (C4-T1, n = 16) and conus medullaris (T12-L1, n = 13), which exhibited distinctive clinical characteristics and molecular features. CONCLUSION: The findings provide guidelines for the evidence-based practice of the specialized management of spinal cord DMGs.