Long noncoding RNA TUG1 promotes cisplatin resistance in ovarian cancer via upregulation of DNA polymerase eta.

Chemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum-based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR-4687-3p and miR-6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin-induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.

Exclusive expression of KANK4 promotes myofibroblast mobility in keloid tissues.

Keloids are characterized by abnormal wound healing with excessive accumulation of extracellular matrix. Myofibroblasts are the primary contributor to extracellular matrix secretion, playing an essential role in the wound healing process. However, the differences between myofibroblasts involved in keloid formation and normal wound healing remain unclear. To identify the specific characteristics of keloid myofibroblasts, we initially assessed the expression levels of well-established myofibroblast markers, α-smooth muscle actin (α-SMA) and transgelin (TAGLN), in scar and keloid tissues (n = 63 and 51, respectively). Although myofibroblasts were present in significant quantities in keloids and immature scars, they were absent in mature scars. Next, we conducted RNA sequencing using myofibroblast-rich areas from keloids and immature scars to investigate the difference in RNA expression profiles among myofibroblasts. Among significantly upregulated 112 genes, KN motif and ankyrin repeat domains 4 (KANK4) was identified as a specifically upregulated gene in keloids. Immunohistochemical analysis showed that KANK4 protein was expressed in myofibroblasts in keloid tissues; however, it was not expressed in any myofibroblasts in immature scar tissues. Overexpression of KANK4 enhanced cell mobility in keloid myofibroblasts. Our results suggest that the KANK4-mediated increase in myofibroblast mobility contributes to keloid pathogenesis.

Astrocyte-induced mGluR1 activates human lung cancer brain metastasis via glutamate-dependent stabilization of EGFR.

There are limited methods to stably analyze the interactions between cancer cells and glial cells in vitro, which hinders our molecular understanding. Here, we develop a simple and stable culture method of mouse glial cells, termed mixed-glial culture on/in soft substrate (MGS), which serves well as a platform to study cancer-glia interactions. Using this method, we find that human lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signaling in the brain microenvironment. Mechanistically, interactions with astrocytes induce mGluR1 in cancer cells through the Wnt-5a/prickle planar cell polarity protein 1 (PRICKLE1)/RE1 silencing transcription factor (REST) axis. Induced mGluR1 directly interacts with and stabilizes the epidermal growth factor receptor (EGFR) in a glutamate-dependent manner, and these cells then become responsive to mGluR1 inhibition. Our results highlight increased dependence on mGluR1 signaling as an adaptive strategy and vulnerability of human lung cancer brain metastasis.

Mutation of the SWI/SNF complex component Smarce1 decreases nucleosome stability in embryonic stem cells and impairs differentiation.

The SWI/SNF chromatin remodeling complex consists of more than ten component proteins that form a large protein complex of >1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem cells (ESCs). Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ESCs, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested that there was an ectopic genomic distribution of the SWI/SNF complex upon disruption of Smarce1, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ESC differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

TUG1-mediated R-loop resolution at microsatellite loci as a prerequisite for cancer cell proliferation.

Oncogene-induced DNA replication stress (RS) and consequent pathogenic R-loop formation are known to impede S phase progression. Nonetheless, cancer cells continuously proliferate under such high-stressed conditions through incompletely understood mechanisms. Here, we report taurine upregulated gene 1 (TUG1) long noncoding RNA (lncRNA), which is highly expressed in many types of cancers, as an important regulator of intrinsic R-loop in cancer cells. Under RS conditions, TUG1 is rapidly upregulated via activation of the ATR-CHK1 signaling pathway, interacts with RPA and DHX9, and engages in resolving R-loops at certain loci, particularly at the CA repeat microsatellite loci. Depletion of TUG1 leads to overabundant R-loops and enhanced RS, leading to substantial inhibition of tumor growth. Our data reveal a role of TUG1 as molecule important for resolving R-loop accumulation in cancer cells and suggest targeting TUG1 as a potent therapeutic approach for cancer treatment.

Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts.

Retrograde menstruation is a widely accepted cause of endometriosis. However, not all women who experience retrograde menstruation develop endometriosis, and the mechanisms underlying these observations are not yet understood. Here, we demonstrated a pathogenic role of Fusobacterium in the formation of ovarian endometriosis. In a cohort of women, 64% of patients with endometriosis but <10% of controls were found to have Fusobacterium infiltration in the endometrium. Immunohistochemical and biochemical analyses revealed that activated transforming growth factor-ß (TGF-ß) signaling resulting from Fusobacterium infection of endometrial cells led to the transition from quiescent fibroblasts to transgelin (TAGLN)-positive myofibroblasts, which gained the ability to proliferate, adhere, and migrate in vitro. Fusobacterium inoculation in a syngeneic mouse model of endometriosis resulted in a marked increase in TAGLN-positive myofibroblasts and increased number and weight of endometriotic lesions. Furthermore, antibiotic treatment largely prevented establishment of endometriosis and reduced the number and weight of established endometriotic lesions in the mouse model. Our data support a mechanism for the pathogenesis of endometriosis via Fusobacterium infection and suggest that eradication of this bacterium could be an approach to treat endometriosis.

Mutation detection of urinary cell-free DNA via catch-and-release isolation on nanowires for liquid biopsy.

Cell-free DNA (cfDNA) and extracellular vesicles (EVs) are molecular biomarkers in liquid biopsies that can be applied for cancer detection, which are known to carry information on the necessary conditions for oncogenesis and cancer cell-specific activities after oncogenesis, respectively. Analyses for both cfDNA and EVs from the same body fluid can provide insights into screening and identifying the molecular subtypes of cancer; however, a major bottleneck is the lack of efficient and standardized techniques for the isolation of cfDNA and EVs from clinical specimens. Here, we achieved catch-and-release isolation by hydrogen bond-mediated binding of cfDNA in urine to zinc oxide (ZnO) nanowires, which also capture EVs by surface charge, and subsequently we identified genetic mutations in urinary cfDNA. The binding strength of hydrogen bonds between single-crystal ZnO nanowires and DNA was found to be equal to or larger than that of conventional hydrophobic interactions, suggesting the possibility of isolating trace amounts of cfDNA. Our results demonstrated that nanowire-based cancer screening assay can screen cancer and can identify the molecular subtypes of cancer in urine from brain tumor patients through EV analysis and cfDNA mutation analysis. We anticipate our method to be a starting point for more sophisticated diagnostic models of cancer screening and identification.

Rapid detection of the MYD88 L265P mutation for pre- and intra-operative diagnosis of primary central nervous system lymphoma.

The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is a disease-specific mutation of primary central nervous system lymphoma (PCNSL) among the central nervous system tumors. Accordingly, this mutation is considered a reliable diagnostic molecular marker of PCNSL. As the intra-operative diagnosis of PCNSL is sometimes difficult to achieve using histological examinations alone, intra-operative detection of the MYD88 L265P mutation could be effective for the accurate diagnosis of PCNSL. Herein, we aimed to develop a novel rapid genotyping system (GeneSoC) using real-time polymerase chain reaction (PCR) based on microfluidic thermal cycling technology. This real-time PCR system shortened the analysis time, which enabled the detection of the MYD88 L265P mutation within 15 min. Rapid detection of the MYD88 L265P mutation was performed intra-operatively using GeneSoC in 24 consecutive cases with suspected malignant brain tumors, including 10 cases with suspected PCNSL before surgery. The MYD88 L265P mutation was detected in eight cases in which tumors were pathologically diagnosed as PCNSL after the operation, while wild-type MYD88 was detected in 16 cases. Although two of the 16 cases with wild-type MYD88 were pathologically diagnosed as PCNSL after the operation, MYD88 L265P could be detected in all eight PCNSL cases harboring MYD88 L265P. The MYD88 L265P mutation could also be detected using cell-free DNA derived from the cerebrospinal fluid of two PCNSL cases. Detection of the MYD88 L265P mutation using GeneSoC might not only improve the accuracy of intra-operative diagnosis of PCNSL but also help the future pre-operative diagnosis through liquid biopsy of cerebrospinal fluid.

CA19-9 in Combination with Methylated HOXA1 and SST Is Useful to Diagnose Stage I Pancreatic Cancer.

INTRODUCTION: We previously developed a novel methylation assay, the combined restriction digital PCR (CORD) assay, consisting of treatment of DNA with methylation-sensitive restriction enzymes and droplet digital PCR. METHODS: In this study, we assessed the diagnostic performance of serum methylated Homeobox A1 (mHOXA1) and methylated somatostatin (mSST) using the CORD assay in combination with CA19-9 for pancreatic cancer using serum samples from 82 healthy individuals, 13 patients with benign pancreatic disease, 3 patients with branched-duct intraductal papillary mucinous neoplasm, and 91 patients with pancreatic cancer. RESULTS: For the single marker tests, sensitivity for all stages of pancreatic cancer, stage I cancer, and specificity were, respectively, 71.4%, 50.0%, and 94.9% for CA19-9; 51.6%, 68.8%, and 90.8% for mHOXA1; and 50.1%, 68.8%, and 94.9% for mSST. Those for the combined marker tests were, respectively, 86.8%, 81.3%, and 85.7% for combined mHOXA1 and CA19-9; 86.8%, 87.5%, and 89.8% for combined mSST and CA19-9; and 89.0%, 87.5%, and 85.7% for all three markers combined. CONCLUSION: The combination of mHOXA1 and mSST with CA19-9 appears to be useful to detect pancreatic cancer even at an early stage.

Characteristic DNA methylation profiles of chorionic villi in recurrent miscarriage.

Dysregulation of transcriptional programs that are tightly regulated by DNA methylation during placental and fetal development at different gestational stages, may cause recurrent miscarriage. Here, we examined genome-wide DNA methylation in chorionic villi and decidual tissues from patients suffering RM and from healthy women who had undergone artificial abortion (n = 5 each). We found that 13,426 and 5816 CpG sites were differentially methylated in chorionic villi and decidua, respectively. DNA methylation profiles of chorionic villi, but not decidua, in RM patients was clearly distinct from AA controls. Among the differentially methylated genes, the enhancer region of SPATS2L was significantly more highly methylated in RM patients (n = 19) than AA controls (n = 19; mean methylation level, 52.0%-vs.-28.9%, P < 0.001), resulting in reduced expression of SPATS2L protein in the former. Functionally, depletion of SPATS2L in extravillous trophoblast cells decreased their invasion and migration abilities. Our data indicate that particularly the chorionic villi in RM patients exhibit distinct DNA methylation profiles compared with normal pregnancies and that this changed DNA methylation status may impede the progression of embryo development via the altered expression of genes such as SPATS2L in the villi.

Annealed ZnO/Al2O3 Core-Shell Nanowire as a Platform to Capture RNA in Blood Plasma.

RNA analytical platforms gained extensive attention recently for RNA-based molecular analysis. However, the major challenge for analyzing RNAs is their low concentration in blood plasma samples, hindering the use of RNAs for diagnostics. Platforms that can enrich RNAs are essential to enhance molecular detection. Here, we developed the annealed ZnO/Al2O3 core-shell nanowire device as a platform to capture RNAs. We showed that the annealed ZnO/Al2O3 core-shell nanowire could capture RNAs with high efficiency compared to that of other circulating nucleic acids, including genomic DNA (gDNA) and cell-free DNA (cfDNA). Moreover, the nanowire was considered to be biocompatible with blood plasma samples due to the crystalline structure of the Al2O3 shell which serves as a protective layer to prevent nanowire degradation. Our developed device has the potential to be a platform for RNA-based extraction and detection.

Newly established patient-derived organoid model of intracranial meningioma.

BACKGROUND: Recent comprehensive studies have revealed several molecular alterations that are frequently found in meningiomas. However, effective treatment reagents targeting specific molecular alterations have not yet been identified because of the limited number of representative research models of meningiomas. METHODS: We performed organoid cultures using meningioma cells and meningioma tumor tissues. Using immunohistochemistry and molecular analyses consisting of whole-exome sequencing, RNA-seq, and DNA methylation analyses, we compared the histological findings and molecular profiling of organoid models with those of parental tumors. Further, using these organoid models together with a public database of meningiomas, we explored molecular alterations, which are a potent treatment target for meningioma. RESULTS: We established 18 organoid models comprising of two malignant meningioma cells (HKBMM and IOMM-Lee), 10 benign meningiomas, four malignant meningiomas, and two solitary fibrous tumors (SFTs). The organoids exhibited consistent histological features and molecular profiles with those of the parental tumors. Using a public database, we identified that upregulated forkhead box M1 (FOXM1) was correlated with increased tumor proliferation. Overexpression of FOXM1 in benign meningioma organoids increased organoid proliferation; depletion of FOXM1 in malignant organoids decreased proliferation. Additionally, thiostrepton, a FOXM1 inhibitor combined with radiation therapy, significantly inhibited the proliferation of malignant meningioma organoid models. CONCLUSIONS: An organoid model for meningioma enabled us to elucidate the tumor biology of meningioma along with potent treatment targets for meningioma.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

Cancer-Specific Targeting of Taurine-Upregulated Gene 1 Enhances the Effects of Chemotherapy in Pancreatic Cancer.

Overcoming drug resistance is one of the biggest challenges in cancer chemotherapy. In this study, we examine whether targeting the long noncoding RNA taurine upregulated gene 1 (TUG1) could be an effective therapeutic approach to overcome drug resistance in pancreatic ductal adenocarcinoma (PDAC). TUG1 was expressed at significantly higher levels across 197 PDAC tissues compared with normal pancreatic tissues. Overall survival of patients with PDAC who had undergone 5-FU-based chemotherapy was shorter in high TUG1 group than in low TUG1 group. Mechanistically, TUG1 antagonized miR-376b-3p and upregulated dihydropyrimidine dehydrogenase (DPD). TUG1 depletion induced susceptibility to 5-FU in BxPC-3 and PK-9 pancreatic cell lines. Consistently, the cellular concentration of 5-FU was significantly higher under TUG1-depleted conditions. In PDAC xenograft models, intravenous treatment with a cancer-specific drug delivery system (TUG1-DDS) and 5-FU significantly suppressed PDAC tumor growth compared with 5-FU treatment alone. This novel approach using TUG1-DDS in combination with 5-FU may serve as an effective therapeutic option to attenuate DPD activity and meet appropriate 5-FU dosage requirements in targeted PDAC cells, which can reduce the systemic adverse effects of chemotherapy. SIGNIFICANCE: Targeting TUG1 coupled with a cancer-specific drug delivery system effectively modulates 5-FU catabolism in TUG1-overexpressing PDAC cells, thus contributing to a new combinatorial strategy for cancer treatment. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1654/F1.large.jpg.

Peritoneal Lavage Tumor DNA as a Novel Biomarker for Predicting Peritoneal Recurrence in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: The clinical role of peritoneal lavage cytology (CY) in pancreatic ductal adenocarcinoma (PDAC) remains controversial, partly due to its low sensitivity. This study aimed to develop a new biomarker, defined as peritoneal lavage tumor DNA (ptDNA), using DNAs extracted from peritoneal lavage samples from patients with PDAC. METHODS: Samples were collected intraoperatively from 89 PDAC patients who underwent pancreatectomy between 2012 and 2017. Droplet digital polymerase chain reaction (PCR) was used to measure ptDNA for detection of KRAS mutations. The ptDNA status and clinical characteristics were retrospectively evaluated. RESULTS: Positive ptDNA was found in 41 patients, including all 9 patients positive for CY (CY+) and 32 patients negative for CY (CY-). The mutant allele frequency was significantly higher in the CY+ patients than in the CY- patients. The disease-free survival (DFS) and overall survival (OS) were significantly poorer in the high-ptDNA group than in the low-ptDNA group (median DFS, 11.0 vs. 18.8 months; p = 0.007; median OS, 28.7 vs not reached; p = 0.001). The survival curves of DFS and OS in the CY+ group were almost equal to those in the CY- and high-ptDNA group. In a multivariable analysis, ptDNA was an independent predictive factor for DFS (p = 0.025) and OS (p = 0.047). The estimated cumulative incidence of peritoneal recurrence was 45.5% in the high-ptDNA group. The ptDNA biomarker had a much higher sensitivity for peritoneal recurrence than CY, whereas CY had higher specificity. CONCLUSIONS: As a promising biomarker, ptDNA may predict poor prognosis and peritoneal recurrence in PDAC, resolving the controversy surrounding CY.

Rare genetic variants in the gene encoding histone lysine demethylase 4C (KDM4C) and their contributions to susceptibility to schizophrenia and autism spectrum disorder.

Dysregulation of epigenetic processes involving histone methylation induces neurodevelopmental impairments and has been implicated in schizophrenia (SCZ) and autism spectrum disorder (ASD). Variants in the gene encoding lysine demethylase 4C (KDM4C) have been suggested to confer a risk for such disorders. However, rare genetic variants in KDM4C have not been fully evaluated, and the functional impact of the variants has not been studied using patient-derived cells. In this study, we conducted copy number variant (CNV) analysis in a Japanese sample set (2605 SCZ and 1141 ASD cases, and 2310 controls). We found evidence for significant associations between CNVs in KDM4C and SCZ (p = 0.003) and ASD (p = 0.04). We also observed a significant association between deletions in KDM4C and SCZ (corrected p = 0.04). Next, to explore the contribution of single nucleotide variants in KDM4C, we sequenced the coding exons in a second sample set (370 SCZ and 192 ASD cases) and detected 18 rare missense variants, including p.D160N within the JmjC domain of KDM4C. We, then, performed association analysis for p.D160N in a third sample set (1751 SCZ and 377 ASD cases, and 2276 controls), but did not find a statistical association with these disorders. Immunoblotting analysis using lymphoblastoid cell lines from a case with KDM4C deletion revealed reduced KDM4C protein expression and altered histone methylation patterns. In conclusion, this study strengthens the evidence for associations between KDM4C CNVs and these two disorders and for their potential functional effect on histone methylation patterns.

The Brain Microenvironment Induces DNMT1 Suppression and Indolence of Metastatic Cancer Cells.

Brain metastasis is an ineffective process, and many cancer cells enter into an indolent state following extravasation in the brain. Single cell RNA sequencing of melanoma brain metastases reveals that non-proliferating brain metastatic melanoma cells exhibit a pattern of gene expression associated with inhibition of DNA methyltransferase 1 (DNMT1). The brain microenvironment, specifically the combination of reactive astrocytes and mechanically soft surroundings, suppressed DNMT1 expression in various cancer types and caused cell cycle delay. Somewhat unexpectedly, we find that DNMT1 suppression not only induces cell cycle delay but also activates pro-survival signals in brain metastatic cancer cells, including L1CAM and CRYAB. Our results demonstrate that transcriptional changes triggered by DNMT1 suppression is a key step for cancer cells to survive in the brain microenvironment and that they also restrict cancer cell proliferation. The dual consequences of DNMT1 suppression can explain the persistence of indolent cancer cells in the brain microenvironment.

Autocrine HGF/c-Met signaling pathway confers aggressiveness in lymph node adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive T-cell neoplasm. While ATL cells in peripheral blood (PB-ATL) are sensitive to anti-CC chemokine receptor 4 treatment, non-PB-ATLs, including lymph node ATLs (LN-ATLs), are more aggressive and resistant. We examined characteristic cytokines and growth factors that allow non-PB-ATLs to proliferate and invade compared with PB-ATLs. Protein array analysis revealed hepatocyte growth factor (HGF) and C-C motif chemokine 2 (CCL2) were significantly upregulated in non-PB-ATLs compared with PB-ATLs. The HGF membrane receptor, c-Met, was expressed in PB-ATL and non-PB-ATL cell lines, but CCR2, a CCL2 receptor, was not. Immunohistochemical analysis in clinical ATLs revealed high HGF expression in LNs, pharynx, bone marrow, and tonsils. The HGF/c-Met signaling pathway was active downstream in non-PB-ATLs. Downregulation of HGF/c-Met by siRNA or chemical inhibitors decreased in vitro and in vivo proliferation and invasion by non-PB-ATLs. Treatment with bromodomain and extra-terminal motif inhibitor suppressed HGF expression and decreased levels of histone H3 lysine 27 acetylation (H3K27Ac) and bromodomain-containing protein 4 (BRD4) binding promoter and enhancer regions, suppressing non-PB-ATL cellular growth. Our data indicate H3K27Ac/BRD4 epigenetics regulates the HGF/c-MET pathway in ATLs; targeting this pathway may improve treatment of aggressive non-PB-ATLs.

A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer.

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

H3F3A mutant allele specific imbalance in an aggressive subtype of diffuse midline glioma, H3 K27M-mutant.

Diffuse midline glioma, H3 K27M-mutant is a lethal brain tumor located in the thalamus, brain stem, or spinal cord. H3 K27M encoded by the mutation of a histone H3 gene such as H3F3A plays a pivotal role in the tumorigenesis of this type of glioma. Although several studies have revealed comprehensive genetic and epigenetic profiling, the prognostic factors of these tumors have not been identified to date. In various cancers, oncogenic driver genes have been found to exhibit characteristic copy number alterations termed mutant allele specific imbalance (MASI). Here, we showed that several diffuse midline glioma, H3 K27M-mutant exhibited high variant allele frequency (VAF) of the mutated H3F3A gene using droplet digital polymerase chain reaction (ddPCR) assays. Whole-genome sequencing (WGS) revealed that these cases had various copy number alterations that affected the mutant and/or wild-type alleles of the H3F3A gene. We also found that these MASI cases showed a significantly higher Ki-67 index and poorer survival compared with those in the lower VAF cases (P < 0.05). Our results indicated that the MASI of the H3F3A K27M mutation was associated with the aggressive phenotype of the diffuse midline glioma, H3 K27M-mutant via upregulation of the H3 K27M mutant protein, resulting in downregulation of H3K27me3 modification.