Whole-transcriptome sequencing in advanced gastric or gastroesophageal cancer: A deep dive into its clinical potential.

Advanced gastric and gastroesophageal junction cancers (GC/GEJCs) harbor diverse molecular signatures, highlighting the need for intricate evaluations to identify potential therapeutic targets. Although whole-transcriptome sequencing (WTS) has emerged as a useful tool for understanding these molecular intricacies, its clinical implications have yet to be fully elucidated. This study evaluated the correlation between immunohistochemistry (IHC) and WTS, compared their clinical significance, and identified potential therapeutic targets undetectable through IHC alone. We enrolled 140 patients with advanced GC/GEJC and assessed them using IHC for six pivotal biomarkers: claudin-18 (CLDN18), human epidermal growth factor receptor 2 (HER2), multiple receptor tyrosine kinases (RTKs), and programmed death ligand 1 (PD-L1). Concurrently, WTS was employed as part of the analyses in MONSTAR-SCREEN-2, a multicenter multiomics study. IHC analysis revealed 16.4% HER2, 39.3% CLDN18 (2+/3 + ≥75%), and 15.8% PD-L1 (combined positive score ≥ 10) positivity, among other molecular markers. Significant correlations were observed between IHC and WTS for all six pivotal biomarkers. Among nineteen HER2 IHC-positive patients treated with anti-HER2 therapeutics, ERBB2 status in WTS was significantly associated with progression-free survival (ERBB2-high vs. -low: median 9.0 vs. 5.6 months, log-rank p = 0.046). IHC-based molecular profiling revealed significantly high expression of CLDN18 in RTK-negative patients, with 78.4% positive for either CLDN18 or PD-L1. Additionally, WTS revealed elevated expression of pivotal biomarkers in patients displaying negative targetable biomarkers via IHC. Our findings highlighted the significant correlation between IHC and WTS, reinforcing the clinical utility of WTS. A subset with IHC-negative but WTS-positive status may benefit from specific biomarker-targeted therapies.

Assessing the efficacy of target adaptive sampling long-read sequencing through hereditary cancer patient genomes.

Innovations in sequencing technology have led to the discovery of novel mutations that cause inherited diseases. However, many patients with suspected genetic diseases remain undiagnosed. Long-read sequencing technologies are expected to significantly improve the diagnostic rate by overcoming the limitations of short-read sequencing. In addition, Oxford Nanopore Technologies (ONT) offers adaptive sampling and computationally driven target enrichment technology. This enables more affordable intensive analysis of target gene regions compared to standard non-selective long-read sequencing. In this study, we developed an efficient computational workflow for target adaptive sampling long-read sequencing (TAS-LRS) and evaluated it through application to 33 genomes collected from suspected hereditary cancer patients. Our workflow can identify single nucleotide variants with nearly the same accuracy as the short-read platform and elucidate complex forms of structural variations. We also newly identified several SINE-R/VNTR/Alu (SVA) elements affecting the APC gene in two patients with familial adenomatous polyposis, as well as their sites of origin. In addition, we demonstrated that off-target reads from adaptive sampling, which is typically discarded, can be effectively used to accurately genotype common single-nucleotide polymorphisms (SNPs) across the entire genome, enabling the calculation of a polygenic risk score. Furthermore, we identified allele-specific MLH1 promoter hypermethylation in a Lynch syndrome patient. In summary, our workflow with TAS-LRS can simultaneously capture monogenic risk variants including complex structural variations, polygenic background as well as epigenetic alterations, and will be an efficient platform for genetic disease research and diagnosis.

Sleeping Beauty transposon mutagenesis in mouse intestinal organoids identifies genes involved in tumor progression and metastasis.

To identify genes important for colorectal cancer (CRC) development and metastasis, we established a new metastatic mouse organoid model using Sleeping Beauty (SB) transposon mutagenesis. Intestinal organoids derived from mice carrying actively mobilizing SB transposons, an activating KrasG12D, and an inactivating ApcΔ716 allele, were transplanted to immunodeficient mice. While 66.7% of mice developed primary tumors, 7.6% also developed metastatic tumors. Analysis of SB insertion sites in tumors identified numerous candidate cancer genes (CCGs) identified previously in intestinal SB screens performed in vivo, in addition to new CCGs, such as Slit2 and Atxn1. Metastatic tumors from the same mouse were clonally related to each other and to primary tumors, as evidenced by the transposon insertion site. To provide functional validation, we knocked out Slit2, Atxn1, and Cdkn2a in mouse tumor organoids and transplanted to mice. Tumor development was promoted when these gene were knocked out, demonstrating that these are potent tumor suppressors. Cdkn2a knockout cells also metastasized to the liver in 100% of the mice, demonstrating that Cdkn2a loss confers metastatic ability. Our organoid model thus provides a new approach that can be used to understand the evolutionary forces driving CRC metastasis and a rich resource to uncover CCGs promoting CRC.

Sleeping Beauty transposon mutagenesis identified genes and pathways involved in inflammation-associated colon tumor development.

Chronic inflammation promotes development and progression of colorectal cancer (CRC). To comprehensively understand the molecular mechanisms underlying the development and progression of inflamed CRC, we perform in vivo screening and identify 142 genes that are frequently mutated in inflammation-associated colon tumors. These genes include senescence and TGFß-activin signaling genes. We find that TNFα can induce stemness and activate senescence signaling by enhancing cell plasticity in colonic epithelial cells, which could act as a selective pressure to mutate senescence-related genes in inflammation-associated colonic tumors. Furthermore, we show the efficacy of the Cdk4/6 inhibitor in vivo for inflammation-associated colonic tumors. Finally, we functionally validate that Arhgap5 and Mecom are tumor suppressor genes, providing possible therapeutic targets for CRC. Thus, we demonstrate the importance of the inactivation of senescence pathways in CRC development and progression in an inflammatory microenvironment, which can help progress toward precision medicine.

Economic costs for outpatient treatment of eating disorders in Japan.

BACKGROUND: Few studies have examined the economic costs of outpatient care for eating disorders in Japan. This study aimed to clarify the reimbursement for outpatient treatment of eating disorders and compare the costs between the departments of Psychosomatic Medicine and Psychiatry in Japan. METHOD: A multicenter, prospective, observational study of patients with an eating disorder was conducted in the Psychosomatic Medicine departments of three centers and the Psychiatry departments of another three centers in Japan. We analyzed medical reimbursement for an outpatient revisit, time of clinical interviews, and the treatment outcome measured by the Eating Disorder Examination Questionnaire (EDE-Q) global scores and body mass index (BMI) at 3 months. Multivariate linear regression models were performed to adjust for covariates. RESULTS: This study included 188 patients in the Psychosomatic Medicine departments and 68 in the Psychiatry departments. The average reimbursement cost for an outpatient revisit was 4670 yen. Even after controlling for covariates, the Psychosomatic Medicine departments had lower reimbursement points per minute of interviews than the Psychiatry departments (coefficient = - 23.86; 95% confidence interval = - 32.09 to - 15.63; P < 0.001). In contrast, EDE-Q global scores and BMI at 3 months were not significantly different between these departments. CONCLUSIONS: This study clarifies the economic costs of treating outpatients with eating disorders in Japan. The medical reimbursement points per interview minute were lower in Psychosomatic Medicine departments than in Psychiatry departments, while there were no apparent differences in the treatment outcomes. Addressing this issue is necessary to provide an adequate healthcare system for patients with eating disorders in Japan.

This study examined the cost of outpatient care for eating disorders in Japan, comparing treatment costs between the Psychosomatic Medicine and Psychiatry departments. The actual cost of outpatient care for eating disorders in Japan was clarified. The results indicate that Psychosomatic Medicine departments have lower reimbursement points per interview time compared to the Psychiatry departments, but there were no noticeable differences in treatment outcomes between the two. This highlights the need to address this cost difference to improve the healthcare system for patients with eating disorders in Japan.

Multiancestry genomic and transcriptomic analysis of gastric cancer.

Gastric cancer is among the most common malignancies worldwide, characterized by geographical, epidemiological and histological heterogeneity. Here, we report an extensive, multiancestral landscape of driver events in gastric cancer, involving 1,335 cases. Seventy-seven significantly mutated genes (SMGs) were identified, including ARHGAP5 and TRIM49C. We also identified subtype-specific drivers, including PIGR and SOX9, which were enriched in the diffuse subtype of the disease. SMGs also varied according to Epstein-Barr virus infection status and ancestry. Non-protein-truncating CDH1 mutations, which are characterized by in-frame splicing alterations, targeted localized extracellular domains and uniquely occurred in sporadic diffuse-type cases. In patients with gastric cancer with East Asian ancestry, our data suggested a link between alcohol consumption or metabolism and the development of RHOA mutations. Moreover, mutations with potential roles in immune evasion were identified. Overall, these data provide comprehensive insights into the molecular landscape of gastric cancer across various subtypes and ancestries.

The subjective assessment of work and social adjustment impairments and associated psychopathologies in Japanese adult female patients with anorexia nervosa.

Aim: Patients with anorexia nervosa (AN) sometimes undergo a chronic course, and they hardly maintain social participation. Work and social adjustment impairments are generally significantly associated with the clinical symptoms of eating disorders. Psychopathologies associated with the subjective social difficulties of patients with AN have been unclear. This study examined the association between AN psychopathologies and work and social adjustment impairments in adult female patients with AN. Methods: This study included 36 Japanese adult female patients with AN who completed the Work and Social Adjustment Scale (WSAS) and the Eating Disorder Inventory-2 (EDI-2). Spearman's rank correlation coefficient was used to assess correlations between WSAS and EDI-2 or demographic variables. Results: The mean age was 31.8 years, the mean current body mass index was 13.4 kg/m2, and the median illness duration was 5 years. Patients demonstrated social difficulties, especially in social leisure activities. The total WSAS scores were significantly correlated with EDI-2 "impulse regulation" and "asceticism." WSAS "social leisure" was significantly correlated with EDI-2 "bulimia," "interoceptive awareness," "impulse regulation," and "asceticism." Conclusion: Psychopathologies, such as impulse regulation, asceticism, and interoceptive awareness, may be related factors to social difficulties. Emotion regulation, such as impulse regulation and emotional awareness, could be an important realm of treatment not only for psychopathology but also for social functioning in patients with AN.

Systematic identification of intron retention associated variants from massive publicly available transcriptome sequencing data.

Many disease-associated genomic variants disrupt gene function through abnormal splicing. With the advancement of genomic medicine, identifying disease-associated splicing associated variants has become more important than ever. Most bioinformatics approaches to detect splicing associated variants require both genome and transcriptomic data. However, there are not many datasets where both of them are available. In this study, we develop a methodology to detect genomic variants that cause splicing changes (more specifically, intron retention), using transcriptome sequencing data alone. After evaluating its sensitivity and precision, we apply it to 230,988 transcriptome sequencing data from the publicly available repository and identified 27,049 intron retention associated variants (IRAVs). In addition, by exploring positional relationships with variants registered in existing disease databases, we extract 3,000 putative disease-associated IRAVs, which range from cancer drivers to variants linked with autosomal recessive disorders. The in-silico screening framework demonstrates the possibility of near-automatically acquiring medical knowledge, making the most of massively accumulated publicly available sequencing data. Collections of IRAVs identified in this study are available through IRAVDB ( https://iravdb.io/ ).

A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy.

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Evaluation of repair activity by quantification of ribonucleotides in the genome.

Ribonucleotides incorporated in the genome are a source of endogenous DNA damage and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated nonuniformly in the genome with a 3'-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.

SAA1 is upregulated in gastric cancer-associated fibroblasts possibly by its enhancer activation.

Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared with that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression.

TET repression and increased DNMT activity synergistically induce aberrant DNA methylation.

Chronic inflammation is deeply involved in various human disorders, such as cancer, neurodegenerative disorders, and metabolic disorders. Induction of epigenetic alterations, especially aberrant DNA methylation, is one of the major mechanisms, but how it is induced is still unclear. Here, we found that expression of TET genes, methylation erasers, was downregulated in inflamed mouse and human tissues, and that this was caused by upregulation of TET-targeting miRNAs such as MIR20A, MIR26B, and MIR29C, likely due to activation of NF-κB signaling downstream of IL-1ß and TNF-α. However, TET knockdown induced only mild aberrant methylation. Nitric oxide (NO), produced by NOS2, enhanced enzymatic activity of DNA methyltransferases (DNMTs), methylation writers, and NO exposure induced minimal aberrant methylation. In contrast, a combination of TET knockdown and NO exposure synergistically induced aberrant methylation, involving genomic regions not methylated by either alone. The results showed that a vicious combination of TET repression, due to NF-κB activation, and DNMT activation, due to NO production, is responsible for aberrant methylation induction in human tissues.

Cancer cell niche factors secreted from cancer-associated fibroblast by loss of H3K27me3.

OBJECTIVE: Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells. DESIGN: Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database. RESULTS: CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration. CONCLUSIONS: H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.

Distinct DNA methylation targets by aging and chronic inflammation: a pilot study using gastric mucosa infected with Helicobacter pylori.

BACKGROUND: Aberrant DNA methylation is induced by aging and chronic inflammation in normal tissues. The induction by inflammation is widely recognized as acceleration of age-related methylation. However, few studies addressed target genomic regions and the responsible factors in a genome-wide manner. Here, we analyzed methylation targets by aging and inflammation, taking advantage of the potent methylation induction in human gastric mucosa by Helicobacter pylori infection-triggered inflammation. RESULTS: DNA methylation microarray analysis of 482,421 CpG probes, grouped into 270,249 genomic blocks, revealed that high levels of methylation were induced in 44,461 (16.5%) genomic blocks by inflammation, even after correction of the influence of leukocyte infiltration. A total of 61.8% of the hypermethylation was acceleration of age-related methylation while 21.6% was specific to inflammation. Regions with H3K27me3 were frequently hypermethylated both by aging and inflammation. Basal methylation levels were essential for age-related hypermethylation while even regions with little basal methylation were hypermethylated by inflammation. When limited to promoter CpG islands, being a microRNA gene and high basal methylation levels strongly enhanced hypermethylation while H3K27me3 strongly enhanced inflammation-induced hypermethylation. Inflammation was capable of overriding active transcription. In young gastric mucosae, genes with high expression and frequent mutations in gastric cancers were more frequently methylated than in old ones. CONCLUSIONS: Methylation by inflammation was not simple acceleration of age-related methylation. Targets of aberrant DNA methylation were different between young and old gastric mucosae, and driver genes were preferentially methylated in young gastric mucosa.

Novel prodrugs of decitabine with greater metabolic stability and less toxicity.

BACKGROUND: DNA demethylation therapy is now used in practice for hematological tumors and is being developed for solid tumors. Nevertheless, it is difficult to achieve stable pharmacokinetics with the current DNA-demethylating agents, azacitidine (AZA) and decitabine (DAC), because of their rapid deamination by cytidine deaminase in vivo and spontaneous hydrolytic cleavage. Here, we aimed to develop metabolically stable prodrugs of AZA and DAC as novel DNA-demethylating agents. RESULTS: Thirty-five 5'-O-trialkylsilylated AZAs/DACs were synthesized with potential resistance to deamination. Out of these, 11 compounds exhibited demethylating activity similar to that of DAC and guadecitabine, and a suitable aqueous solubility. Pharmacokinetic analysis in mice showed that OR-2003 displayed the highest serum concentration and the area under the curve in an intraperitoneal experiment, whereas OR-2100 exhibited high stability to cytidine deaminase. Treatment of cells with OR-2003 and OR-2100 depleted DNA methyltransferase 1 completely and induced both gene-specific and genome-wide demethylation. The treatment suppressed the growth of multiple types of cancer cells and induced re-expression of tumor suppressor genes. The anti-tumor effect and DNA demethylation effect of OR-2003 and OR-2100 were comparable to that of DAC with fewer adverse effects in vivo. CONCLUSIONS: We developed two novel prodrugs of DAC that exhibited greater stability, comparable DNA demethylation activity, and less toxicity. These compounds are expected to overcome the difficulty in achieving stable pharmacokinetics in patients, leading to maximum DNA demethylation activity with minimum adverse effects.

Effectiveness of parent training in improving stress-coping capability, anxiety, and depression in mothers raising children with autism spectrum disorder.

PURPOSE: Raising a child with autism spectrum disorder (ASD) can be a stressor, and mothers of ASD children often present with high levels of stress and depression. Interventional steps to enhance parental coping skills and resiliency are more important for parental mental health and the family-centered care of children with ASD than merely reducing parental stress. Although the importance of stress-coping skills is well established, only a few studies have investigated interventional steps to improve parental coping or resiliency. Parent training (PT) is known to improve a mother's mental health. Here, we aimed to assess the effectiveness of PT in improving the stress-coping style of mothers raising children with ASD. PATIENTS AND METHODS: Thirty mothers of children with ASD aged 4-11 years participated in this study. The mothers underwent PT based on the Hizen Parenting Skills Training in Japan, which comprised seven sessions. Each session included education on behavior therapy, individual consultation, and workshops in small groups. Sixteen mothers completed psychological assessment, including the Stress Coping Inventory, the Beck Depression Inventory Second Edition, the State-Trait Anxiety Inventory, and the Child Behavior Checklist conducted before and after 2 months of PT. RESULTS: The outcomes before and after the PT program were compared using the paired t-test and Pearson's correlation. After the PT program, the mothers' stress-coping strategy "positive appraisal" significantly increased (P<0.01) and "escape/avoidance" significantly decreased (P<0.01). The Beck Depression Inventory Second Edition (P<0.05) and the trait anxiety scores (P<0.01) also significantly decreased. The change in the stress-coping strategy "distancing" had a significantly negative correlation with the change in the externalizing Child Behavior Checklist T-scores of children with ASD (Pearson r=-0.518, P<0.05). CONCLUSION: PT may be effective for mothers of children with ASD to improve their stress-coping style and to decrease their depression and trait anxiety.

Novel epigenetic markers for gastric cancer risk stratification in individuals after Helicobacter pylori eradication.

BACKGROUND: The risk stratification of healthy individuals after Helicobacter pylori eradication is an urgent issue. The assessment of aberrant DNA methylation accumulated in gastric tissues with normal appearance, which can reflect overall epigenomic damage, is a promising strategy. We aimed to establish novel epigenetic cancer risk markers for H. pylori-eradicated individuals. METHODS: Gastric mucosa was collected from eight healthy volunteers without H. pylori infection (G1), 75 healthy individuals with gastric atrophy (G2), and 94 gastric cancer patients (G3) after H. pylori eradication. Genome-wide analysis was conducted using Infinium 450 K and differentially methylated probes were screened using large difference and iEVORA-based methods. Bisulfite pyrosequencing was used for validation. RESULTS: Screening, using 8 G1, 12 G2, and 12 G3 samples, isolated 57 candidates unmethylated in G1 and differentially methylated in G3 compared with G2. Validation for nine candidate markers (FLT3, LINC00643, RPRM, JAM2, ELMO1, BHLHE22, RIMS1, GUSBP5, and ZNF3) in 63 G2 and 82 G3 samples showed that all of them had significantly higher methylation levels in G3 than in G2 (P < 0.0001). Their methylation levels were highly correlated, which indicated that they reflect overall epigenomic damage. The candidates had sufficient performance (AUC: 0.70-0. 80) and high odds ratios (5.43-23.41), some of which were superior to a previous marker, miR-124a-3. The methylation levels of our novel markers were not associated with gastric atrophy, gender, or age. CONCLUSIONS: Novel epigenetic markers for gastric cancer risk optimized for H. pylori-eradicated individuals were established.

MACON: a web tool for computing DNA methylation data obtained by the Illumina Infinium Human DNA methylation BeadArray.

AIM: Bioinformatics analysis for Illumina Infinium Human DNA methylation BeadArray is essential, but still remains difficult task for many experimental researchers. We here aimed to develop a browser-accessible bioinformatics tool for analyzing the BeadArray data. MATERIALS & METHODS: The tool was established as an analytical pipeline using R, Perl and Python programming languages. RESULTS: We introduced a method that groups neighboring probes into a genomic block, which facilitated efficient identification of densely methylated/unmethylated regions. The tool, MACON, provided probe filtering, ß-mixture quantile normalization, grouping into genomic blocks, annotation and production of a data subset. CONCLUSION: MACON allows researchers to analyze the BeadArray data using a web browser ( http://epigenome.ncc.go.jp/macon ).

Genetic and epigenetic alterations in normal tissues have differential impacts on cancer risk among tissues.

Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69-89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52-23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.

Targeting of super-enhancers and mutant BRAF can suppress growth of BRAF-mutant colon cancer cells via repression of MAPK signaling pathway.

Bromodomain and extra-terminal (BET) inhibitors suppress super-enhancers and show cytotoxicity against multiple types of tumors. However, early clinical trials with BET inhibitors showed severe hematopoietic toxicities, highlighting the need for sensitive tumors and rational combination strategies to enhance their therapeutic potential. Here, we identified colon cancer-specific super-enhancers that were associated with multiple oncogenic pathways, including the mitogen-activated protein kinase (MAPK) signaling pathway. Among the 14 colon cancer cell lines tested, their sensitivity to JQ1, a BET inhibitor, was not correlated with c-MYC expression. Three of four BRAFV600E-mutant cell lines were sensitive. Addition of JQ1 to vemurafenib, a specific mutant BRAF inhibitor, suppressed cell growth by arresting cell cycle progression and inducing apoptosis in the BRAFV600E-mutant cells. Mechanistically, the feedback activation of MAPK signaling pathway by vemurafenib was repressed by JQ1. Further, the addition of JQ1 to a BRAF inhibitor enhanced the in vivo anti-tumor effect. Thus, this study indicates the therapeutic potential of targeting of super-enhancers and mutant BRAF in patients with BRAFV600E-mutant colorectal cancer.