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PurposeThis study aimed to present the methodology of the text data analysis to establish marketing strategies for fintech companies in a practical way. Specifically, the methodology was presented to convert customers' review data, which consisted of the text data (unstructured data), to the numerical data (structured data) by using a text mining algorithm “Global Vectors for Word Representation,” abbreviated as “GloVe”; additionally, the authors presented the methodology to deploy the numerical data for marketing strategies with eliminate-reduce-raise-create (ERRC) value factor analytics.Design/methodology/approachFirst, the authors defined the background, features and contents of fintech services based on a review of related literature review. Additionally, they examined business strategies, the importance of social media for fintech services and fintech technology trends based on the literature review. Next, they analyzed the similarity between fintech-related keywords, which represent the trends in fintech services, and the text data related to fintech corporations and their services posted on Facebook and Twitter, which are two of the most popular social media globally, during the period 2017–2019. The similarity was then quantified and categorized in terms of the representative global fintech companies and the status of each fintech service sector. Furthermore, the similarity was visualized, and value elements were rebuilt using ERRC strategy analytics.FindingsThis study is meaningful in that it quantifies the degree of similarity between customers' responses, experiences and expectations regarding the rapidly growing global fintech firms' services and trends in fintech services.Originality/valueThis study suggests a practical way to apply in business by providing a method for transforming unstructured text data into structured numerical data it is measurable. It is expected that this study can be used as the basis for exploring sustainable development strategies for the fintech industry.

Introduction: In the Phase 3 IMbrave150 trial (NCT03434379), atezolizumab + bevacizumab demonstrated a clinically meaningful survival benefit over sorafenib in patients with unresectable hepatocellular carcinoma (HCC), including those with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. We used IMbrave150 data to investigate the safety and risk of viral reactivation or flare in infected patients treated with atezolizumab + bevacizumab or sorafenib. Methods: Patients with unresectable HCC not previously treated with systemic therapy were randomized 2:1 to atezolizumab + bevacizumab or sorafenib. In this exploratory analysis, safety was continually evaluated, including for hepatic adverse events. Patients were monitored for HBV and HCV reactivation and flare at screening, the beginning of Cycles 5 and 9, and at treatment discontinuation. Results: Of 501 enrolled patients, 485 were included in the safety population; 329 (68%) received atezolizumab + bevacizumab, and 156 (32%) received sorafenib. Overall, 150 (31%) and 58 (12%) patients had HBV and HCV infections, respectively. The safety profiles of atezolizumab + bevacizumab and sorafenib were consistent across patients, regardless of viral infection. Overall, hepatic serious adverse events occurred in 11% of patients receiving atezolizumab + bevacizumab and 8% receiving sorafenib. HBV or HCV reactivation occurred in 2% or 16% of atezolizumab + bevacizumab-treated patients, respectively, versus 7% or 14% with sorafenib. There were no instances of hepatitis flare with atezolizumab + bevacizumab. Conclusions: Atezolizumab + bevacizumab had a similar hepatic safety profile in patients with and without HBV or HCV infection. Viral reactivation rates were similar between arms. Overall, these data support the use of atezolizumab + bevacizumab in patients with HCC and HBV or HCV infection without any special precaution.

Traditional tissue-based assessments of genomic alterations in castration-resistant prostate cancer (CRPC) can be challenging. To evaluate the real-world clinical utility of liquid biopsies for the evaluation of genomic alterations in CRPC, we preemptively collected available plasma samples and archival tissue samples from patients that were being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) and tumor tissue DNA were analyzed using the AlphaLiquid®100-HRR panel. Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) patients, regardless of the presence of overt metastasis or concomitant treatment given at the time of plasma sample collection. Twenty-three patients were found to have known deleterious somatic or germline mutations in HRR genes from their cfDNA. Archival tissue samples from 33 (37.9%) patients were available for comparative analysis. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations in tissues was 71.8%, but important somatic/germline mutations in HRR genes were found to have a higher concordance (100%). Liquid biopsies can be a reasonable substitute for tissue biopsies in CRPC patients when evaluating genomic alterations.

BACKGROUND: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. METHODS: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. RESULTS: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10–0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. CONCLUSION: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

PurposeThe value of the genomic profiling by targeted gene-sequencing on radiation therapy response prediction was evaluated through integrated analysis including clinical information. Radiation response prediction model was constructed based on the analyzed findings.Materials and MethodsPatients who had the tumor sequenced using institutional cancer panel after informed consent and received radiotherapy for the measurable disease served as the target cohort. Patients with irradiated tumor locally controlled for more than 6 months after radiotherapy were defined as the durable local control (DLC) group, otherwise, non-durable local control (NDLC) group. Significant genomic factors and domain knowledge were used to develop the Bayesian Network model to predict radiotherapy response.ResultsAltogether, 88 patients were collected for analysis. Of those, 41 (43.6%) and 47 (54.4%) patients were classified as the NDLC and DLC group, respectively. Somatic mutations of NOTCH2 and BCL were enriched in the NDLC group, whereas, mutations of CHEK2, MSH2, and NOTCH1 were more frequently found in the DLC group. Altered DNA repair pathway was associated with better local failure–free survival (hazard ratio, 0.40; 95% confidence interval, 0.19 to 0.86; p=0.014). Smoking somatic signature was found more frequently in the DLC group. Area under the receiver operating characteristic curve of the Bayesian network model predicting probability of 6-month local control was 0.83.ConclusionDurable radiation response was associated with alterations of DNA repair pathway and smoking somatic signature. Bayesian network model could provide helpful insights for high precision radiotherapy. However, these findings should be verified in prospective cohort for further individualization.

Lymphatic invasion, vascular invasion, and perineural invasion are prognostic factors for colon cancer. However, the prognostic significance of those factors according to the location of permeation (intramural and extramural invasion) in stage II colon cancer is still unclear.This study aims to clarify whether the location of lymphatic invasion, vascular invasion, and perineural invasion could affect survival of stage II colon cancer patients.This was a retrospective cohort study.This study took place at a university teaching hospital.A total of 1130 patients with stage II colon cancers who underwent radical surgery at the Seoul National University Hospital between July 2003 and December 2015 were included.Patients were classified according to the location of lymphatic invasion, vascular invasion, and perineural invasion. Survival outcomes were compared among those without invasion, and those with intramural and extramural invasion. Primary end point is overall survival and secondary end point is disease free survival.Disease-free survival and overall survival of patients with extramural invasion were worse than those of patients without invasion and those with intramural invasion. Multivariate analysis for survival outcomes confirmed that extramural invasion were significant independent prognostic factors. However, both disease free survival and overall survival were not significantly different between patients without invasion and those with intramural invasion.This study was limited by its retrospective design.Extramural invasion was associated with worse prognosis in stage II colon cancer, but intramural invasion was not. Therefore, pathologic reports about the location of lymphatic invasion, vascular invasion, and perineural invasion might be helpful for predicting prognosis and for determining the need of adjuvant chemotherapy in stage II colon cancers. See Video Abstract at http://links.lww.com/DCR/B939.

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

PDF file - 114K, Chemical structures of investigated molecules in this article

PDF file - 135K, Treatment of saracatinib in LS174T and SKBR3 cells

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PDF file - 173K, Induction of Bim by saracatinib treatment

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

Supplementary Table 1. The IC50 values of bosutinib, gemcitabine, and cisplatin in biliary tract cancer cell lines.

PDF file - 96K, Downregulation of thymidylate synthase (TS) by saracatinib

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PDF file - 3250K, Figure S1 Olaparib has variable levels of anti-tumor activity in human cancer cell lines. Figure S2 Olaparib sensitive cell lines have different patterns of gene expression compared to insensitive cell lines. Figure S3 PTEN expression does not affect olaparib sensitivity. Figure S4 RAD51C expression affects olaparib sensitivity in human cancer cell lines. Figure S5 RAD51C over-expression decreases sensitivity to olaparib in the BT-549 cell line. Figure S6 Olaparib induces G2/M cell cycle arrest and apoptosis in sensitive cell lines. Figure S7 RAD51C depletion increases the accumulation of DNA damage. Figure S8 RAD51C expression decreases in tumor tissues. Figure S9 RAD51C expression is down-regulated in gastric tumor tissues via DNA methylation. Table S1. Sequences of the primers for PCR and real-time PCR analyses. Table S2. BRCA1 and BRCA2 mutations in the gastric cancer cell lines.

PDF file - 215K, mRNA and protein expression level of integrin �8 in gastric cancer cell lines

Supplementary Tables 1-2. Table S1. HER2/CEP17 ratio and HER2 gene copy number by HER2 IHC; Table S2: Univariateand multivariate Cox analysis of overall survival

Prognostic role of tumor mutation burden (TMB) stratified by microsatellite instability (MSI) status.

EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526–36]

Supplementary Data from Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

Purpose:Recent sequencing studies revealed that a subset of colorectal cancer harbors a significantly higher number of somatic mutations. These hypermutated tumors show distinct clinicopathologic features. However, the prognostic impact of the hypermutated tumors is not clearly established.Experimental Design:We analyzed tumor mutation burden (TMB) from targeted next-generation sequencing data of 40 major genes in 516 patients with colorectal cancer. TMB was defined as total number of nonsynonymous mutations per tumor. Cutoff value for TMB-high was chosen by which best discriminated relapse-free survival (RFS) using the Contal and O'Quigley method.Results:In the TCGA data, mutation count of the selected 40 genes reflected the whole exome mutation burden (Pearson correlation = 0.873, P < 0.001). In our patient cohort, 8 or more mutations in the 40 genes was defined as TMB-high, which best discriminated RFS. A total of 55 patients (10.7%) had TMB-high. TMB-high tumors were more frequently found in a proximal location (63.6%) and had a higher proportion of N0 disease (30.9%) and MSI-H (49.1%) compared with TMB-low. Most importantly, TMB-high was associated with better 5-year RFS compared with TMB-low (96.3% vs. 79.8%, P = 0.005). Although there was significant overlap between TMB-high and MSI-H, MSI-H status was not significantly associated with RFS. Multivariate analysis revealed TMB-high as an independent positive prognostic factor for RFS [adjusted HR, 0.16 (95% confidence interval, 0.04–0.66), P = 0.011].Conclusions:TMB-high is associated with better prognosis in patients with colorectal cancer treated with curative surgery followed by adjuvant fluoropyrimidine and oxaliplatin chemotherapy.

Supplementary Figure 2. Representative images of HER2 IHC and HER2 FISH

Purpose:Despite the well-known prognostic value of the tumor–immune microenvironment (TIME) in colorectal cancers, objective and readily applicable methods for quantifying tumor-infiltrating lymphocytes (TIL) and the tumor–stroma ratio (TSR) are not yet available.Experimental Design:We established an open-source software-based analytic pipeline for quantifying TILs and the TSR from whole-slide images obtained after CD3 and CD8 IHC staining. Using a random forest classifier, the method separately quantified intraepithelial TILs (iTIL) and stromal TILs (sTIL). We applied this method to discovery and validation cohorts of 578 and 283 stage III or high-risk stage II colorectal cancers patients, respectively, who were subjected to curative surgical resection and oxlaliplatin-based adjuvant chemotherapy.Results:Automatic quantification of iTILs and sTILs showed a moderate concordance with that obtained after visual inspection by a pathologist. The K-means–based consensus clustering of 197 TIME parameters that showed robustness against interobserver variations caused colorectal cancers to be grouped into five distinctive subgroups, reminiscent of those for consensus molecular subtypes (CMS1-4 and mixed/intermediate group). In accordance with the original CMS report, the CMS4-like subgroup (cluster 4) was significantly associated with a worse 5-year relapse-free survival and proved to be an independent prognostic factor. The clinicopathologic and prognostic features of the TIME subgroups have been validated in an independent validation cohort.Conclusions:Machine-learning–based image analysis can be useful for extracting quantitative information about the TIME, using whole-slide histopathologic images. This information can classify colorectal cancers into clinicopathologically relevant subgroups without performing a molecular analysis of the tumors.

Supplementary Figure 1. CONSORT diagram

Supplementary data

Supplementary Figure 3. Receiver operating characteristic curves of HER2 gene copy number according to HER2 IHC status.

Supplementary Figure 4. Overall survival and objective response rate according to HER2 gene copy number.

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Tumor mutation burden in MSI-H patients

Purpose: Chemotherapy plus trastuzumab is standard of care for HER2-positive advanced gastric cancer (AGC). However, not all patients with HER2-positive AGC seem to benefit from trastuzumab. We evaluated the association between treatment outcomes with trastuzumab and HER2 status in patients with HER2-positive AGC.Experimental Design: We enrolled 126 patients with HER2-positive AGC treated with trastuzumab plus chemotherapy in a training cohort. HER2 IHC (N = 126), HER2/CEP17 ratio (N = 66), and HER2 gene copy number (GCN; N = 59) were analyzed, and the optimal values for discriminating overall survival (OS) were determined using receiver operating characteristic (ROC) curve analysis. We validated the findings from the training cohort using an independent validation cohort (N = 72).Results: Patients with HER2 IHC 3+ showed significantly longer OS (29 vs. 15.3 months; P = 0.025) than patients with IHC ≤ 2+. An HER2/CEP17 ratio of 4.48 was the optimal cutoff for predicting longer OS (26.9 vs. 14.7 months; P = 0.027). In subgroup analysis, treatment outcomes of patients with IHC 3+ were not influenced by the level of HER2 gene amplification. However, in patients with IHC ≤ 2+, an HER2/CEP17 ratio more than 3.69 and HER2 GCN more than 7.75 were positive predictive factors for better outcomes with trastuzumab-based chemotherapy. These findings were confirmed in both the validation cohort and the combined cohort.Conclusions: HER2 IHC status, HER2/CEP17 ratio, and HER2 GCN were correlated with clinical outcomes of trastuzumab-based treatment in HER2-positive AGC. Clinical outcomes of patients with IHC ≤ 2+ were strongly dependent on the HER2/CEP17 ratio and HER2 GCN. Clin Cancer Res; 21(11); 2520–9. ©2015 AACR.

Supplementary Figure 5. Survival analysis according to HER2 gene copy number in validation cohort

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2′-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes. (Cancer Res 2006; 66(15): 7490-501)

Cystatin M is a secreted inhibitor of lysosomal cysteine proteases. Several lines of evidence indicate that cystatin M is a tumor suppressor important in breast malignancy; however, the mechanism(s) that leads to inactivation of cystatin M during cancer progression is unknown. Inspection of the human cystatin M locus uncovered a large and dense CpG island within the 5′ region of this gene (termed CST6). Analysis of cultured human breast tumor lines indicated that cystatin M expression is either undetectable or in low abundance in several lines; however, enhanced gene expression was measured in cells cultured on the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC). Increased cystatin M expression does not correlate with a cytotoxic response to 5-aza-dC; rather, various molecular approaches indicated that the CST6 gene was aberrantly methylated in these tumor lines as well as in primary breast tumors. Moreover, 60% (12 of 20) of primary tumors analyzed displayed CST6 hypermethylation, indicating that this aberrant characteristic is common in breast malignancies. Finally, preinvasive and invasive breast tumor cells were microdissected from nine archival breast cancer specimens. Of the five tumors displaying CST6 gene methylation, four tumors displayed methylation in both ductal carcinoma in situ and invasive breast carcinoma lesions and reduced expression of cystatin M in these tumors was confirmed by immunohistochemistry. In summary, this study establishes that the tumor suppressor cystatin M is a novel target for epigenetic silencing during mammary tumorigenesis and that this aberrant event can occur before development of invasive breast cancer. (Cancer Res 2006; 66(16): 7899-909)

Supplementary Figures 1-2 from Enzastaurin, a Protein Kinase Cβ Inhibitor, Suppresses Signaling through the Ribosomal S6 Kinase and Bad Pathways and Induces Apoptosis in Human Gastric Cancer Cells

Supplementary Figure 3 from Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression

Supplementary Figure 1 from Epigenetic Silencing of the Tumor Suppressor Cystatin M Occurs during Breast Cancer Progression

Supplementary Figures 1-3, Table 1 from Epigenomic Profiling Reveals Novel and Frequent Targets of Aberrant DNA Methylation-Mediated Silencing in Malignant Glioma

Activation of protein kinase C (PKC) has been implicated in gastric carcinogenesis. Enzastaurin is an oral ATP-competitive inhibitor of the PKCβ isozyme. Although enzastaurin was initially advanced to the clinic based on its antiangiogenic activity, it is also known to have a direct effect on a variety of human cancer cells, inducing apoptosis by inhibiting the Akt signal pathway. However, data on enzastaurin for gastric cancer are limited. Therefore, this study was performed to assess the antitumor activity of enzastaurin on gastric cancer cells and to investigate the underlying antitumor mechanisms. Enzastaurin suppressed the proliferation of cultured gastric cancer cells and the growth of gastric carcinoma xenografts. Enzastaurin did not have an effect on gastric cancer cell cycle progression; however, it had a direct apoptosis-inducing effect through the caspase-mediated mitochondrial pathway. Glycogen synthase kinase 3β phosphorylation, a reliable pharmacodynamic marker of enzastaurin activity, and Akt phosphorylation were both decreased after treatment with enzastaurin. Although the p90 ribosomal S6 kinase (Rsk) was also dephosphorylated, Erk phosphorylation was not affected in the enzastaurin-treated gastric cancer cells. Enzastaurin activated Bad, one of the Bcl-2 proapoptotic proteins, through dephosphorylation at Ser112, and depletion of Bad activity resulted in resistance to enzastaurin-induced apoptosis and cytotoxicity in gastric cancer cells. These data suggest that enzastaurin induces apoptosis through Rsk-mediated and Bad-mediated pathways, besides inhibiting the Akt signal cascade. Furthermore, enzastaurin had synergistic or additive effects when combined with 5-fluorouracil, cisplatin, paclitaxel, or irinotecan. These results warrant further clinical investigation of enzastaurin for gastric cancer treatment. [Cancer Res 2008;68(6):1916–26]

Background A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods. Results EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples. Conclusions EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.

Background:Assessment of genomic alterations in castration-resistant prostate cancer (CRPC) patients has become an important process for treatment decisions, but traditional tissue-based evaluation can be challenging. Methods:To evaluate the clinical utility of liquid biopsy for the evaluation of genomic alterations in CRPC, we have prospectively collected plasma samples from patients being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) extracted from the plasma samples were analyzed using AlphaLiquid®100-HRR panel. Available archival tumor tissue samples were also collected and analyzed using the same panel. Results:Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) of all 87 CRPC patients. Somatic mutations were detectable regardless of the presence of overt metastasis, or concomitant treatment given at the time of plasma sample collection. 23 patients were found to have known deleterious mutations in HRR genes from their cfDNA. 11 patients were additionally found to have possibly pathogenic mutations calculated by pathogenicity prediction algorithm. Archival tissue samples were available in 33 (37.9%) of the patients, 31 from prostate and 2 from metastatic bone lesions, collected a median of 2 years before the blood sample collection. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations discovered from tissues was 71.8%, but important somatic/germline mutations in HRR genes were found in higher concordance (100%). Conclusions:Liquid biopsy can be a reasonable substitute for tissue biopsy in CRPC patients for evaluating genomic alterations.