The Molecular Landscape of Gastric Cancers for Novel Targeted Therapies from Real-World Genomic Profiling.

BACKGROUND: Panel-based comprehensive genomic profiling is used in clinical practice worldwide; however, large real-world datasets of patients with advanced gastric cancer are not well known. OBJECTIVE: We investigated what differences exist in clinically relevant alterations for molecularly defined or age-stratified subgroups. METHODS: This was a collaborative biomarker study of a real-world dataset from comprehensive genomic profiling testing (Foundation Medicine, Inc.). Hybrid capture was carried out on at least 324 cancer-related genes and select introns from 31 genes frequently rearranged in cancer. Overall, 4634 patients were available for analyses and were stratified by age (≥ 40/< 40 years), microsatellite instability status, tumor mutational burden status (high 10 ≥ /low < 10 Muts/Mb), Epstein-Barr virus status, and select gene alterations. We analyzed the frequency of alterations with a chi-square test with Yate's correction. RESULTS: Genes with frequent alterations included TP53 (60.1%), ARID1A (19.6%), CDKN2A (18.2%), KRAS (16.6%), and CDH1 (15.8%). Differences in comprehensive genomic profiling were observed according to molecularly defined or age-stratified subgroups. Druggable genomic alterations were detected in 31.4% of patients; ATM (4.4%), BRAF V600E (0.4%), BRCA1 (1.5%), BRCA2 (2.9%), ERBB2 amplification (9.2%), IDH1 (0.2%), KRAS G12C (0.7%), microsatellite instability-high (4.8%), NTRK1/2/3 fusion (0.13%), PIK3CA mutation (11.4%), and tumor mutational burden-high (9.4%). CDH1 alterations and MET amplification were significantly more frequent in patients aged < 40 years (27.7 and 6.2%) than in those aged ≥ 40 years (14.7 and 4.0%). CONCLUSIONS: Real-world datasets from clinical panel testing revealed the genomic landscape in gastric cancer by subgroup. These findings provide insights for the current therapeutic strategies and future development of treatments in gastric cancer.

Impact of mixed-infection rate of clarithromycin-susceptible and clarithromycin-resistant Helicobacter pylori strains on the success rate of clarithromycin-based eradication treatment.

BACKGROUND: Clarithromycin (CAM) resistance is a major contributor to the failure to eradicate Helicobacter pylori (H. pylori). The mixed-infection ratio of CAM-susceptible and CAM-resistant H. pylori strains differs among individuals. Pyrosequencing analysis can be used to quantify gene mutations at position each 2142 and 2143 of the H. pylori 23S rRNA gene in intragastric fluid samples. Herein, we aimed to clarify the impact of the rate of mixed infection with CAM-susceptible and CAM-resistant H. pylori strains on the success rate of CAM-containing eradication therapy. MATERIALS AND METHODS: Sixty-four H. pylori-positive participants who received CAM-based eradication therapy, also comprising vonoprazan and amoxicillin, were enrolled in this prospective cohort study. Biopsy and intragastric fluid samples were collected during esophagogastroduodenoscopy. H. pylori culture and CAM-susceptibility tests were performed on the biopsy samples, and real-time PCR and pyrosequencing analyses were performed on the intragastric fluid samples. The mutation rates and eradication success rates were compared. RESULTS: The overall CAM-based eradication success rate was 84% (54/64): 62% (13/21) for CAM-resistant strains, and 95% (39/41) for CAM-sensitive strains. When the mutation rate of the 23S rRNA gene was 20% or lower for both positions (2142 and 2143), the eradication success rate was 90% or more. However, when the mutation rate was 20% or higher, the eradication success rate was lower (60%). CONCLUSIONS: The mutation rate of the CAM-resistance gene was related to the success of eradication therapy, as determined via pyrosequencing analysis.

Time-series transcriptome analysis of peripheral blood mononuclear cells obtained from individuals who received the SARS-CoV-2 mRNA vaccine.

Messenger ribonucleic acid (mRNA) vaccination against coronavirus disease 2019 (COVID-19) is an effective prevention strategy, despite a limited understanding of the molecular mechanisms underlying the host immune system and individual heterogeneity of the variable effects of mRNA vaccination. We assessed the time-series changes in the comprehensive gene expression profiles of 200 vaccinated healthcare workers by performing bulk transcriptome and bioinformatics analyses, including dimensionality reduction utilizing the uniform manifold approximation and projection (UMAP) technique. For these analyses, blood samples, including peripheral blood mononuclear cells (PBMCs), were collected from 214 vaccine recipients before vaccination (T1) and on Days 22 (T2, after second dose), 90, 180 (T3, before a booster dose), and 360 (T4, after a booster dose) after receiving the first dose of BNT162b2 vaccine (UMIN000043851). UMAP successfully visualized the main cluster of gene expression at each time point in PBMC samples (T1-T4). Through differentially expressed gene (DEG) analysis, we identified genes that showed fluctuating expression levels and gradual increases in expression levels from T1 to T4, as well as genes with increased expression levels at T4 alone. We also succeeded in dividing these cases into five types based on the changes in gene expression levels. High-throughput and temporal bulk RNA-based transcriptome analysis is a useful approach for inclusive, diverse, and cost-effective large-scale clinical studies.

Clinical evaluation of a novel molecular diagnosis kit for detecting Helicobacter pylori and clarithromycin-resistant using intragastric fluid.

BACKGROUND: Although there are many Helicobacter pylori (H. pylori) diagnostic methods, the culture and antibiotic susceptibility test is an important method for selecting the most effective H. pylori eradication regimen. However, this diagnostic method is complicated and takes several days; therefore, the development of a rapid and simple diagnostic method is required. Eradication failure due to clarithromycin (CAM) resistance should also be considered. In this study, we report the clinical evaluation of point-of-care testing (POCT) kit using intragastric fluid, a novel kit for detecting H. pylori and CAM resistance. MATERIALS AND METHODS: The study participants were 143 patients suspected of H. pylori infection and had an endoscopic examination. The novel diagnostic kit diagnosed H. pylori infection and CAM resistance-associated mutation using intragastric fluid. To diagnose H. pylori infection, the relationship between the diagnostic kit and conventional diagnostic methods (urea breath test, stool antigen test, culture test, and real-time polymerase chain reaction [PCR]) was evaluated. For CAM resistance-associated mutation detection, the concordance between the diagnostic kit and antibiotic susceptibility test was evaluated. RESULTS: The diagnosis of H. pylori infection with the novel molecular diagnostic kit using intragastric fluid showed significant relationship with conventional diagnostic methods. Especially when the culture was control, the sensitivity was 100% (67/67), the specificity was 95.9% (71/74), and the overall concordance was 97.9% (138/141). The detection of CAM resistance-associated mutations had a concordance rate of 97.0% (65/67) when compared with the antibiotic susceptibility test. CONCLUSIONS: The H. pylori molecular POCT kit uses intragastric fluid as a sample and can diagnose H. pylori infection and detect CAM resistance-associated mutations within an hour. This novel kit is expected to prove useful in selecting the most effective eradication regimen for H. pylori.

DNA methylation at hepatitis B virus integrants and flanking host mitochondrially encoded cytochrome C oxidase III.

It is widely accepted that hepatitis B virus (HBV) integrants in the human genome are one of the key factors in liver carcinogenesis. Although it is difficult to observe pre/post-HBV infection genomic-level changes in the same clinical sample pairs, they can be observed using artificially infected HBV cell lines such as HepG2.2.15. A detailed HBV integration analysis comparing HepG2.2.15 with HepG2 cells, especially their mitochondrial (mt) DNA, was conducted using next-generation sequencing (NGS)-based integration analysis. Following target DNA enrichment for elements of the HBV genome, NGS was used to identify HBV integration sites in the mtDNA and DNA methylation was analyzed using semi-quantitative pyrosequencing at the boundaries of the integrated region. The results revealed the HBV integration site in the mtDNA of HepG2.215, most notably the insertion of the HBV preCore, X gene fragment in exon 1 of mitochondrially encoded cytochrome C oxidase III (MT-CO3; ChrM 9652), along with a 'CACCA' microhomology sequence. Both boundaries of the integrated region were concordant and highly methylated (HBV side, 92.3%; MT-CO3 side, 95.5%) relative to those observed in nonintegrated HepG2 (4.3%), HepG2.2.15 (3.0%) and PLC/PRF/5 (4.0%) cells. In conclusion, HBV integration sites were successfully identified in the MT-CO3 gene along with a 'CACCA' microhomology sequence using NGS-based analysis and mitochondrial heteroplasmy was identified. The present study also revealed that the HBV/MT-CO3-integrated boundary DNA was hypermethylated at both the HBV and MT-CO3 sides.

Long-term changes in aberrant DNA methylation and gastritis after Helicobacter pylori eradication focused on metachronous gastric cancer.

BACKGROUND: A persistently high methylation level in gastric mucosa after Helicobacter pylori (H. pylori) eradication is presumed to be a risk for metachronous gastric cancer (MGC); however, long-term changes in aberrant DNA methylation and histological gastritis have been unclear. Our aim was to examine changes in DNA methylation and histological gastritis according to the occurrence of MGC. METHODS: Subjects were classified into three groups: 25 patients in whom MGCs occurred after the initial endoscopic resection (ER) for early gastric cancer and H. pylori eradication (MGC group), 17 patients in whom MGC did not occur for more than 5 years after the initial ER and H. pylori eradication (non-MGC group) and 29 patients without a history of gastric cancer who succeeded in eradication more than 5 years ago (HP group). Aberrance of DNA methylation in three genes (miR-124a-3, EMX1, NKX6-1) and histological score of atrophy and intestinal metaplasia (IM) were evaluated using biopsy samples before and more than a mean of 5 years after H. pylori eradication. Also, the mean Z-score was calculated using Z-score values of the three genes. RESULTS: The methylation level of miR-124a-3 in the HP group and non-MGC group and that of EMX1 in the HP group significantly decreased in the long term after eradication. In the MGC group, H. pylori eradication did not improve aberrant methylation, and the mean Z-score significantly increased. There were significant positive correlations between methylation levels in miR-124a-3 and EMX1 and histological findings after eradication. CONCLUSIONS: A persistently high methylation level after H. pylori eradication reflected precancerous mucosal conditions and led to long-term MGC.

Heterogeneity assessment of vaccine-induced effects using point-of-care surrogate neutralization test for severe acute respiratory syndrome coronavirus 2.

INTRODUCTION: Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic even after vaccination. We aimed to identify immunological heterogeneity over time in vaccinated healthcare workers using neutralization antibodies and neutralizing activity tests. METHODS: Serum samples were collected from 214 healthcare workers before vaccination (pre) and on days 22, 90, and 180 after receiving the first dose of BNT162b2 vaccine (day 0). Neutralization antibody (NAb, SARS-CoV-2 S-RBD IgM/IgG) titers and two kinds of surrogate virus neutralization tests (sVNTs) were analyzed (UMIN000043851). RESULTS: The NAb (SARS-CoV-2 S-RBD IgG) titer peaked on day 90 after vaccination (30,808.0 µg/ml ± 35,211; p < 0.0001) and declined on day 180 (11,678.0 µg/ml ± 33,770.0; p < 0.0001). The neutralizing activity also peaked on day 90 and declined with larger individual differences than those of IgG titer on day 180 (88.9% ± 15.0%, 64.8% ± 23.7%, p < 0.0001). We also found that the results of POCT-sVNT (immunochromatography) were highly correlated with those of conventional sVNT (ELISA). CONCLUSIONS: Neutralizing activity is the gold standard for vaccine efficacy evaluation. Our results using conventional sVNT showed large individual differences in neutralizing activity reduction on day 180 (64.8% ± 23.7%), suggesting an association with the difference in vaccine efficacy. POCT-sVNT is rapid and user-friendly; it might be used for triage in homes, isolation facilities, and event venues without restrictions on the medical testing environment.

The Molecular Landscape of Pancreatobiliary Cancers for Novel Targeted Therapies From Real-World Genomic Profiling.

BACKGROUND: Chemotherapies have limited efficacy in pancreatic cancer (PC) and biliary tract cancer (BTC), underscoring the need for new regimens. Recently, tumor-agnostic approaches have been developed for some targeted therapies in advanced solid tumors; however, the frequency of alterations by clinical and genomic background is unclear in PC and BTC. METHODS: To assess the frequencies of druggable gene alterations and investigate new potential therapeutic targetable genomic alterations, advanced PC and BTC patients were tested with comprehensive genomic profiling at Foundation Medicine during the course of clinical care. RESULTS: A total of 16 913 PC patients and 3031 BTC patients were available for analyses, and frequencies of genomic alterations were stratified by age (≥40 years or <40 years), microsatellite instability status, tumor mutational burden status (high ≥10 or low <10 Muts/Mb), and select genomic alterations. Alterations in BRCA2, BRAF, ERBB2, CDK12, PIK3CA, FGFR2, EGFR, and other potential targets were seen across cohorts, with enrichment observed within particular subsets such as in PC patients lacking a KRAS mutation. In BTC patients, the rate of ERBB2 amplification was statistically significantly higher in the tumor mutational burden-high population (23.3% vs 13.7%). Interestingly, CDK12 rearrangement was observed in BTC patients with ERBB2 amplification tumors. In patients younger than 40 years, FGFR2 rearrangement (4%) was observed in PC: GATA6 amplification (11.1%) and rearrangement of BRAF (2.8%)FGFR2 (5.6%) was observed in BTC patients. CONCLUSIONS: We identified an appreciable frequency of immunotherapy biomarkers and targetable gene alterations in both PC and BTC, with notable frequencies in PC samples lacking KRAS mutations and children or adolescent and young adult populations, that should encourage comprehensive genomic profiling testing.

Visceral disseminated varicella-zoster virus infection in an immunocompetent host.

Varicella-zoster virus (VZV) can cause visceral disseminated VZV infection in immunocompromised patients. We experienced visceral disseminated VZV infection in an immunocompetent host. A 78-year-old woman visited our hospital complaining of abdominal pain that had persisted for 7 days. On day 3 after admission, a skin rash with blisters appeared mainly on her head and trunk that was diagnosed as generalized zoster via rapid skin VZV diagnostic kit. Esophagogastroduodenoscopy showed gastric erosions, and VZV was detected by real-time polymerase chain reaction testing of the gastric mucosal biopsy specimen. Computed tomography imaging also revealed pancreatitis and colitis, and she was diagnosed as having visceral disseminated VZV infection involving multiple organs. She was treated with acyclovir intravenously, after which her skin rash and abdominal pain disappeared. Because visceral disseminated VZV infection can occur in immunocompetent patients, this disease should be considered in patients with unexplained inflammatory lesions of the gastrointestinal tract or pancreas.

Combination of artificial intelligence-based endoscopy and miR148a methylation for gastric indefinite dysplasia diagnosis.

BACKGROUND AND AIM: Gastrointestinal endoscopy and biopsy-based pathological findings are needed to diagnose early gastric cancer. However, the information of biopsy specimen is limited because of the topical procedure; therefore, pathology doctors sometimes diagnose as gastric indefinite for dysplasia (GIN). METHODS: We compared the accuracy of physician-performed endoscopy (trainee, n = 3; specialists, n = 3), artificial intelligence (AI)-based endoscopy, and/or molecular markers (DNA methylation: BARHL2, MINT31, TET1, miR-148a, miR-124a-3, NKX6-1; mutations: TP53; and microsatellite instability) in diagnosing GIN lesions. We enrolled 24,388 patients who underwent endoscopy, and 71 patients were diagnosed with GIN lesions. Thirty-two cases of endoscopic submucosal dissection (ESD) in 71 GIN lesions and 32 endoscopically resected tissues were assessed by endoscopists, AI, and molecular markers to identify benign or malignant lesions. RESULTS: The board-certified endoscopic physicians group showed the highest accuracy in the receiver operative characteristic curve (area under the curve [AUC]: 0.931), followed by a combination of AI and miR148a DNA methylation (AUC: 0.825), and finally trainee endoscopists (AUC: 0.588). CONCLUSION: AI with miR148s DNA methylation-based diagnosis is a potential modality for diagnosing GIN.

Evaluation of a new point-of-care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID-19), has become a global pandemic. Although there are multiple methods for detecting SARS-CoV-2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for detecting SARS-CoV-2. METHODS: Samples from 96 patients with suspected SARS-CoV-2 infection were assessed using the real-time qRT-PCR-based POCT and the conventional real-time qRT-PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). RESULTS: The real-time qRT-PCR-based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR. All patients could be triaged within 1 day using the real-time qRT-PCR-based POCT without invalid reports. CONCLUSIONS: The real-time qRT-PCR-based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.

Cancer-related genetic variants of Helicobacter pylori strains determined using gastric wash-based whole-genome analysis with single-molecule real-time technology.

Helicobacter pylori (H. pylori) are a primary factor in the pathogenesis of gastric cancer (GC); GC ranks third among cancer-related mortality. A clear understanding of the H. pylori genome factors underlying GC is necessary to develop more effective methods to prevent GC. A single-molecule real-time DNA sequencing-based H. pylori genome-wide association study analysis was performed using the H. pylori genome present in five early-stage GC (EGC) and five non-GC clinical DNA samples recovered from gastric washes. A total of 275 genes with 702 nucleotide variants (NVs) were found to be common to three or more patients with EGC but no non-GC patients (single-NV: 654/702, 93.2%; multi-NV: 40/702, 5.7%; deletion: 3/702, 0.4%; insertion: 3/702, 0.7%). Gene ontology analysis of H. pylori revealed that genes involved in the mitochondrial electron transport system, glycolytic processes and the TCA cycle were highly enriched. Cancer-related NVs were most frequently found in a member of the Helicobacter outer membrane protein family, hopL. In particular, one of the NVs in hopL was a novel six-nucleotide insertion (1159095̂1159096, TACTTC); this mutant was detected more frequently in a validation set of 50 additional EGC samples (22/50, 44.0%) than in 18 non-GC samples (3/18, 16.7%, P = .04). These results suggest that the hopL variant is associated with the development of GC and may serve as a genetic biomarker of H. pylori virulence and GC risk. Our assay can serve as a potent tool to expand our understanding of bacteria-associated tumorigenesis.

Mouthwash-Based Highly Sensitive Pyro-Genotyping for Nine Sexually Transmitted Human Papilloma Virus Genotypes.

Human papillomavirus (HPV) is a common sexually transmitted infection worldwide, which spreads via contact with infected genital, anal, and oral/pharyngeal areas (oral sex) owing to diverse manners of sexual intercourse. In this study, we devised an oral HPV detection method using mouthwash waste fluids that causes less psychological resistance to visiting the outpatient otolaryngology departments. We successfully detected only the specific unique reverse sequencing probe (using pyro-genotyping) and identified the nine genotypes of HPV targeted for vaccination by pyrosequencing the mouthwash waste fluids of non-head and neck cancer patient volunteers (n = 52). A relatively large number (11/52) of mouthwash waste fluids tested positive for HPV (21.2%; genotype 6, n = 1; 11, n = 1; 16, n = 1; and 18, n = 8). These results surpassed the sensitivity observed testing the same specimens using the conventional method (1/52, 1.9%). Our method (pyro-genotyping) was developed using nine HPV genotypes targeted for vaccination and the results were highly sensitive compared to those of the conventional method. This less expensive, high-throughput, and simple method can be used for detecting oral HPV infection with fewer socio-psychological barriers.

Dysregulation of miRNA in chronic hepatitis B is associated with hepatocellular carcinoma risk after nucleos(t)ide analogue treatment.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk.

GNAS-mutated carcinoma arising from gastric foveolar metaplasia in the duodenum after 9 years of observation.

This case involved an 80-year-old man. Screening with esophagogastroduodenoscopy (EGD) in 2004 revealed Brunner's gland hyperplasia (BGH), 5 mm in size, in the duodenal bulb. The size of the lesion increased and its shape has changed since then, as detected in subsequent EGDs. The lesion had increased in size to 15 mm with a depression and biopsy specimens revealed an adenocarcinoma. The patient underwent endoscopic mucosal resection. Histopathological assessments indicated an adenocarcinoma arising from gastric foveolar metaplasia (GFM) adjacent to BGH. BGH stained positive for MUC6, and GFM and the adenocarcinoma stained positive for MUC5AC. Mutations of the GNAS gene were not detected in the GFM biopsied in 2007. On the other hand, common GNAS mutations (R201H) were detected in GFM and the adenocarcinoma in the endoscopically resected specimen in 2013. Moreover, mutant allele frequencies were higher in the carcinoma than in GFM. The patient remains disease-free for 4 years after endoscopic treatment. This case report further supports the notion that GFM may be a precursor lesion in the process of GNAS-mutated, gastric-type duodenal carcinogenesis.

Enrichment of Helicobacter pylori mutant strains after eradication therapy analyzed by gastric wash-based quantitative pyrosequencing.

The eradication of Helicobacter pylori reduces the risk of gastric cancer. A clear understanding of the factors underlying mixed infection with multiple clarithromycin-susceptible and clarithromycin-resistant H. pylori strains is necessary to design more effective therapies against H. pylori. We aimed to assess how the abundance and prevalence of H. pylori strains vary after clarithromycin-based eradication therapy. Using gastric wash samples, which represent the entire stomach, we sequentially analyzed the abundance and prevalence of H. pylori DNA by 23S ribosomal RNA pyrosequencing before and 1, 2, and 3 years after eradication therapy. Low levels of H. pylori DNA were still detectable at the first-year follow-up in all samples with negative post-treatment urea breath test results. The abundance of H. pylori DNA decreased significantly until the 2-year follow-up, but it switched to an increase at the 3-year follow-up. Importantly, the ratio of the prevalence of mutant strains to the prevalence of wild-type strains had already increased at the first-year follow-up and continued to increase, suggesting the selection and growth of clarithromycin-resistant strains during the follow-up periods. Being sensitive and representative, our assay will be useful in effectively addressing gastric cancer development by enhancing the long-term success of intervention strategies and consecutive surveillance for H. pylori eradication.

Serum monomeric laminin-γ2 as a novel biomarker for hepatocellular carcinoma.

The diagnosis of hepatocellular carcinoma (HCC) in the early stages is important for successful clinical management. Laminin (Ln)-γ2 expression has been reported in various types of malignant carcinomas. We recently developed a highly sensitive method to measure serum monomeric Ln-γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). Using our CLIA, we evaluated its diagnostic value in sera from patients with chronic liver disease (CLD) and patients with hepatocellular carcinoma (HCC). Serum alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) were also examined in these subjects. Median levels of Ln-γ2 were significantly higher in patients with HCC (173.2 pg/mL; range: 39.5-986 pg/mL) compared with patients with CLD (76.7 pg/mL; range: 38.7-215.9 pg/mL) and with healthy volunteers (41.1 pg/mL; range: 10.9-79.0 pg/mL). The optimal cutoff value for Ln-γ2 that allowed us to distinguish between HCC and nonmalignant CLD was 116.6 pg/mL. Elevated Ln-γ2 levels were observed in 0% of healthy volunteers, 17% of patients with CLD, and 63% of patients with HCC. The positivity rate in patients with HCC for the combination of Ln-γ2 and DCP was 89.5%, which was better than that for either of the two markers alone (63% and 68%, respectively). Among patients with early-stage HCC (T1 or T2), the positivity rates for monomeric Ln-γ2, AFP and DCP were 61%, 39% and 57%, respectively. Serum Ln-γ2 may be a potential biomarker for HCC surveillance. The combination of Ln-γ2 and DCP may be more sensitive for laboratory diagnosis of HCC than the combination of AFP and DCP.

Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

BARHL2 Methylation Using Gastric Wash DNA or Gastric Juice Exosomal DNA is a Useful Marker For Early Detection of Gastric Cancer in an H. pylori-Independent Manner.

OBJECTIVES: The main purpose of this study was to develop a methylation analysis pipeline by using gastric wash-derived DNA and/or gastric juice-derived exosomal DNA (exoDNA), and to evaluate its suitability for the early detection of gastric cancer (GC) in clinical settings. METHODS: We analyzed alterations of BarH-like 2 homeobox protein (BARHL2) in GC cell lines and tissues, as well as in DNA obtained from 128 gastric washes and 30 gastric juice-derived exosomes. GC cell lines were transfected with plasmids encoding BARHL2 and subjected to proliferation, colony formation, and gene expression analyses. RESULTS: High levels of BARHL2 methylation were detected in three of seven GC cell lines; consistent with this, these cell lines expressed low levels of BARHL2. Treatment of these cell lines with 5-aza-2'-deoxycytidine restored BARHL2 expression. Levels of BARHL2 methylation in 18 normal and 14 atrophic gastritis samples were low irrespective of Helicobacter pylori infection. High levels of BARHL2 methylation were observed in gastric wash-derived DNA obtained from early GC patients before endoscopic resection (ER), but methylation was significantly lower after curative ER. Analysis using gastric juice-derived exoDNA samples revealed that BARHL2 methylation yielded an area under the curve of 0.923 with 90% sensitivity and 100% specificity with respect to discriminating GC patients from non-GC controls. BARHL2 nuclear immunoreactivity was found in all normal gastric epithelial cells and in cells from patients with gastritis and adenoma. In contrast, loss of BARHL2 expression was observed in the vast majority of the GC tissues. Finally, transfection of BARHL2 into MKN7 and MKN45 cell lines significantly inhibited their proliferation and ability to form colonies. CONCLUSIONS: Methylation analysis of BARHL2 using gastric wash-derived DNA and/or gastric juice-derived exoDNA could be useful for early detection of GC in clinical settings.

Analysis of Helicobacter pylori genotypes in clinical gastric wash samples.

Helicobacter pylori is a key factor in the development of gastric cancer; indeed, clearance of H. pylori helps prevent gastric cancer. However, the relationship between gastric cancer and the abundance and diversity of H. pylori genotypes in the stomach remains unknown. Here, we present, for the first time, a quantitative analysis of H. pylori genotypes in gastric washes. A method was first developed to assess diversity and abundance by pyrosequencing and analysis of single nucleotide polymorphisms in 23S ribosomal RNA (rRNA), a gene associated with clarithromycin resistance. This method was then validated using arbitrarily mixed plasmids carrying 23S rRNA with single nucleotide polymorphisms. Multiple strains were detected in many of 34 clinical samples, with frequency 24.3 ± 24.2 and 26.3 ± 33.8 % for the A2143G and A2144G strains, respectively. Importantly, results obtained from gastric washes were similar to those obtained from biopsy samples. The method provides opportunities to investigate drug resistance in H. pylori and assess potential biomarkers of gastric cancer risk, and should thus be validated in large-scale clinical trials.