Role of magnifying endoscopy with narrow-band imaging in the diagnosis of noninvasive gastric neoplasia.

BACKGROUND AND AIM: There are no globally approved, distinguishing criteria enabling the classification of gastric adenomas and intramucosal carcinomas for differential diagnosis of noninvasive neoplasia (NIN). METHODS: Next-generation sequencing of 50 cancer-related genes was undertaken on 68 pathologically diagnosed microdissected gastric neoplasms (25 adenomas, 27 intramucosal carcinomas, and 16 submucosal carcinomas) obtained during endoscopic submucosal dissection. Findings from magnifying endoscopy with narrow-band imaging (M-NBI) of 52 NINs (the 25 adenomas and 27 intramucosal carcinomas) were compared with these data. RESULTS: Among all 68 neoplasms, the most frequently mutated genes were APC (76% in adenoma, 11.1% in intramucosal carcinoma, and 0% in submucosal carcinoma; P < 0.001) and TP53 in intramucosal and submucosal carcinomas (8% in adenoma, 48.1% in intramucosal carcinoma, and 75% in submucosal carcinoma; P < 0.001). Dividing the NIN neoplasms into five groups according to their mutational status (A1: APC mutation, A2: APC + α mutation, B: APC + TP53 mutation, C: TP53 mutation, D: no mutation in either APC or TP53) resulted in almost identical diagnoses by pathology and M-NBI for groups A1 (12/13, 92%), C (12/13, 92%), and D (16/17, 94%) but not for groups A2 (3/7, 43%) or B (0/2, 0%). This finding implies that NINs with the APC + α mutation have carcinoma-like endoscopic features despite most being judged as adenomas by pathology. CONCLUSION: A diagnosis of NINs by pathology or M-NBI in the subset of gastric tumors classified by cancer-related mutations is not completely identical, suggesting the possible additional role of M-NBI in diagnosing NINs. Further studies are needed to confirm this.

Identification of early genetic changes in well-differentiated intramucosal gastric carcinoma by target deep sequencing.

BACKGROUND AND AIMS: The recent advancement of next-generation sequencing (NGS) has enabled the identification of cancer-related somatic aberrations in advanced gastric cancer. However, these remain unclear in early gastric cancers, especially in intramucosal gastric cancers. PATIENTS AND METHODS: Thirty-one well-differentiated (tub1) intramucosal gastric cancers obtained by endoscopic submucosal dissection (ESD) from 29 patients were analyzed. After precise collection of tumors and non-tumors from formalin-fixed paraffin-embedded tissues using laser-captured microdissection (LCM), target sequencing analysis of 50 cancer-related genes was performed using an Ion-Proton sequencer. RESULTS: The most frequent hotspot mutation was found in TP53 (17 lesions, 54.8%) followed by the Wnt-signaling pathway genes, APC and CTNNB1 (6 lesions, 19.4%). The mutations in TP53 and the Wnt-signaling genes were mutually exclusive (p = 0.004). There was a tendency that H. pylori infection (p = 0.082) and macroscopic protrusion (p = 0.095) was associated with the presence of these mutations. Only 10 lesions (59%) among 17 lesions with proven TP53 mutations were positive for p53 immunostaining demonstrating the superiority of the mutational analysis. In addition, focal gene amplification of ERBB2 (16%) was found frequently in these early stage lesions. CONCLUSIONS: Using LCM and NGS, mutations in TP53 and the Wnt-signaling pathway were frequently found and were mutually exclusive in the earliest stage of gastric carcinogenesis.

Target sequencing of cancer-related genes in early esophageal squamous neoplasia resected by endoscopic resection in Japanese patients.

BACKGROUND AND AIMS: Next generation sequencing (NGS) has revealed a great deal about cancer-related somatic changes in esophageal squamous cell neoplasia; however, the changes in the very early stages remain unclear. RESULTS: TP53 (87%) and CDKN2A (20%) hot spot mutations were frequently found in early lesions. TP53 was the most common mutation (LGIN/HGIN, 86%; EP, 83%; LPM, 95%; MM/SM1, 80%), followed by CDKN2A (29%, 28%, 16% and 10%, respectively); the frequency of other mutations increased as the disease advanced (p < 0.01). Copy number variation analysis revealed copy number aberrations in multiple genes, including PIK3CA amplification (48%). NGS was superior to p53 immunostaining for detecting TP53 mutations (74% vs. 87%); in combination, the two tests improved detectability to 94%. Clinically, smoking was associated with the occurrence of TP53 mutations in these early lesions (p = 0.049). MATERIALS AND METHODS: Fifty-four early esophageal neoplasia lesions from 47 patients treated by endoscopic resection (low-grade intraepithelial neoplasia [LGIN], n = 1; high-grade intraepithelial neoplasia [HGIN] n = 7; invasion limited to epithelium [EP/M1], n = 18; lamina propria mucosae [LPM/M2], n = 19; muscularis mucosae [MM/M3], n = 8; and upper third of the SM [SM1], n = 2) were isolated from formalin-fixed paraffin-embedded tissue specimens by laser-capture microdissection. Target sequencing of 50 cancer-related genes was performed with an Ion Proton sequencer; their association with the clinical characteristics was investigated. CONCLUSIONS: Mutations of TP53 and CDKN2A, and PIK3CA amplification were common in early esophageal squamous neoplasia, while other mutations accumulated with disease progression. An understanding of these molecular events might provide a molecular basis for early lesion treatment.

Semiannual Imaging Surveillance Is Associated with Better Survival in Patients with Non-B, Non-C Hepatocellular Carcinoma.

Since it remains elusive whether and how the imaging surveillance affects the survival in patients with non-B, non-C hepatocellular carcinoma (NBNC-HCC), we conducted this retrospective study which investigated the association between the semiannual surveillance prior to HCC diagnosis and the survival in patients with the initial diagnosis of HCC induced by hepatitis B virus (HBV) and/or hepatitis C virus (HCV) infections (N = 141) and non-B, non-C etiology (N = 30). It was demonstrated that surveillance was less frequently performed in the NBNC-HCC patients compared to that in HCC patients with HBV and/or HCV infections (B/C-HCC patients), and the survival was unfavorable in NBNC-HCC patients. On the other hand, the survival of NBNC-HCC patients with semiannual surveillance was significantly favorable than those patients without semiannual surveillance, and the survival was similar between B/C-HCCs and NBNC-HCCs with semiannual surveillance. In conclusion, though NBNC-HCC patients compared to B/C-HCC patients had poorer prognosis overall, these NBNC-HCC patients with semiannual surveillance had a better survival almost equivalent to the survival of B/C-HCC patients with semiannual surveillance, demonstrating the clinical utility of the semiannual imaging surveillance program for NBNC-HCCs.

Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.

UNLABELLED: Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. IMPORTANCE: In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Stratifying the risk of lymph node metastasis in undifferentiated-type early gastric cancer.

AIM: To study how lymph node metastasis (LNM) risk is stratified in undifferentiated-type early gastric cancer (undiff-EGC) dependent on combinations of risk factors. METHODS: Five hundred and sixty-seven cases with undiff-EGC undergoing gastrectomy with lymphadenectomy were examined retrospectively. Using clinicopathological factors of patient age, location, size, an endoscopic macroscopic tumor form, ulceration, depth, histology, lymphatic involvement (LI) and venous involvement (VI), LNM risk was examined and stratified by conventional statistical analysis and data-mining analysis. RESULTS: LNM was positive in 44 of 567 cases (7.8%). Univariate analysis revealed > 2 cm, protrusion, submucosal (sm), mixed type, LI and VI as significant prognostic factors and > 2 cm and LI-positive were independent factors by multivariate analysis. In preoperatively evaluable factors excluding LVI, sm and > 2 cm were independent factors. According to the depth and size, cases were categorized into the low-risk group [m and ≤ 2 cm, 0% (LNM incidence)], the moderate-risk group (m and > 2 cm, 5.6%; and sm and ≤ 2 cm, 6.0%), and the high-risk group (sm and > 2 cm, 19.3%). On the other hand, LNM occurred in 1.4% in all LI-negative cases, greatly lower than 28.2% in all LI-positive cases, and LNM incidence was low in LI-negative cases even in the moderate- and high-risk groups. CONCLUSION: LNM-related factors in undiff-EGC were depth and size preoperatively while those were LI and size postoperatively. Among these factors, LI was the most significantly correlated factor.

Hepatocellular carcinoma risk assessment using gadoxetic acid-enhanced hepatocyte phase magnetic resonance imaging.

AIM: To investigate whether the patients with hypovascular liver nodules determined on the arterial phase and hypointensity on the hepatocyte phase gadoxetic acid-enhanced magnetic resonance imaging (hypovascular hypointense nodules) are at increased risk of hepatocarcinogenesis, we assessed subsequent typical hepatocellular carcinoma (HCC) development at any sites of the liver with and without such nodules. METHODS: One hundred and twenty-seven patients with chronic hepatitis B or C and without a history of HCC, including 68 with liver cirrhosis, were divided into those with (non-clean liver group, n = 18) and without (clean liver group, n = 109) hypovascular hypointense nodules. All the patients were followed up for 3 years, and HCC development rates and risk factors were analyzed with the Kaplan-Meier method and the Cox proportional hazard model, respectively. RESULTS: A total of 17 patients (10 in the non-clean liver group and seven in the clean liver group) developed typical HCC. Cumulative 3-year rates of HCC development were 55.5% in the non-clean liver group and 6.4% in the clean liver group (P < 0.001), and those at the different sites from the initial nodules was also higher in the non-clean liver group (22.2%) than the clean liver group (6.4%) (P = 0.003). Multivariate analysis identified older age (P = 0.024), low platelet counts (P = 0.017) and a non-clean liver (P < 0.001) as independent risk factors for subsequent HCC development. CONCLUSION: Patients with hypovascular hypointense liver nodules are at a higher risk for HCC development at any sites of the liver than those without such nodules.

Stage IV intramucosal gastric marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type.

A 45-year-old woman with no symptoms underwent upper gastrointestinal endoscopy. A discolored area was noted at the greater curvature of the gastric upper body. Endoscopic ultrasonography demonstrated thickening of the second sonographic layer indicating that the depth of invasion was confined to the mucosa. A urea breath test and anti-Helicobacter pylori antibody test were negative. A computed tomography scan showed a consolidation at the right lung. Gastric biopsy and transbronchial lung biopsy (TBLB) demonstrated a monotonous proliferation of atypical small lymphocytes. A diagnosis of gastric marginal zone B cell lymphoma of mucosa-associated lymphoid tissue type (MALT lymphoma) was made. The clinical stage was stage IV. A genetic analysis showed rearrangement of the joining region of the immunoglobulin heavy chain gene and identical clones in both lesions. An API2-MALT1 fusion gene was detected in the gastric lesion. After H. pylori eradication treatment, combination treatment with rituximab plus CHOP (R-CHOP) was performed; 6 months later an endoscopy revealed complete disappearance of the lesion. Multiple gastric biopsies showed no infiltrating atypical lymphocytes. Similarly, the lesion in the lung showed complete remission (CR) on CT and TBLB. This report shows that a gastric MALT lymphoma located in the mucosa and disseminated to the lung maintained CR by R-CHOP.

Correlation between pretreatment viral sequences and the emergence of lamivudine resistance in hepatitis B virus infection.

The emergence of amino acid or nucleotide substitutions leads to lamivudine resistance in hepatitis B virus (HBV) infected patients. The aim of this study was to investigate whether viral sequences help predict the emergence of lamivudine resistance. The study subjects comprised 59 consecutive patients infected with HBV treated with daily therapy of 100 mg lamivudine. Among those, 32 patients with adequate pretreatment serum preservation were investigated for the correlation between viral amino acid substitutions and the appearance of lamivudine resistance with consideration of clinical background by determining dominant HBV full open reading frames. Viral resistance to lamivudine emerged in 28 of 59 patients (47%) in a median period of 2.45 years. Sequence comparisons of HBV genomes between patients who later developed lamivudine resistance and patients who did not revealed the existence of significant differences between the two groups in the pre-S1 84 (P = 0.042), pre-S2 1 (P = 0.017) and 22 (P = 0.015), and polymerase tp 95 (P = 0.046), judged by a log-rank test. Viral sequence analyses revealed the presence of amino acid substitutions in HBV pre-S1 and pre-S2 that may be associated with the emergence of lamivudine resistance during chronic HBV infection.

Complete remission of gastric plasmacytoma following eradication of "Candidatus Helicobacter heilmannii".

This is the first case report of gastric plasmacytoma associated with "Candidatus Helicobacter heilmannii" ('H. heilmannii') infection. The patient was a 40-year-old woman with epigastric discomfort. Upper gastrointestinal endoscopy demonstrated a white granular lesion on the wall of the gastric body. Histological studies showed numerous eosinophilic globules expanding the lamina propria mucosae. Immunohistochemically, the cells with these globules stained positive for CD138, CD79a, immunoglobulin (Ig) M, and kappa light chain, but negative for CD20, IgG, IgA, and lambda light chain. A diagnosis of plasmacytoma was made. Although a Helicobacter pylori infection was not detected, the patient received H. pylori eradication treatment. Two months after H. pylori eradication treatment, an upper gastrointestinal endoscopy showed a reduction of the white granular lesion. Eighteen months after eradication treatment, endoscopy, endoscopic ultrasonography and histological studies revealed complete remission of the lesion. No relapse has been documented 30 months after the initial diagnosis of plasmacytoma. Retrospectively, analysis of biopsy specimens removed before eradication treatment demonstrated that this patient had 'H. heilmannii' infection. Therefore, H. pylori eradication therapy should be considered as a potential first-line therapy for early-stage gastric plasmacytoma with or without H. pylori infection.

Analysis of viral amino acids sequences and the IL28B SNP influencing the development of hepatocellular carcinoma in chronic hepatitis C.

BACKGROUND AND AIMS: The association between hepatitis C virus (HCV) sequences with interleukin 28B (IL28B) single-nucleotide polymorphism (SNP) in the development of hepatocellular carcinoma (HCC) has not been well clarified. METHODS: Complete HCV open-reading frame sequences were determined in 20 patients developing HCC and 23 non-HCC patients with HCV-1b infection in two distant time points. An additional 230 patients were studied cross-sectionally for core and NS5A sequences with HCC development. Among them, 98 patients with available samples were investigated for changes in viral core sequences over time. Finally, IL28B SNPs and HCC development were investigated in 228 patients. RESULTS: During observation period (HCC for 10.8 years, and non-HCC for 11.1 years), changes in core a.a. 70 and three amino acid positions in NS5A were characteristics of the patients developing HCC. In 230 patients, Q (glutamine) or H (histidine) to R (arginine) ratio at core a.a. 70 was significantly higher in the HCC group (HCC group 43:22 vs. non-HCC group 66:99, p = 0.001). A change in core R70Q was observed over time in 11 patients associated with a decrease in platelets (p = 0.005) and albumin (p = 0.005), while a Q70R change was observed in 4 patients without associated changes in platelets (nonsignificant) and albumin (nonsignificant). IL28B SNP showed significant correlation with the core a.a. 70 residue. There was no evident link between IL28B SNPs and the occurrence of HCC. CONCLUSIONS: Hepatitis C virus core a.a. 70 residue is associated with liver disease progression and is independent factor for HCC development in genotype-1b infection. IL28B SNPs are related to core a.a. 70 residue, but not to HCC. The functional relevance of core a.a. 70 residue in hepatitis C pathogenesis should be further investigated.

Analysis of the complete open reading frame of genotype 2b hepatitis C virus in association with the response to peginterferon and ribavirin therapy.

BACKGROUND AND AIMS: Patients infected with genotype 2b hepatitis C virus (HCV) generally can achieve favorable responses to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV). However, a proportion of patients show poorer responses and the correlation between viral sequence variation and treatment outcome remains unclear. METHODS: The pretreatment complete open reading frame (ORF) sequences of genotype 2b HCV determined by direct sequencing were investigated for correlation with the final outcome in a total of 60 patients. RESULTS: In this study group, 87.5% (14/16) of non-sustained virological response (non-SVR) patients (n = 16) were relapsers. Compared to sustained virological response (SVR) patients (n = 44), non-SVR patients were older and could not achieve prompt viral clearance after the therapy induction. Comparing each viral protein between the two groups, viral sequences were more diverse in SVR patients and that diversity was found primarily in the E1, p7, and NS5A proteins. In searching for specific viral regions associated with the final outcome, several regions in E2, p7, NS2, NS5A, and NS5B were extracted. Among these regions, part of the interferon sensitivity determining region (ISDR) was included. In these regions, amino acid substitutions were associated with the final outcome in an incremental manner, depending upon the number of substitutions. CONCLUSIONS: Viral sequences are more diverse in SVR patients than non-SVR patients receiving PEG-IFN/RBV therapy for genotype-2b HCV infection. Through systematic comparison of viral sequences, several specific regions, including part of the ISDR, were extracted as having significant correlation with the final outcome.

[A family with attenuated familial adenomatous polyposis identified because of fundic gland polyposis].

We report a 41-year-old woman who had fundic gland polyposis without a colorectal polyp. Because her father had colon cancer, multiple colorectal polyps and the gastric polyposis, we suspected AFAP and researched the APC gene. There was 1 base pair deletion of guanine at codon 99-100 in exon 3 of the APC gene, and its frame shift mutation made stop codon at codon 124. It was proved that the gene carrier of AFAP can be discovered based on the family-history and the presence of fundic gland polyposis, even when no colorectal polyps exist.

Analysis of the complete open reading frame of hepatitis C virus in genotype 2a infection reveals critical sites influencing the response to peginterferon and ribavirin therapy.

PURPOSE: A proportion of patients infected with genotype 2a hepatitis C virus (HCV) cannot achieve a sustained virological response (SVR) to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV) but the reason remains unclear. The present study aimed to clarify the possible correlation between viral sequence variations and final outcome. METHODS: The pretreatment complete open reading frame (ORF) sequences of genotype 2a HCV were determined by direct sequencing for two independent groups of patients (43 patients as test; group 1 and 35 as validation; group 2), and the correlation with the final outcome was explored. RESULTS: Patients with SVR (n = 58) and with non-SVR (n = 20) differed significantly in pretreatment HCV RNA level (p = 0.002), fibrosis score (p = 0.047), and cumulative RBV dosage (p = 0.003). By comparison of all amino acid positions in the complete HCV ORFs, threonine at amino acid (aa) 110 in the core region was remarkably frequent in SVR (p = 0.01 for group 1, p = 0.004 for group 2, and p = 5E-05 for combined). A sliding window analysis revealed that the total number of amino acid variations within the NS5A aa 2258-2306 region were significantly high in SVR compared to non-SVR patients (p = 0.01 for group 1, p = 0.006 for group 2, and p = 0.0006 for combined). Multivariate analyses revealed that core aa 110 (p = 0.02), NS5A aa 2258-2306 (p = 0.03), and cumulative RBV dosage (p = 0.02) were identified as independent variables associated with the final outcome. CONCLUSIONS: The outcome of PEG-IFN/RBV therapy is significantly influenced by variation in the core and NS5A regions in genotype 2a HCV infection.

DICKKOPF-4 and -2 genes are upregulated in human colorectal cancer.

To comprehensively screen for genetic events underlying colorectal cancer, we performed suppression subtraction hybridization analysis on an advanced colon cancer. Because Dickkopf-4, a member of the Dickkopf family acting as a Wnt-signaling modulator, was identified as one of the upregulated genes in this specimen, we investigated expression profiles of all the Dickkopf family members in 55 colorectal tumors (21 cancers and 34 adenomas). We also investigated mechanisms regulating the expression of Dickkopf-4 in these cancers in vitro and in vivo. Compared with normal adjacent mucosae, Dickkopf-4 (median 27.4, P < 0.01) and -2 (median 51.4, P < 0.01) were strongly expressed in colorectal cancers. The level of Dickkopf-4 was positively correlated with fibroblast growth factor-20 (r(s) = 0.61, P = 0.00017), a representative beta-catenin transcriptional target gene, and with the degree of nuclear accumulation of beta-catenin in colorectal tumors. Dickkopf-4 was induced by activated beta-catenin in vitro. Reciprocally, recombinant Dickkopf-4 significantly inhibited T-cell factor/lymphocyte enhancer factor reporter activity stimulated by recombinant Wnt3a in human embryonic kidney 293 cells. We conclude that Dickkopf-4 and -2 are significantly upregulated in most colorectal tumors, and that Dickkopf-4 upregulation reflects activation of the Wnt/canonical pathway.

[Osteoporosis in elderly patients].

Recent reports indicate an increased prevalence of reflux esophagitis (RE) in Japan. There are many factors causing RE, and many kinds of changes associating aging are important in the cause of RE in elderly patients. We reported that the osteoporosis and the orthopedics degenerative disease in elderly people had relation in development of symptoms of RE of our country. The elderly are complicated with posture change (kyphosis) due to osteoporosis caused by menopause; some pharmaceutical agents such as bisphosphonates (BPs) or NSAIDs. Furthermore, it is necessary to provide elderly RE treatment with the all-inclusive treatment not only the conventional medical treatment which made the PPI maintenance therapy but a living environment, complications, an oral medicine including BPs and NSAIDs.

[Pathophysiology of reflux esophagitis in the elderly patients].

Recent reports indicate an increased prevalence of reflux esophagitis(RE) in Japan. There are many factors causing RE, and many kinds of changes associating aging are important in the causes of RE in the elderly patients. Characteristic features of the causes of RE in elderly patients are summarized here. Within the elderly patients, there are cases with persistent gastric acid secretion. Aging affections lead to esophageal motor dysfunctions and to failure of LES function(presbyesophagus). The elderly are complicated by orthopedic degenerative diseases with posture change due to osteoporosis; some pharmaceutical agents such as Ca-channel blockers or NSAIDs. Hiatal hernia is also an aggravating factor. In the future, elderly people with persistent gastric acid secretion will be increased based on declining prevalence of Helicobacter pylori. Therefore further increase in the prevalence and development of RE is foreseen in our country. Pathophysiology of RE in the elderly patients is expected to show various changes in the future.