Hiroko Shindo

Deep sequencing of cancer-related genes revealed GNAS mutations to be associated with intraductal papillary mucinous neoplasms and its main pancreatic duct dilation.

Background To clarify the genetic mutations associated with intraductal papillary mucinous neoplasms (IPMN) and IPMN-related pancreatic tumours, we conducted cancer-related gene profiling analyses using pure pancreatic juice and resected pancreatic tissues. Methods Pure pancreatic juice was collected from 152 patients [nine with a normal pancreas, 22 with chronic pancreatitis (CP), 39 with pancreatic ductal adenocarcinoma (PDAC), and 82 with IPMN], and resected tissues from the pancreas were collected from 48 patients (six IPMNs and 42 PDACs). The extracted DNA was amplified by multiplexed polymerase chain reaction (PCR) targeting 46 cancer-related genes containing 739 mutational hotspots. The mutations were analysed using a semiconductor-based DNA sequencer. Results Among the 46 cancer-related genes, KRAS and GNAS mutations were most frequently detected in both PDAC and IPMN cases. In pure pancreatic juice, GNAS mutations were detected in 7.7% of PDAC cases and 41.5% of IPMN cases (p<0.001 vs. others). All PDAC cases with GNAS mutations (n = 3) were accompanied by IPMN. Multivariate analysis revealed that GNAS mutations in IPMN cases were associated with dilated main pancreatic ducts (MPD, p = 0.016), while no statistically independent associations with clinical variables were observed for KRAS mutations. In the resected pancreatic tissues, GNAS mutations were detected in 50% of PDAC cases concomitant with IPMN, 33.3% of PDAC cases derived from IPMN, and 66.7% of IPMN cases, while no GNAS mutations were detected in cases of PDAC without IPMN. Conclusions The GNAS mutation was specifically found in the cases with IPMN and it was speculated that some PDACs might be influenced by the concomitant but separately-located IPMN in their pathogenic mechanism. Furthermore, the GNAS mutation was significantly associated with MPD dilatation in IPMN cases, suggesting its role in mucus hypersecretion.

IL-28B (IFN-λ3) and IFN-α synergistically inhibit HCV replication

Genetic variation in the IL‐28B (interleukin‐28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon‐α and ribavirin. However, the mechanisms by which polymorphisms in the IL‐28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN‐λs and IFN‐α on HCV RNA replication. The anti‐HCV effect of IFN‐λ3 and IFN‐α in combination was also assessed. Changes in gene expression induced by IFN‐λ3 and IFN‐α were compared using cDNA microarray analysis. IFN‐λs at concentrations of 1 ng/mL or more exhibited concentration‐ and time‐dependent HCV inhibition. In combination, IFN‐λ3 and IFN‐α had a synergistic anti‐HCV effect; however, no synergistic enhancement was observed for interferon‐stimulated response element (ISRE) activity or upregulation of interferon‐stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN‐λ3‐induced gene expression occurred later and lasted longer than that induced by IFN‐α. In addition, although the genes upregulated by IFN‐α and IFN‐λ3 were similar to microarray analysis, interferon‐stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN‐α and IFN‐λ3 in combination showed synergistic anti‐HCV activity in vitro. Differences in time‐dependent upregulation of these genes might contribute to the synergistic antiviral activity.

Comprehensive analysis for viral elements and interleukin‐28B polymorphisms in response to pegylated interferon plus ribavirin therapy in hepatitis C virus 1B infection

To comprehensively characterize the contribution of virological factors as well as interleukin‐28B (IL28B) single‐nucleotide polymorphisms (SNPs) in determining treatment responses in pegylated‐interferon plus ribavirin (Peg‐IFN/RBV) therapy for chronic hepatitis C virus (HCV)‐1b infection, we undertook a retrospective cohort analysis for the pretreatment dominant complete HCV open reading frame (ORF) amino‐acid (aa) sequence study in 103 consecutive HCV‐1b Japanese patients. The dominant HCV sequences classified by the response were subjected to systematic sliding‐window comparison analysis to characterize response‐specific viral sequences, along with IL28B SNP analyses (rs8099917). In each comparison of the patients between with and without rapid viral response (RVR), nonearly viral response (nEVR), sustained virological response (SVR), or relapse, the following regions were extracted as most significantly associated with the different responses respectively: nonstructural protein 5A (NS5A) aa.2224‐2248 (P = 1.2E‐07); core aa.70 (P = 4E‐04); NS5A aa.2340‐2382 (P = 7.0E‐08); and NS5A aa.2360‐2377 (P = 1.1E‐05). Those NS5A regions nearly coincided with the interferon (IFN) sensitivity‐determining region (NS5A aa.2209‐2248) and the IFN/RBV resistance‐determining region (NS5A aa.2339‐2379). In a multivariate analysis, the IL28B SNP (odds ratio [OR] = 16.8; P = 0.009) and NS5A aa.2340‐2382 (OR = 13.8; P = 0.0003) were extracted as the two most‐significant independent variables contributing to the final outcome. Conclusion: In Peg‐IFN/RBV therapy, polymorphisms in IL28B, NS5A aa.2224‐2248, core aa.70, and, most important, NS5A aa.2340‐2382 have a tremendous influence on treatment response in association with viral kinetics, resulting in significantly different outcomes in chronic HCV‐1b infection. (HEPATOLOGY 2012;56:1611–1621)

Inhibitory effects of caffeic acid phenethyl ester derivatives on replication of hepatitis C virus.

Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC50 values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC50 value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.

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.

Performance status and neutrophil-lymphocyte ratio are important prognostic factors in elderly patients with unresectable pancreatic cancer

BACKGROUND The usefulness of various prognostic factors for pancreatic cancer (PC) has been reported, but the number of elderly patients in these studies is disproportionately fewer compared with those in everyday practice. The purpose of this study was to investigate the prognostic factors for unresectable PC in elderly patients. METHODS We retrospectively analyzed 67 elderly (age ≥75 years) patients with unresectable PC who underwent chemotherapy between January 2006 and December 2014 at our hospital. Univariate and multivariate Cox regression models were applied to investigate independent prognostic factors. RESULTS Multivariate analysis revealed that an increased neutrophil-lymphocyte ratio (NLR) [hazard ratio (HR) 1.91, P=0.03] and Eastern Cooperative Oncology Group (ECOG) performance status (PS) 2 (HR 2.74, P=0.01) were independent negative prognostic factors. CONCLUSIONS The two prognostic factors identified herein are useful in the identification of patients with a poor prognosis and subsequent administration of supportive care alone, which may help avoid the unnecessary adverse effects and complications of systemic chemotherapy.

Biliary drainage strategy of unresectable malignant hilar strictures by computed tomography volumetry.

AIM To identify criteria for predicting successful drainage of unresectable malignant hilar biliary strictures (UMHBS) because no ideal strategy currently exists. METHODS We examined 78 patients with UMHBS who underwent biliary drainage. Drainage was considered effective when the serum bilirubin level decreased by ≥ 50% from the value before stent placement within 2 wk after drainage, without additional intervention. Complications that occurred within 7 d after stent placement were considered as early complications. Before drainage, the liver volume of each section (lateral and medial sections of the left liver and anterior and posterior sections of the right liver) was measured using computed tomography (CT) volumetry. Drained liver volume was calculated based on the volume of each liver section and the type of bile duct stricture (according to the Bismuth classification). Tumor volume, which was calculated by using CT volumetry, was excluded from the volume of each section. Receiver operating characteristic (ROC) analysis was performed to identify the optimal cutoff values for drained liver volume. In addition, factors associated with the effectiveness of drainage and early complications were evaluated. RESULTS Multivariate analysis showed that drained liver volume [odds ratio (OR) = 2.92, 95%CI: 1.648-5.197; P < 0.001] and impaired liver function (with decompensated liver cirrhosis) (OR = 0.06, 95%CI: 0.009-0.426; P = 0.005) were independent factors contributing to the effectiveness of drainage. ROC analysis for effective drainage showed cutoff values of 33% of liver volume for patients with preserved liver function (with normal liver or compensated liver cirrhosis) and 50% for patients with impaired liver function (with decompensated liver cirrhosis). The sensitivity and specificity of these cutoff values were 82% and 80% for preserved liver function, and 100% and 67% for impaired liver function, respectively. Among patients who met these criteria, the rate of effective drainage among those with preserved liver function and impaired liver function was 90% and 80%, respectively. The rates of effective drainage in both groups were significantly higher than in those who did not fulfill these criteria (P < 0.001 and P = 0.02, respectively). Drainage-associated cholangitis occurred in 9 patients (12%). A smaller drained liver volume was associated with drainage-associated cholangitis (P < 0.01). CONCLUSION Liver volume drainage ≥ 33% in patients with preserved liver function and ≥ 50% in patients with impaired liver function correlates with effective biliary drainage in UMHBS.

Next-Generation Sequencing Revealed TP53 Mutations to Be Malignant Marker for Intraductal Papillary Mucinous Neoplasms That Could Be Detected Using Pancreatic Juice

Objectives The aims of this study were to identify the genetic mutations associated with malignant intraductal papillary mucinous neoplasms (IPMNs) and evaluate the possibility of detecting mutations in pure pancreatic juice by next-generation sequencing. Methods Resected tissues were collected from 50 patients with IPMN, and pure pancreatic juice samples were collected from 19 patients who had a resection. The extracted DNA was amplified by multiplex polymerase chain reaction targeting 52 cancer-related genes, including KRAS, GNAS, RNF43, and TP53; the mutations were then detected by next-generation sequencing and then analyzed for correlations with the clinicopathological characteristics. Results In the resected tissues, the most frequently detected mutations were in KRAS, GNAS, TP53, and RNF43, in 88%, 76%, 36%, and 30% of cases, respectively. Univariate and multivariate analyses revealed that only TP53 mutations were associated with malignant IPMNs (P = 0.023). In the pure pancreatic juice, TP53 mutations were detected in 5 of 10 resected samples with malignant IPMN and in 4 of 5 pancreatic juice samples with mutation in resected samples. Conclusions From 52 cancer-related gene analysis, only TP53 mutation was associated with malignant IPMNs. TP53 mutation could also be detected in pure pancreatic juice, potentially making it a useful tool to diagnose malignant IPMNs preoperatively.

Hypoechoic Lesions in Submandibular Glands Are Diagnostic Markers of Type 1 Autoimmune Pancreatitis.

Routine laboratory evaluation is often unrevealing, but possible abnormalities include the following: hypercalcemia, hypercalcuria, elevated alkaline phosphatase level, elevated angiotensin-converting enzyme levels. Regarding this case, the whole body F-FDG positron emission tomography/CTand a biopsy result stands strongly for the diagnosis of pancreatic sarcoidosis. In this case, the confirmation of AIP is based on clinical, laboratory test results, radiographic, and histological findings. The pancreatic biopsy performed by EUS-FNA had sufficient tissue for both cytological and histological diagnosis. Despite radiological advances, pancreatic masses are often difficult to diagnose until operative exploration and histological examination of biopsies. There are 25 cases of pancreatic sarcoidosiswhichwere reported having performed surgical operation as a combined diagnostic and therapeutic measure. Also, such a comprehensive surgical resection is much harmful to a patient if the final result shows a benign lesion. Compared with open biopsy, EUS-FNA biopsy has a lower risk of postoperative complications. In addition, the patient in our case did not take any medication to treat his illness, sowhether the patient would react well to therapeutic diagnosis may be another way to differentiating pancreatic sarcoidosis and other pancreatic diseases. Conclusively, we report here a case showing very uncommon combination of a pancreatic sarcoidosis and type 1 AIP. They sometimes have similar presentation and possibly share the same cause but large numbers of studies are still needed. The EUS-FNAmay present an effective and relatively secure method for differentiating pancreatic sarcoidosis and chronic pancreatitis. However, whether the sensitivity and specificity of this technique in preoperative diagnosis of pancreatic diseases are higher than those of other imaging techniques, and open biopsies still need more researches to verify.

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. J. Med. Virol. 84:1360–1368, 2012.