Growth Arrest of HPV-Positive Cells after Histone Deacetylase Inhibition Is Independent of E6/E7 Oncogene Expression

AbstractInhibitors of histone deacetylase (HDAC) are capable of arresting growth in cervical carcinoma cells in the G1 phase of the cell cycle. Although HPV E6/E7 mRNA steady-state levels appeared to be constant after prolonged treatment, time-course experiments revealed that viral transcription was transiently down-regulated between 7–10 h prior to cdk2 suppression. To test whether transitory suppression was a prerequisite for the biological outcome after HDAC inhibition, we took advantage of two immortalized human keratinocyte cell lines in which E6/E7 oncogene expression was controlled by different regulatory regions. After treatment with sodium butyrate (NaB) or trichostatin A (TSA), HPV16 upstream regulatory region (URR)-directed transcription was down-regulated, showing kinetics similar to those in cervical carcinoma cells. In contrast, β-actin promoter controlled E6/E7 transcription was even temporarily increased and finally declined to levels initially detected in the untreated controls. Both cell lines, however, were arrested in G1 and showed complete suppression of cdk2 activity that was preceded by a strong up-regulation of the cdk2 inhibitors p21CIP1 and p27KIP1. These results demonstrate that growth of HPV16/18-positive cells can be arrested by HDAC inhibitors despite ongoing HPV transcription and thus independently of any potential position effects uncoupling URR-directed gene expression by adjacent cellular promoters or by downstream 3′-polyadenylation sites after viral integration into the host genome during multistep carcinogenesis

Diagnostic approach to Helicobacter pylori-related gastric oncogenesis.

Helicobacter pylori (H. pylori) is a causative agent of peptic ulcer disease and plays an important role in the development of various other upper and lower gastrointestinal tract and systemic diseases; in addition to carcinogenesis and the development of mucosa-associated lymphoid tissue lymphoma, extragastric manifestations of H. pylori are increasingly being unraveled. Therefore, prompt and accurate diagnosis is essential. Within this narrative review we present an overview of the current trend in the diagnosis of H. pylori infection and its potential oncogenic sequelae, including gastric mucosa atrophy, intestinal metaplasia, dysplasia and gastric cancer. Signs of H. pylori-related gastric cancer risk can be assessed by endoscopy using the Kyoto classification score. New technology, such as optical or digital chromoendoscopy, improves diagnostic accuracy and provides information regarding H. pylori-related gastric preneoplastic and malignant lesions. In addition, a rapid urease test or histological examination should be performed, as these offer a high diagnostic sensitivity; both are also useful for the diagnosis of sequelae including gastric and colon neoplasms. Culture is necessary for resistance testing and detecting H. pylori-related gastric dysbiosis involved in gastric oncogenesis. Likewise, molecular methods can be utilized for resistance testing and detecting H. pylori-related gastric cancer development and progression. Noninvasive tests, such as the urea breath and stool antigen tests, can also be implemented; these are also suitable for monitoring eradication success and possibly for detecting H. pylori-related gastric malignancy. Serological tests may help to exclude infection in specific populations and detect gastric and colon cancers. Finally, there are emerging potential diagnostic biomarkers for H. pylori-related gastric cancer

Assessment of hepatic fibrosis and inflammation with look-locker T1 mapping and magnetic resonance elastography with histopathology as reference standard.

PURPOSE To compare the diagnostic performance of T1 mapping and MR elastography (MRE) for staging of hepatic fibrosis and grading inflammation with histopathology as standard of reference. METHODS 68 patients with various liver diseases undergoing liver biopsy for suspected fibrosis or with an established diagnosis of cirrhosis prospectively underwent look-locker inversion recovery T1 mapping and MRE. T1 relaxation time and liver stiffness (LS) were measured by two readers. Hepatic fibrosis and inflammation were histopathologically staged according to a standardized fibrosis (F0-F4) and inflammation (A0-A2) score. For statistical analysis, independent t test, and Mann-Whitney U test and ROC analysis were performed, the latter to determine the performance of T1 mapping and MRE for fibrosis staging and inflammation grading, as compared to histopathology. RESULTS Histopathological analysis diagnosed 9 patients with F0 (13.2%), 21 with F1 (30.9%), 11 with F2 (16.2%), 10 with F3 (14.7%), and 17 with F4 (25.0%). Both T1 mapping and MRE showed significantly higher values for patients with significant fibrosis (F0-1 vs. F2-4; T1 mapping p < 0.0001, MRE p < 0.0001) as well as for patients with severe fibrosis or cirrhosis (F0-2 vs. F3-4; T1 mapping p < 0.0001, MRE p < 0.0001). T1 values and MRE LS were significantly higher in patients with inflammation (A0 vs. A1-2, both p = 0.01). T1 mapping showed a tendency toward lower diagnostic performance without statistical significance for significant fibrosis (F2-4) (AUC 0.79 vs. 0.91, p = 0.06) and with a significant difference compared to MRE for severe fibrosis (F3-4) (AUC 0.79 vs. 0.94, p = 0.03). For both T1 mapping and MRE, diagnostic performance for diagnosing hepatic inflammation (A1-2) was low (AUC 0.72 vs. 0.71, respectively). CONCLUSION T1 mapping is able to diagnose hepatic fibrosis, however, with a tendency toward lower diagnostic performance compared to MRE and thus may be used as an alternative to MRE for diagnosing hepatic fibrosis, whenever MRE is not available or likely to fail due to intrinsic factors of the patient. Both T1 mapping and MRE are probably not sufficient as standalone methods to diagnose hepatic inflammation with relatively low diagnostic accuracy

Assessment of hepatic fibrosis and inflammation with look-locker T1 mapping and magnetic resonance elastography with histopathology as reference standard

Purpose: To compare the diagnostic performance of T1 mapping and MR elastography (MRE) for staging of hepatic fibrosis and grading inflammation with histopathology as standard of reference. Methods: 68 patients with various liver diseases undergoing liver biopsy for suspected fibrosis or with an established diagnosis of cirrhosis prospectively underwent look-locker inversion recovery T1 mapping and MRE. T1 relaxation time and liver stiffness (LS) were measured by two readers. Hepatic fibrosis and inflammation were histopathologically staged according to a standardized fibrosis (F0-F4) and inflammation (A0-A2) score. For statistical analysis, independent t test, and Mann-Whitney U test and ROC analysis were performed, the latter to determine the performance of T1 mapping and MRE for fibrosis staging and inflammation grading, as compared to histopathology. Results: Histopathological analysis diagnosed 9 patients with F0 (13.2%), 21 with F1 (30.9%), 11 with F2 (16.2%), 10 with F3 (14.7%), and 17 with F4 (25.0%). Both T1 mapping and MRE showed significantly higher values for patients with significant fibrosis (F0-1 vs. F2-4; T1 mapping p < 0.0001, MRE p < 0.0001) as well as for patients with severe fibrosis or cirrhosis (F0-2 vs. F3-4; T1 mapping p < 0.0001, MRE p < 0.0001). T1 values and MRE LS were significantly higher in patients with inflammation (A0 vs. A1-2, both p = 0.01). T1 mapping showed a tendency toward lower diagnostic performance without statistical significance for significant fibrosis (F2-4) (AUC 0.79 vs. 0.91, p = 0.06) and with a significant difference compared to MRE for severe fibrosis (F3-4) (AUC 0.79 vs. 0.94, p = 0.03). For both T1 mapping and MRE, diagnostic performance for diagnosing hepatic inflammation (A1-2) was low (AUC 0.72 vs. 0.71, respectively). Conclusion: T1 mapping is able to diagnose hepatic fibrosis, however, with a tendency toward lower diagnostic performance compared to MRE and thus may be used as an alternative to MRE for diagnosing hepatic fibrosis, whenever MRE is not available or likely to fail due to intrinsic factors of the patient. Both T1 mapping and MRE are probably not sufficient as standalone methods to diagnose hepatic inflammation with relatively low diagnostic accuracy. Keywords: Biopsy; Fibrosis; Liver; MR elastography; T1 mappin

Widely differing screening and treatment practice for osteoporosis in patients with inflammatory bowel diseases in the Swiss IBD cohort study.

Low bone mineral density (BMD) and osteoporosis remain frequent problems in patients with inflammatory bowel diseases (IBDs). Several guidelines with nonidentical recommendations exist and there is no general agreement regarding the optimal approach for osteoporosis screening in IBD patients. Clinical practice of osteoporosis screening and treatment remains insufficiently investigated.In the year 2014, a chart review of 877 patients included in the Swiss IBD Cohort study was performed to assess details of osteoporosis diagnostics and treatment. BMD measurements, osteoporosis treatment, and IBD medication were recorded.Our chart review revealed 253 dual-energy x-ray absorptiometry (DXA) scans in 877 IBD patients; osteoporosis was prevalent in 20% of tested patients. We identified widely differing osteoporosis screening rates among centers (11%-62%). A multivariate logistic regression analysis identified predictive factors for screening including steroid usage, long disease duration, and perianal disease; even after correction for all risk factors, the study center remained a strong independent predictor (odds ratio 2.3-21 compared to the center with the lowest screening rate). Treatment rates for patients with osteoporosis were suboptimal (55% for calcium, 65% for vitamin D) at the time of chart review. Similarly, a significant fraction of patients with current steroid medication were not treated with vitamin D or calcium (treatment rates 53% for calcium, 58% for vitamin D). For only 29% of patients with osteoporosis bisphosphonate treatment was started. Treatment rates also differed among centers, generally following screening rates. In patients with longitudinal DXA scans, calcium and vitamin D usage was significantly associated with improvement of BMD over time.Our analysis identified inconsistent usage of osteoporosis screening and underuse of osteoporosis treatment in IBD patients. Increasing awareness of osteoporosis as a significant clinical problem in IBD patients might improve patient care

Effect of Immune Pressure on Hepatitis C Virus Evolution: Insights From a Single-Source Outbreak

The host's immune response to hepatitis C virus (HCV) can result in the selection of characteristic mutations (adaptations) that enable the virus to escape this response. The ability of the virus to mutate at these sites is dependent on the incoming virus, the fitness cost incurred by the mutation, and the benefit to the virus in escaping the response. Studies examining viral adaptation in chronic HCV infection have shown that these characteristic immune escape mutations can be observed at the population level as human leukocyte antigen (HLA)–specific viral polymorphisms. We examined 63 individuals with chronic HCV infection who were infected from a single HCV genotype 1b source. Our aim was to determine the extent to which the host's immune pressure affects HCV diversity and the ways in which the sequence of the incoming virus, including preexisting escape mutations, can influence subsequent mutations in recipients and infection outcomes. Conclusion: HCV sequences from these individuals revealed 29 significant associations between specific HLA types within the new hosts and variations within their viruses, which likely represent new viral adaptations. These associations did not overlap with previously reported adaptations for genotypes 1a and 3a and possibly reflected a combination of constraint due to the incoming virus and genetic distance between the strains. However, these sites accounted for only a portion of the sites in which viral diversity was observed in the new hosts. Furthermore, preexisting viral adaptations in the incoming (source) virus likely influenced the outcomes in the new hosts. (Hepatology 2011;53:396-405

Whole Genome Pyrosequencing of Rare Hepatitis C Virus Genotypes Enhances Subtype Classification and Identification of Naturally Occurring Drug Resistance Variants

Background. Infection with hepatitis C virus (HCV) is a burgeoning worldwide public health problem, with 170 million infected individuals and an estimated 20 million deaths in the coming decades. While 6 main genotypes generally distinguish the global geographic diversity of HCV, a multitude of closely related subtypes within these genotypes are poorly defined and may influence clinical outcome and treatment options. Unfortunately, the paucity of genetic data from many of these subtypes makes time-consuming primer walking the limiting step for sequencing understudied subtypes. Methods. Here we combined long-range polymerase chain reaction amplification with pyrosequencing for a rapid approach to generate the complete viral coding region of 31 samples representing poorly defined HCV subtypes. Results. Phylogenetic classification based on full genome sequences validated previously identified HCV subtypes, identified a recombinant sequence, and identified a new distinct subtype of genotype 4. Unlike conventional sequencing methods, use of deep sequencing also facilitated characterization of minor drug resistance variants within these uncommon or, in some cases, previously uncharacterized HCV subtypes. Conclusions. These data aid in the classification of uncommon HCV subtypes while also providing a high-resolution view of viral diversity within infected patients, which may be relevant to the development of therapeutic regimens to minimize drug resistanc

Sequencing of the Hepatitis C Virus: A Systematic Review

Since the identification of hepatitis C virus (HCV), viral sequencing has been important in understanding HCV classification, epidemiology, evolution, transmission clustering, treatment response and natural history. The length and diversity of the HCV genome has resulted in analysis of certain regions of the virus, however there has been little standardisation of protocols. This systematic review was undertaken to map the location and frequency of sequencing on the HCV genome in peer reviewed publications, with the aim to produce a database of sequencing primers and amplicons to inform future research. Medline and Scopus databases were searched for English language publications based on keyword/MeSH terms related to sequence analysis (9 terms) or HCV (3 terms), plus "primer" as a general search term. Exclusion criteria included non-HCV research, review articles, duplicate records, and incomplete description of HCV sequencing methods. The PCR primer locations of accepted publications were noted, and purpose of sequencing was determined. A total of 450 studies were accepted from the 2099 identified, with 629 HCV sequencing amplicons identified and mapped on the HCV genome. The most commonly sequenced region was the HVR-1 region, often utilised for studies of natural history, clustering/transmission, evolution and treatment response. Studies related to genotyping/classification or epidemiology of HCV genotype generally targeted the 5'UTR, Core and NS5B regions, while treatment response/resistance was assessed mainly in the NS3-NS5B region with emphasis on the Interferon sensitivity determining region (ISDR) region of NS5A. While the sequencing of HCV is generally constricted to certain regions of the HCV genome there is little consistency in the positioning of sequencing primers, with the exception of a few highly referenced manuscripts. This study demonstrates the heterogeneity of HCV sequencing, providing a comprehensive database of previously published primer sets to be utilised in future sequencing studies

High Erk-1 activation and Gadd45a expression as prognostic markers in high risk pediatric haemolymphoproliferative diseases

Studies on activated cell-signaling pathways responsible for neoplastic transformation are numerous in solid tumors and in adult leukemias. Despite of positive results in the evolution of pediatric hematopoietic neoplasias, there are some high-risk subtypes at worse prognosis. The aim of this study was to asses the expression and activation status of crucial proteins involved in cell-signaling pathways in order to identify molecular alterations responsible for the proliferation and/or escape from apoptosis of leukemic blasts. The quantitative and qualitative expression and activation of Erk-1, c-Jun, Caspase8, and Gadd45a was analyzed, by immunocytochemical (ICC) and western blotting methods, in bone marrow blasts of 72 patients affected by acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (ALL) and stage IV non-Hodgkin Lymphoma (NHL). We found an upregulation of Erk-1, Caspase8, c-Jun, and Gadd45a proteins with a constitutive activation in 95.8%, 91.7%, 86.2%, 83.4% of analyzed specimens, respectively. It is worth noting that all AML patients showed an upregulation of all proteins studied and the high expression of GADD45a was associated to the lowest DFS median (p = 0.04). On univariate analysis, only Erk-1 phosphorylation status was found to be correlated with a significantly shorter 5-years DFS in all disease subgroups (p = 0.033) and the lowest DFS median in ALL/NHL subgroup (p = 0.04). Moreover, the simultaneous activation of multiple kinases, as we found for c-Jun and Erk-1 (r = 0.26; p = 0.025), might synergistically enhance survival and proliferation potential of leukemic cells. These results demonstrate an involvement of these proteins in survival of blast cells and, consequently, on relapse percentages of the different subgroups of patients