Application of card blot as a semi-quantitative detection genes expression

The purpose of this research is to invent a break-trough methods of genes expression which are based on RNA-DNA hybridization, through a sensitive, efficient and cost effective way. In this research, biotin labeled probe were used to measure bc1-2 and c-myc niRNA genes expressions of peripheral blood lymphocyte from patients of Infectious Mononucleosis (IM), Nasopharyngeal Carcinoma (NPC) and Non-diagnostic asymptomatic (NDA). The blotting process was brought about to attach the total RNA sample at the nitrocellulose membrane on card blot through vacuum process, continued by washing and baking at 80 ° C degrees for a period of 1 hour. Hybridization was undergone using bc1-2 and c-myc genes DNA fragment biotin labeled probe. Indirect detection system utilizing Streptavidin-Avidin - Horse Raddish Peroxidase (SA-HRP) to bind the biotin, was amplified with Biotynil-Tyramide (BT) and Diaminobenzidine (DAB) chromogenic substrate that exists in Tyramide Signal AmplificationTM-Indirect (ISH). Result of this research showed that card blot can be used in detecting gene on the RNA level with a sum of 20 ng/sample rapidly and with an effective cost. Keywords: card blot dot blot â TS

Interleukin-4 and interferon- γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma

Background: Cytokines have been suggested to participate in the pathogenesis of infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC). METHODS: Serum levels and gene expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) were assessed by immunologic and PCR assays, respectively in patients with Epstein-Barr virus (EBV)-associated IM and NPC and EBV nega-tive controls. RESULTS: The serum levels of IFN- γ were elevated, but those of IL-4 were decreased in IM and NPC patients as compared with those of the control group (p < 0.05). CONCLUSIONS: These results suggest that serum levels of IFN-γ may be predominant over those of IL-4 during the cours of IM and NPC

CD4, CD8 and MHC Class I Expression in Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma: An Immunohistochemical Study

Aim: The exact immunopathogenesis of Epstein-Barr virus (EBV)- associated nasopharyngeal carcinoma (NPC) remains unclear. The aim of the present study was to assess the expression of CD4, CD8, and MHC class I molecules in NPC. Method: Biopsies were obtained from patients with NPC as well as the Epstein Barr virus (EBV)-seronegative patients as a control. Nasopharyngeal carcinoma patients were classified using the World Health Organization (WHO) pathological assessment and clinical stag- ing of NPC. The expression of CD4, CD8, and MHC class I in the biop- sies were assessed immunohistochemically. Result: The results showed that the number of CD4 positive, CD8 pos- itive, and MHC class I positive cells in NPC patients were higher than those in EBV-negative subjects (p<0.05). The number of these positive cells in NPC patients with WHO Type II or early clinical stage was not significantly differences with those with WHO Type III or late clinical stage, respectively (p>0.05). No statistical differences between the number of CD4 positive and CD8 positive cells in NPC patients could be found (p>0.05). Conclusion: The results of the present study suggest, therefore, that the expression of CD4, CD8 and MHC class I molecules may not be as- sociated with the pathologic classification and clinical staging of NPC and that the CD4:CD8 ratio in nasopharyngeal carcinoma may indicate decreased functions of these infiltrating T cell subsets. Key words: CD4; CD8; MHC class I; NP

Short Communication Interleukin-4 and interferon-γ levels in Epstein-Barr virus-associated infectious mononucleosis and nasopharyngeal carcinoma

Abstract BACKGROUND: Cytokines have been suggested to participate in the pathogenesis of infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC)

Epigenetic markers for early detection of nasopharyngeal carcinoma in a high risk population

<p>Abstract</p> <p>Background</p> <p>Undifferentiated nasopharyngeal carcinoma (NPC) is strongly related to Epstein-Barr virus (EBV) infection, allowing aberrant antibodies against EBV and viral DNA load as screening tools in high risk populations. Methylation analysis in the promoter of tumor suppressor genes (TSGs) may serve as a complementary marker for identifying early cases. This study determined methylation status of multiple TSGs and evaluated whether it may improve early detection.</p> <p>Methods</p> <p>Nasopharyngeal brushings were taken from 53 NPC patients, 22 high risk subjects and 25 healthy EBV carriers. Corresponding NPC paraffin tissue was included. DNA was bisulfite-modified preceding analysis by methylation-specific PCR (MSP). Ten TSGs were studied.</p> <p>Results</p> <p>NPC paraffin and brushing DNA revealed an 81.8% concordance so that MSP analysis was done using either one of both specimens. NPC samples showed methylation for individual TSGs (DAPK1 79.2%, CDH13 77.4%, DLC1 76.9%, RASSF1A 75.5%, CADM1 69.8%, p16 66.0%, WIF1 61.2%, CHFR 58.5%, RIZ1 56.6% and RASSF2A 29.2%). High risk individuals, having elevated EBV IgA and viral load, showed high frequency of methylation of CDH13, DAPK1, DLC1 and CADM1, but low frequency of methylation of p16 and WIF1 and undetectable methylation of RASSF1A, CHFR, RIZ1 and RASSF2A. Healthy subjects showed similar patterns as high risk individuals. A combination of RASSF1A and p16 gave good discrimination between NPC and non-NPC, but best results were combined analysis of five methylation markers (RASSF1A, p16, WIF1, CHFR and RIZ1) with detection rate of 98%.</p> <p>Conclusion</p> <p>Multiple marker MSP is proposed as a complementary test for NPC risk assessment in combination with EBV-based markers.</p

Mutational heterogeneity between different regional tumour grades of clear cell renal cell carcinoma

Only a limited number of studies have explored the possible associations between tumour grade and mutated genes in clear cell renal cell carcinoma (ccRCC), and we set out to investigate this further using a multiple sampling and next generation sequencing (NGS) approach in a series of ccRCCs. Multiple regions were sampled from formalin-fixated paraffin-embedded ccRCC tumour blocks from seven patients. In 27 samples from six patients, we performed targeted NGS using a custom 42-gene panel based on the most frequently mutated genes in ccRCC reported in public databases. In four samples from the seventh patient, we performed whole exome sequencing (WES) and array comparative genomic hybridisation for detection of copy number variants (CNVs). Mutated genes and the tumour grades of the samples in which they had been identified were compared both within and between all individual tumours. CNVs were compared across all samples from patient 7. We identified clear genetic heterogeneity within and across tumours, but VHL mutations were seen in all patients. Looking across all samples, we identified eleven genes that were only mutated in samples with one particular tumour grade. However, these genes were never mutated in all samples with that tumour grade. Increasing chromosomal instability corresponded with increasing tumour grade, but we observed minimal association between tumour grade and total mutational load in the WES data. Our study confirms the genetic heterogeneity and tumour grade heterogeneity of ccRCC. Although a relatively small number of samples was analysed, genes were identified that could potentially be specific, though insensitive, markers of higher ccRCC tumour grades

Knowledge of general practitioners about nasopharyngeal cancer at the Puskesmas in Yogyakarta, Indonesia

<p>Abstract</p> <p>Background</p> <p>Nasopharyngeal carcinoma (NPC) is one of the leading causes of cancer death in Indonesia. At initial diagnosis, 80% of the patients present with advanced stage disease. In Indonesia, primary medical care is generally provided by the health care centres; named Puskesmas. The lack of knowledge of various aspects of NPC of the General practitioners (GPs) working in these centers might contribute to the diagnostic delay. The aim of this study was to assess the knowledge of these GPs on different aspects of NPC including symptoms, risk factors and incidence.</p> <p>Methods</p> <p>One hundred six GPs in the Puskesmas in the Yogyakarta province were subjected to a questionnaire on different aspects of NPC based on literature and interviews with Head and Neck Surgeons.</p> <p>Results</p> <p>All GPs approached participated and in total 106 questionnaires were filled in. All participants were aware of NPC as a disease and 89% confirmed that it is a serious problem in Indonesia. However, 50% of the participants believed NPC has a low incidence in their region. The question on early symptoms gave a mean 4.2 answers of which 50% were incorrect.</p> <p>The GPs provided a total of 318 answers when asked for the risk factors of NPC, 75% of which were incorrect. Fifty seven GPs (54%) stated that they did not receive sufficient education on NPC at the university and insufficient knowledge was gained during daily practice. Ninety-two percent of the GPs were interested in additional education, preferably in form of lectures, meetings or folders.</p> <p>Conclusion</p> <p>This study revealed that GPs in the Puskesmas in Yogyakarta lack knowledge on all aspects of NPC. This is an important finding as NPC is endemic in Indonesia and the Puskesmas are the institutions which provide primary medical health care in the country. Further education of the GPs in these endemic areas could be a first step to increase the rate of early detection. Therefore, we suggest 1) to conduct a medical awareness campaign for GPs on the most important subjects concerning NPC, and 2) as soon as NPC awareness among GPs has risen, provide further education on the risk factors, the early symptoms and the incidence, education to the community. We propose to extend this study to other areas in Indonesia (i.e. Jakarta, Surabaya, Central Java), using models that have been developed in Yogyakarta.</p

Effectiveness of a multicentre nasopharyngeal carcinoma awareness programme in Indonesia

Objective: To evaluate the effectiveness of a nasopharyngeal carcinoma (NPC) awareness programme on the short-term and long-term improvement of knowledge and referral of patients with NPC by primary healthcare centres (PHCCs) staff in Indonesia. Design: The NPC awareness programme consisted of 12 symposia including a Train-The-Trainer component, containing lectures about early symptoms and risk factors of NPC, practical examination and the referral system for NPC suspects. Before and after training participants completed a questionnaire. The Indonesian Doctors Association accredited all activities. Participants: 1 representative general practitioner (GP) from each PHCC attended an NPC awareness symposium. On the basis of the Train-The-Trainer principle, GPs received training material and were obligated to train their colleagues in the PHCC. Results: 703 GPs attended the symposia and trained 1349 staff members: 314 other GPs, 685 nurses and 350 midwives. After the training, respondents’ average score regarding the knowledge of NPC symptoms increased from 47 points (of the 100) to 74 points (p<0.001); this increase was similar between symposium and Train-The-Trainer component (p=0.88). At 1½ years after the training, this knowledge remained significantly increased at 59 points (p<0.001). Conclusions: The initial results of this NPC awareness programme indicate that the programme effectively increases NPC knowledge in the short and long term and therefore should be continued. Effects of the improved knowledge on the stage at diagnoses of the patients with NPC will still need to be scrutinised. This awareness programme can serve as a blueprint for other cancer types in Indonesia and for other developing countries

Primary treatment results of Nasopharyngeal Carcinoma (NPC) in Yogyakarta, Indonesia

INTRODUCTION Nasopharyngeal Carcinoma (NPC) is a major health problem in southern and eastern Asia. In Indonesia NPC is the most frequent cancer in the head and neck area. NPC is very sensitive to radiotherapy resulting in 3-year disease-free and overall survival of approximately 70% and 80%, respectively. Here we present routine treatment results in a prospective study on NPC in a top referral; university hospital in Indonesia. METHODS All NPC patients presenting from September 2008 till January 2011 at the ear, nose and throat (ENT) department of the Dr. Sardjito General Hospital, Universitas Gadjah Mada, Yogyakarta, Indonesia, were possible candidates. Patients were included if the biopsy was a histological proven NPC without distant metastasis and were assessed during counselling sessions prior to treatment, as being able to complete the entire treatment. RESULTS In total 78 patients were included for treatment analysis. The median time between diagnosis and start of radiotherapy is 120 days. Forty-eight (62%) patients eventually finished all fractions of radiotherapy. The median duration of the radiotherapy is 62 days for 66 Gy. Median overall survival is 21 months (95% CI 18–35) from day of diagnosis. CONCLUSION The results presented here reveal that currently the treatment of NPC at an Indonesian hospital is not sufficient and cannot be compared to the treatment results in literature. Main reasons for these poor treatment results are (1) a long waiting time prior to the start of radiotherapy, (2) the extended overall duration of radiotherapy and (3) the advanced stage of disease at presentation.Maarten A. Wildeman, Renske Fles, Camelia Herdini, Rai S. Indrasari, Andrew D. Vincent, Maesadji Tjokronagoro, Sharon Stoker, Johan Kurnianda, Baris Karakullukcu, Kartika W. Taroeno- Hariadi, Olga Hamming-Vrieze, Jaap M. Middeldorp, Bambang Hariwiyanto, Sofia M. Haryana, I. Bing Ta

The HOX-11 gene is expressed in leukemia cells from myeloid lineage

[no abstract available