APC Hypermethylation as a Prostate Cancer Indicator

APC Hypermethylation as a Prostate Cancer Indicator Evaluation of APC Hypermethylation From Serum Samples as an Indicator for Presence of Prostate Cancer and Higher Gleason Score Abstract Purpose In this prospective study it is aimed to identify role of hypermethlation analysis of APC gene from serum samples on detection of prostate cancer in patients with PSA level 2,5-10 ng/ml and its correlation with GS of the patients Materials and Methods Data from 142 patients underwent prostate biopsy with PSA level of 2,5-10 ng/ml was analyzed. Hypermethlation analysis of promoter region of APC gene from serum samples was done via methlation specific PCR. Patients were grouped as positive or negative depending on results of methylation analysis and compared for presence of prostate cancer and GS distrubution. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of methylation analysis were calculated. P value of 0.05 is used for statistical significance. Results There were 48 patients (33.8%) in methylation (+) group and 94 patients (66,2%) in methylation (-) group. Prostate cancer was detected in 43 of 142 (30,2%) patients after the first biopsy and 32 of these patients were in methylation (+) group. sensitivity and specificity of methylation analysis were 74,4% and 83,8%. PPV and NPV were 66.6% and 88.30 %. When GS distrubutions of the two groups analyzed patients in methylation (+) group were more likely to have higher GS compared to methylation (-) group(p=0,03). Conclusions Hypermethlation analysis APC gene from serum samples have shown promising results with its correlation with prostate cancer and higher GS. Further studies on prediciton of survival with long term follow up are needed to verify its value on prediction of survival Introduction Prostate Cancer (PCa) is one of the most important medical problems of the male population as it is the most common solid neoplasm in Europe having the highest incidence of 202,100 cases in 2004 (1). It is not only common but also a dangerous disease that it is the second most common cause of cancer death in men (2). These findings prove the importance of detection of PCa. However identifiying patients with clinically significant disease is an important issue, that is to treat patients that really need treatment while sparing ones that have silent disease. The European Randomized Study of Screening for Prostate Cancer (ERSPC) trial concluded that 1,410 men would need to be screened and 48 additional cases of PCa would need to be treated to prevent one death from PCa (ERSPC-3). These findings point out that current diagnostic tools are inadequate for screening and detection of clinically significant PCa (3). Epigenetic studies evolved in the era of urooncology in the last decade and DNA methylation is widely studied for certain urological malignancies especialy for PCa (4). Hypermethylation of promoter regions of tumor supresor genes results in gene silencing. This epigenetic change in APC gene promoter regions has been shown in cancerous and precancerous prostate tissues,serum, and urine of PCa patients and sensitivity and specificity rates of 27-100% and 50-100% have been reported (5,6). However the current literature lacks studies that investigate corrolation of results of methylation analysis from serum samples and clinicopathologic properties (grade and stage of disease) of patients. This point should be verified to identify applicability of this method in clinical practice. In this prospective study it is aimed to identify role of hypermethlation analysis of promoter region of APC gene from serum samples on detection of prostate cancer in a population of patients with PSA level 2,5-10 ng/ml and its correlation with Gleason score (GS) of the patients. Patients and Methods. This study was approved by ethical committe of Turkish Ministry of Health. All patients signed informed consent. The patients admitted to urology outpatient clinics of Ankara University Schol of Medicine with PSA level of 2,5-10 ng/ml and who were decided to undergo prostate biopsy were involved in the study. Serum samples were obtained from all patients prior to biopsy and 12 cores transrectal ultrasound guided prostate biopsy was performed by a single radiologist. Age, serum PSA level, prostate biopsy results were recorded and patients were followed regardless of the results of methylation analysis. Rebiopsy was suggested to patients with benign histology and persistant elevated PSA levels 6 months after the first biopsy. Patients with biopsy results of PCa were evaluted for staging and treated based on their stage. GS of the patients were evaluated in 3 groups GS:6 , GS: 7 and GS:8-10. The patients were grouped as positive (methylation +) and negative (methylation -) based on methylation analysis results and compared with results of prostate biopsy. The primary outcome measures were sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of methylation analysis. As a secondary outcome measure the results were also compared with the GS groups. Genetic analysis DNA extraction Genomic DNA was extracted from peripheral blood samples using a PureLink â„ ¢ Genomic DNA Mini Kit (Invitrogen â„ ¢, catalog No. K1820-02) according to the manufacturer’s instructions and were stored at -20 °C until modification and MSPCR amplification. DNA integrity and quantity was assessed with agarose gel electrophoresis and spectrophotometry, respectively. For this purpose, 1% agarose gel was used and 260 nm and 280 nm optical density ratio values (OD260/OD280) were determined. Bisulfite Modification and Methylation-Specific PCR of APC and GSTPI promoters The genomic DNA was modified by sodium bisulfite treatment using the CpGenomeTM DNA Modification Kit (S7820, Chemicon, Temecula, CA). The DNA promoter methylation status of APC and GSTPI genes were investigated by MSPCR using the specific primers to methylated and unmethylated promoters. The methylated and unmethylated control DNA were also subjected to bisulfite DNA modification and PCR analysis, and serves as positive controls for methylated and unmethylated DNA respectively. H2O was used as negative control for the PCR reaction. The PCR reaction was started with denaturation at 95 °C for 13 min, followed by 40 cycles at 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s; and 72 °C for 5 min. Statistical analysis Continuous variables were compared using the t –test. Categorical variables were compared by the chi-square test..Sensitivity, specificity, PPV and NPV were calculated. Results Prostate biopsy was performed in 142 patients. The mean age of the patients were 68,3  ± 8,2 and mean ages of the methylated and unmethylated groups were not significantly different (67,8 vs 68,6 , p=0,47). There were 48 patients (33.8%) in group 1 and 94 patients (66,2%) in group 2. Prostate cancer was detected in 43 of 142 (30,2%) patients after the first biopsy and 32 of these patients were in methylation (+) group and 11 of them were in methylation (-) group. Based on these results, sensitivity and specificity of methylation analysis were 74,4% (95% CI: 58.83 % 86.46 %) and 83,8% (95% CI: 75.08 % to 90.47 %). PPV and NPV were 66.6% (95% CI: 51.59 % to 79.59 %) and 88.30 % (95% CI: 80.02 % to 94.00 %). When GS distrubutions of the two groups analyzed patients in methylation (+) group were more likely to have higher GS compared to methylation (-) group (p=0,041) and results are summarized in table 1. Second biopsy was performed in 12 (6 patients in each group) patients totally. At these second biopsies, PCa was observed in 4 patients, and all of them were in methylation (+) group. Based on the total results of first and second biopsies, sensitivity and specificity , PPV and NPV were reevaluated. Sensitivity and specificity of methylation analysis were 76.6 % (95% CI: 61.97 % to 87.68 %) and 87.3 % (95% CI: 78.97 % to 93.29 %). PPV and NPV were 75.0 % (95% CI: 60.4 % to 86.3 %) and 88.30 % (95% CI: 80.02 % to 94.00 %). Discussion Epigenetic alterations, such as hypermethylation of prompter regions of tumor supressor genes are associated with many human tumor types and this type of alteration have also emerged as markers for cancer risk assessment, cancer diagnosis, and therapy monitoring in several different types of cancer (7-10). APC is a well-characterized tumor suppressor gene and it downregulates Wnt signaling by targeting the transcriptional coactivator beta-catenin for proteasomal degradation and this prevents its association with the nuclear transcription factor Tcell factor/lymphoid enhancer factor (11). The activation of Wnt pathway leads to deregulation of cell proliferation and differentiation and is a common feature of many human cancers including prostate cancer (12-14). Detection of clinically significant PCa at an early stage is still a great challenge and current imaging modalities are often not able to detect a tumor and biopsies may miss the tumor focus. Role of gene promoter hypermethylation in prostate carcinogenesis, has been studied recently and promising results to improve PCa diagnosis has been established (5-7). In this study methylation analysis of promoter region of APC gene is performed prior to prostate biopsy in serum samples in order to evaluate the the role of this analysis on prostate cancer detection. Serum samples were obtained from all patients prior to biopsy and PCa was detected in siginificantly higher rates in patients with positive methylation analysis. In their study Yoon et al. showed that APC methylation levels were significantly higher in PCa patients than in benign prostate hyperplasia patients. Moreover, APC hypermethylation was not only associated with the increased incidence of PCa but was also positively correlated with increased GS (15). This has also been shown in other studies indicating APC hypermethylation as a reliable predictor of PCa and of its aggressive feature (12,13). Similarly, in our study, methylation positive patients were found to have higher GS and GS>7 was not observed in any patient with negative methylation analysis. Sensitivity and specificity of APC hypermethylation has been studied on prostate tissue samples and high sensitivity (89.3%) and specificity (98.1%) rates have been reported (15). In our study, sensitivity of 74,4% and specificity of 83,8% was found. These results are slightly lower compared to results of Yoon et al. and this may be due to source of DNA, that is higher rates might be reached when analysis is made through prostate tissue samples compared to serum samples. Additionally the results may be effected from the detection method, including the primer design, reagents, detectors, equipment, and protocols, all of which potentially influence the sensitivity and specificity. The important point of our study is making the analysis from serum samples to evaluate its role in clinical practice. The lack of such studies has been emphasized in previous trials conducted on tissue samples (15). Repat biopsies is an important issue in PCa diagnosis in the study of Trock et al. (16) APC methylation provided a very high NPV with a low percentage of false negatives, in a clinical cohort of men undergoing repeat biopsy.In our study, repeat biopsy was performed in 12 patients (6 patients in each group) and all of the 4 patients with PCa detected in second biopsy were in the methylation positive group. Our study involves low number of repeat biopsies therefore further studies with higher number of patients evaluating role of hypermethylation analysis from body fluids should be conducted on the topic of repeat biopsies. Conclusion Prediction of clinical significance of PCa is of clinicial importance and hypermethlation analysis of promoter region of APC gene from serum samples have shown promising results with its correlation with prostate cancer and higher GS. Further studies on prediciton of survival with long term follow up are needed to verify its value on prediction of survival. Conflict of interest: None of the authors have any conflict of interest Ethical standarts: The study was approwed by ethical committe of Turkish Ministry of Health. References 1. Boyle P, Ferlay J. Cancer incidence and mortality in Europe 2004. Ann Oncol 2005 Mar;16(3):481-8. 2. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008 Mar-Apr;58(2):71-96. 3. Schrà ¶der FH, Hugosson J, Roobol MJ, et al.Screening and prostate-cancer mortality in a randomized European study. N Engl J Med 2009 Mar 26;360(13):1320-8. 4. Veeck J, Esteller M J Mammary Gland Biol Neoplasia. Breast cancer epigenetics: from DNA methylation to microRNAs. J Mammary Gland Biol Neoplasia. 2010 Mar;15(1):5-17. 5. Kang GH, Lee S, Lee HJ, et al. Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia. J Pathol 2004; 202: 233–40 6. Yegnasubramanian S, Kowalski J, Gonzalgo ML et al. Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res 2004; 64: 1975–86 7. Moritz R, Ellinger J, Nuhn P, Haese A, Muller SC, Graefen M, Schlomm T, Bastian PJ. DNA hypermethylation as a predictor of PSA recurrence in patients with low- and intermediate-grade prostate cancer. Anticancer Res 2013 Dec;33(12):5249-54. 8. Kim WJ, Kim YJ. Epigenetic biomarkers in urothelial bladder cancer. Expert Rev Mol Diagn 2009;9:259-69. 9. Hoque MO. DNA methylation changes in prostate cancer: current developments and future clinical implementation. Expert Rev Mol Diagn 2009;9:243-57. 10. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer 2005;5:223-31. 11. Rai K, Sarkar S, Broadbent TJ, et al. DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC. Cell 2010;142: 930-42. 12. Jeronimo C, Henrique R, Hoque MO, et al. A quantitative promoter methylation profile of prostate cancer. Clin Cancer Res 2004;10:8472-8. 13. Bastian PJ, Ellinger J, Wellmann A, et al. Diagnostic and prognostic information in prostate cancer with the help of a small set of hypermethylated gene loci. Clin Cancer Res 2005;11:4097-106. 14. Gavert N, Ben-Zeev A. beta-Catenin signaling in biological control and cancer. J Cell Biochem 2007;102:820-8. 15. Hyung-Yoon Yoon, Young-Won Kim, Ho Won Kang, et al. Pyrosequencing Analysis of APC Methylation Level in Human Prostate Tissues: A Molecular Marker for Prostate Cancer. Korean J Urol. 2013 Mar;54(3):194-198. 16. Trock BJ, Brotzman MJ, Mangold LA, et al. Evaluation of GSTP1 and APC methylation as indicators for repeat biopsy in a high-risk cohort of men with negative initial prostate biopsies. BJU Int. 2012 Jul;110(1):56-62 Table 1. GS distrubutions of the two groups

Of Mice and Men Emotions

In the following essay I will be writing about and exploring what emotions Steinbeck conveys in his novella ‘of mice and men’. The novella was the second of Steinbeck’s ‘dustbowl’ trilogy. These books were based on Steinbeck’s own first hand experience against the back drop of depression-era America where he became concerned with the exploitation of migrant workers and exposing the institutionalized prejudices ( racism, ageism and sexism) of the migrant workers themselves, hence the trilogy.The novella shows an effective state of consciousness in which joy, fear, sorrow and hate is experienced as distinguished from cognitive and volitional state of consciousness. The main theme/ emotion that are conveyed are loneliness. Steinbeck uses many different linguistic language techniques to depict this. These include: adverbs, speech verbs, imperative verbs, adjectives, abstract nouns, rhetorical questions and many more. However the opening of the nove lla displays a bond of friendship and mutual understanding shown by George and lennie.Steinbeck writes â€Å"he pulled his hat down a little more over his eyes the way George’s hat was. † Our immediate interpretation is that Lennie looks up to George as a role model, the quote â€Å"way George’s hat was† infers that Lennie looks up to George as someone who can guide him and teach him much like a younger brother’s attitude would be towards an older sibling. Steinbeck writes â€Å"Lennie looked timidly over to him. â€Å"George? † â€Å"Yeah, what ya want? † â€Å"Where we goin’ George? †Ã¢â‚¬ ¦ â€Å"So you forgot that already did you? † this implies that without George, Lennie would be hopeless and would get no where because he would always be lost. When Lennie says â€Å"where we goin’† he sounds slightly worried, this will be because he knows that George will get angry at him because he is constan tly having to remind him on the objective. The word â€Å"already† suggests that Lennie is always forgetting things and George is getting more and more fed up. The adverb â€Å"timidly† implies that George often gets angry with Lennie and now Lennie has learnt to approach him differently when he is angry so he won’t get as upset.This implies their friendship is strong as they know how to approach each other showing an almost brotherly relationship. This also depicts Georges frustration and anger towards lennie however, unlike other migrant workers at the time, it does not come from the face the he is lonely and isolated but because of the fact the he someone there for him that he can talk to. George says â€Å"where the hell would you go? † the fact that this question is rhetorical shows that George knows that it’s an empty threat.However the taboo language â€Å"hell† gives the impression that George is slightly apprehensive and not entirel y convinced. This shows the worry and compassion that he has for lennie, and also showing that not only can lennie not cope without George but George cannot cope without lennie. Steinbeck’s purpose was to show George and Lennie’s bond of friendship and how unlike other migrant workers, their negative emotions does not stem from the fact that they are lonely and isolated but the fact there is a companionship and the sense of a bond between them, showing how strong their bond of friendship really is.

Best Learning Resources For Self-Studying French

If you dont want to or cannot study French with a tutor,  in a class or in immersion, youll be going it alone. This is known as self-study. There are ways to make self-study effective, but its essential that you pick the right self-study  method for you. After all, you want to spend your time doing something that actually works. So spend some time analyzing whats out there, and dont just take the first self-study path that comes to your attention. Audio Training Is Essential If you want to communicate in French (and not only pass exams or read in French), learning with audio is a must. Theres a huge difference between book French and spoken French, and traditional methods will not prepare you for the way French people actually speak today. French Language  Books French-language books such as childrens books, bilingual books, and audiobooks are a great and relatively inexpensive way to improve your French, in conjunction with audio courses. With Amazon delivering to your doorstep, its easy to order French-language books these days. Hard-copy paper books are still the best way to train on a specific point of grammar  and to do exercises. For all the rest, youll need audio. Childrens Books Reading Le Petit Prince is, for more advanced students, a wonderful way to expand your vocabulary. It is a myth that all French-language childrens books are easy. They are not. Children books are easier than most French books written for the French because they use short sentences, but the language is some French childrens books can be quite difficult. Consider the language used in the Dr. Seuss books. They definitely would not be an easy read for a beginner in English. Bilingual Books Most bilingual-book  series  are taken from free-copyright books and translated into English. They were not typically books written for students. So they are still very difficult and will often feature older French vocabulary and expressions: Find out  when your book  was written, and take this into account when learning the vocabulary. French Audiobooks and Audio Magazines Both of these are a fantastic resource, even though most have been created for the French student. Much of what has been developed for the French is going to be difficult for a beginning or intermediate student of French, so difficult that they could be overwhelming and discouraging. There are, however,  audio magazines that can be used to good effect by beginning and intermediate students of  French. Among the better audio magazines are  Think French,  Bien Dire,  and  Fluent French Audio (although the latter is probably better suited to high-intermediate students). There are also level-adapted French audiobooks and audio novels  with English translations,  such as the À Moi Paris series and Une Semaine à   Paris. French Audio Courses French audio courses are the ideal tool for the self-learner. A good audio course should teach you vocabulary and grammar, if possible in context, and, of course, pronunciation. It should be fun to use, direct you through a well-proven learning path  and nurture your self-confidence. Because they involve a lot of work, these courses are usually quite expensive, so look for a 100 percent money-back guarantee disclaimer, a trial period or extensive samples. Among the good French audio courses:  Michel Thomas,  Assimil,  and French Today. Rosetta Stone language books are a great, fun tool to develop your vocabulary, but they are very light on grammar. This may be fine for other languages, but it is a true problem for French. Do Your Research and Find Whats Best for You There are, of course, still more  methods to learn French. Do your research and find out what methods best fit your needs, goals, time and budget. You wont be sorry.

The Effects Of Climate Change On Our Lives - 1037 Words

Global climate change has been a highly debated topic for decades. Climate change is a change in usual weather patterns found globally or in certain regions. This could be a change in a place’s normal temperature for its time or it could be how much rain a place gets in a year. Normally, climate change is considered normal on planet Earth. Things evolve and change all the time on our home, ever since it formed. Mother nature always goes through different phases. Our planet was a lot different millions of years ago, it was even different decades ago compared to today! It all ranges from culture to society to technology to everything in general. Change is just a part of everyday life. However, abnormalities such as rising global temperatures, melting ice caps and ocean acidification can pose a threat to humanity’s way of living. Our current society can thrive because of Earth’s conditions today and changes, even small ones, can deeply affect us. Our planet, Earth, has evolved alongside with our species. Researchers theorize that humanity is causing massive negative changes to our planet. Many sources, backed up by many scientists, claim that global climate change is real. Unfortunately, the majority of the general public still does not know whether it is actually real or not. This is because of greed and politics; people with higher authority help attribute to claiming that it is a hoax to benefit their own agendas. Whether or not anyone believes global climate change is realShow MoreRelatedClimate Change Is A Serious Problem1123 Words   |  5 PagesClimate Change is a Serious Problem to the Planet â€Å"Climate change is happening, humans are causing it, and I think it is perhaps the most serious environmental issue facing us.† This quote from Bill Nye illustrates the serious nature of climate change. Climate change is the most serious issue that is plaguing the world. Global temperatures have been increasing in recent years and it is clear that our climate is changing. Climate change is the change of temperature and weather in our environmentRead MoreClimate Change Is A Real Threat1105 Words   |  5 PagesClimate Change is a Real Threat â€Å"Climate change is happening, humans are causing it, and I think it is perhaps the most serious environmental issue facing us.† This quote from Bill Nye illustrates the serious nature of climate change. Climate change is the most serious issue that is plaguing the world. Global temperatures have been increasing in recent years and it is clear that our climate is changing. Climate change is the change of temperature of our environment. A majority of scientists suspectRead MoreGlobal Climate Change And Global Warming1054 Words   |  5 Pagespart of our daily lives and over the years we have seen energy consumption rates raise significantly. It really isn t much of a secret that global climate change is happening. From the melting of the polar ice caps, to record severe temperatures, rise in natural disasters, rise in pollution, greater number of vector-borne and water borne illnesses, and much more. Unless there is something done to change the current technology being us ed to provide energy to the human population global climate changeRead MoreThe Effects Of Climate Change On People s Habitat And Safety790 Words   |  4 PagesIntroduction Climate changing is a serious issue for humanity and all the living creature on the earth. These days, more and more scientists and climatologists indicate the climate and our environment suffer the huge change. For all the humanity, the influences become more and more significant. Climate’s change definitely impacts People’s habitat and safety. Why is the climate important to us? Because of the climate change, people will live in danger. Climate will affect the whole ecosystems, notRead MoreProblems Related to Climate Change Essay examples1267 Words   |  6 PagesClimate change has been a topic of concern for humanity over the past two decades. In my opinion, it is the best example of the consequences of our actions on our planet. Most countries have taken some efforts and precautions to save the Earth from experiencing further damage. Decline of forests and global warming has affected our lives in more ways than one and in the future, the Earth and its inhabitants might see a more crucial period in their lives, if we neglect this issue. At this point inRead MoreClimate Change Is A Change Of Weather Patterns That Lasts For A Long Duration Essay1372 Words   |  6 PagesClimate Change is a change of weather patterns that lasts for a long duration. Climate change can be caused by the ‘greenhouse effect’. Humans create greenhouse gases from burning fossil fuels, electricity production and more. These gases trap the sun’s heat in the atmosphere and increase the earth’s overall temperature. This process is also called Global Warming which can cause more natural disasters like floods and droughts. Climate change is an existent global issue that started since 1880 whenRead MoreEarth Science : Critical Thinking Exercise Questions1702 Words   |  7 Pageswhy it is a conflict. The climate is changing and this comes with the result of temperatures rising, polar ice caps melting, and water levels rising. Furthermore, if these types effects continue without being checked the end result for human and animal life on earth could be catastrophic. With this in mind, there are several people that have argued that this change has occurred as of natural causes. The people from school of thought believe that the current climate change is a result of the naturalRead MoreClimate Change Affects People s Habitat And Safety775 Words   |  4 PagesClimate change is a serious issue for humanity and all the living creatures on the earth. These days, more and more scientists and climatologists indicate that the climate and our environment are suffering from huge changes. For all humanity, the influences become more and more significant. Climate change affects people’s habitat and safety. Why is climate important to us? Because of climate change, people may live in an unfavorable situation. Climate may affect whole ecosystems, not only human,Read MoreIs Global Climate Change Man Made? Global Temperature?1310 Words   |  6 Pagesglobal climate change man-made? Global temperature has been changing for the past couple of decades. This leaves researchers to believe that mankind is to blame for this abrupt change. Life on earth depends upon the average climate that has been around for thousands of years. Without this favorable climate, life on earth will be unsustainable. Those who argue that climate change is not affected by humans state that the emissions humans put into the atmosphere are too small to have an effect on theRead MoreEssay about The Impact of Climate Change on Africa1479 Words   |  6 Pages Scientists, economists, and policy makers all agree the world is facing threat from climate warming. Climate warming is caused by excess greenhouse gases such as carbon dioxide which are heat trapping gasses. Human use of fossil fuels i s a significant source of these gasses. When we drive our cars, heat our homes with oil or natural gas, or use electricity from coal fired power plants, we contribute to global warming. Including any loss of trees or forests also contributes, considering trees