Frequent Promoter Hypermethylation of the APC and RASSF1A Tumour Suppressors in Parathyroid Tumours

BACKGROUND: Parathyroid adenomas constitute the most common entity in primary hyperparathyroidism, and although recent advances have been made regarding the underlying genetic cause of these lesions, very little data on epigenetic alterations in this tumour type exists. In this study, we have determined the levels of promoter methylation regarding the four tumour suppressor genes APC, RASSF1A, p16(INK4A) and RAR-beta in parathyroid adenomas. In addition, the levels of global methylation were assessed by analyzing LINE-1 repeats. METHODOLOGY/PRINCIPAL FINDINGS: The sample collection consisted of 55 parathyroid tumours with known HRPT2 and/or MEN1 genotypes. Using Pyrosequencing analysis, we demonstrate APC promoter 1A and RASSF1A promoter hypermethylation in the majority of parathyroid tumours (71% and 98%, respectively). Using TaqMan qRT-PCR, all tumours analyzed displayed lower RASSF1A mRNA expression and higher levels of total APC mRNA than normal parathyroid, the latter of which was largely conferred by augmented APC 1B transcription levels. Hypermethylation of p16(INK4A) was demonstrated in a single adenoma, whereas RAR-beta hypermethylation was not observed in any sample. Moreover, based on LINE-1 analyses, parathyroid tumours exhibited global methylation levels within the range of non-neoplastic parathyroid tissues. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that APC and RASSF1A promoter hypermethylation are common events in parathyroid tumours. While RASSF1A mRNA levels were found downregulated in all tumours investigated, APC gene expression was retained through APC 1B mRNA levels. These findings suggest the involvement of the Ras signaling pathway in parathyroid tumorigenesis. Additionally, in contrast to most other human cancers, parathyroid tumours were not characterized by global hypomethylation, as parathyroid tumours exhibited LINE-1 methylation levels similar to that of normal parathyroid tissues

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The design, fate and impact of a hospital-wide training program in evidence-based medicine for physicians : An observational study

Background: Many doctors fail to practice Evidence-Based Medicine (EBM) effectively, in part due to insufficient training. We report on the design, fate and impact of a short learner-centered EBM train-the-trainer program aimed at all 2400 doctors at the Karolinska University Hospital in Sweden on the heels of a tumultuous merger, focusing particularly on whether it affected the doctors' knowledge, attitudes and skills regarding EBM.  Methods: We used a validated EBM instrument in a before-and-after design to assess the impact of the training. Changes in responses were analyzed at the individual level using the Wilcoxon matched pairs test. We also reviewed documentation from the program - including the modular EBM training schedule and the template for participants' Critically Appraised Topic reports - to describe the training's content, design, conduct, and fate.  Results: The training, designed to be delivered in modules of 45 min totaling 1.5 days, failed to reach most doctors at the hospital, due to cost cutting pressures and competing demands. Among study participants (n = 174), many reported suboptimal EBM knowledge and skills before the training. Respondents' strategies for solving clinical problems changed after the training: the proportion of respondents reporting to use (or intend to use) secondary sources "Often/very often" changed from 5 % before the training to 76 % after the training; in parallel, reliance on textbooks and on colleagues fell (48 to 23 % and 79 to 65 %, respectively). Participants' confidence in assessing scientific articles increased and their attitudes toward EBM became more positive. The proportion of correct answers in the EBM knowledge test increased from 52 to 71 %. All these changes were statistically significant at p < 0.05.  Conclusions: Many study participants, despite working at a university hospital, lacked basic EBM knowledge and skills and used the scientific literature suboptimally. The kind of short learner-centered EBM training evaluated here brought significant improvements among the minority of hospital doctors who were able to participate and, if applied widely, could contribute to better, safer and more cost-effective care

Additional file 1: of The design, fate and impact of a hospital-wide training program in evidence-based medicine for physicians – an observational study

The Critically Appraised Topic template. (DOCX 16 kb

Genome-wide and locus specific alterations in CDC73/HRPT2-mutated parathyroid tumors.

Mutations in the hyperparathyroidism type 2 (HRPT2/CDC73) gene and alterations in the parafibromin protein have been established in the majority of parathyroid carcinomas and in subsets of parathyroid adenomas. While it is known that CDC73-mutated parathyroid tumors display specific gene expression changes compared to CDC73 wild-type cases, the molecular cytogenetic profile in CDC73-mutated cases compared to unselected adenomas (with an expected very low frequency of CDC73 mutations) remains unknown. For this purpose, nine parathyroid tumors with established CDC73 gene inactivating mutations (three carcinomas, one atypical adenoma and five adenomas) were analyzed for copy number alterations and loss of heterozygosity using array-comparative genomic hybridization (a-CGH) and single nucleotide polymorphism (SNP) microarrays, respectively. Furthermore, CDC73 gene promoter methylation levels were assessed using bisulfite Pyrosequencing. The panel included seven tumors with single mutation and three with double mutations of the CDC73 gene. The carcinomas displayed copy number alterations in agreement with previous studies, whereas the CDC73-mutated adenomas did not display the same pattern of alterations at loci frequently deleted in unselected parathyroid tumors. Furthermore, gross losses of chromosomal material at 1p and 13 were significantly (p = 0.012) associated with parathyroid carcinomas as opposed to adenomas. Quantitative PCR-based copy number loss regarding CDC73 was observed in three adenomas, while all the carcinomas were diploid or showed copy number gain for CDC73 gene. Hypermethylation of the CDC73 gene promoter was not observed. Our data could suggest that CDC73-mutated parathyroid adenomas exhibit a partly unique cytogenetic profile in addition to that of carcinomas and unselected adenomas. Furthermore, CDC73-mutated carcinomas displayed losses at 1p and 13 which are not seen in CDC73-mutated adenomas, making these regions of interest for further studies regarding malignant properties in tumors from CDC73-mutated cases. However, due to the small sample size, validation of the results in a larger cohort is warranted

Figure 2

<p>(A) Bar graph showing predicted DNA copy number of the <i>CDC73</i> gene using TaqMan DNA copy number analysis. Each bar represents one parathyroid sample and the height of the bar represents the predicted <i>CDC73</i> gene DNA copy number. T2, T3 and T4 had 1 copy of <i>CDC73</i> gene, while T6 and T8 had more than 2 copies. N1, N2 and N3 refer to the normal parathyroid samples used as calibrators. (B) Individual value plot illustrating the methylation density at the three analyzed CpG dinucleotides of the <i>HPRT2</i> promoter. Each red dot represents one CpG site in one parathyroid tumor numbered from T2–T8 along with the three normal references denoted as N1, N2 and N3.</p