Elevation of methylated DNA in KILLIN/PTEN in the plasma of patients with thyroid and/or breast cancer

© 2014 Ng et al. Around 80% of mutations in the PTEN gene have been reported to be associated with diseases such as Cowden syndrome, which is an autosomal dominant disorder associated with an increased risk of developing breast, thyroid, and endometrial neoplasms. Recent studies have also demonstrated that KILLIN, which is located proximally to PTEN, shares the same transcription start site, and is assumed to be regulated by the same promoter, but is transcribed in the opposite direction. In this regard, we postulate that there may be a connection between KILLIN/PTEN genes and breast and thyroid cancers. Using real-time quantitative polymerase chain reaction (qPCR), we found that expression of KILLIN, but not PTEN, was significantly decreased in 23 Chinese women with a personal history of breast and thyroid cancer or a personal history of breast cancer and a family history of thyroid cancer, or vice versa, and at least two persons in the family with thyroid cancer or at a young age ,40 years, when compared with healthy controls (P<0.0001). No PTEN mutations were found in these 23 patients. We then developed a simple methylation-sensitive restriction enzyme digestion followed by real-time quantitative assay to quantify plasma methylated KILLIN/PTEN DNA in these patients. Plasma levels of methylated KILLIN/PTEN DNA were significantly increased in these patients when compared with healthy controls (P<0.05). This study shows that plasma methylated KILLIN/PTEN DNA was significantly elevated, suggesting hypermethylation of the KILLIN/PTEN promoter in breast and thyroid cancer patients.published_or_final_versio

Australian and New Zealand Pulmonary Rehabilitation Guidelines

Background and objective: The aim of the Pulmonary Rehabilitation Guidelines (Guidelines) is to provide evidence-based recommendations for the practice of pulmonary rehabilitation (PR) specific to Australian and New Zealand healthcare contexts. Methods: The Guideline methodology adhered to the Appraisal of Guidelines for Research and Evaluation (AGREE) II criteria. Nine key questions were constructed in accordance with the PICO (Population, Intervention, Comparator, Outcome) format and reviewed by a COPD consumer group for appropriateness. Systematic reviews were undertaken for each question and recommendations made with the strength of each recommendation based on the GRADE (Gradings of Recommendations, Assessment, Development and Evaluation) criteria. The Guidelines were externally reviewed by a panel of experts. Results: The Guideline panel recommended that patients with mild-to-severe COPD should undergo PR to improve quality of life and exercise capacity and to reduce hospital admissions; that PR could be offered in hospital gyms, community centres or at home and could be provided irrespective of the availability of a structured education programme; that PR should be offered to patients with bronchiectasis, interstitial lung disease and pulmonary hypertension, with the latter in specialized centres. The Guideline panel was unable to make recommendations relating to PR programme length beyond 8 weeks, the optimal model for maintenance after PR, or the use of supplemental oxygen during exercise training. The strength of each recommendation and the quality of the evidence are presented in the summary. Conclusion: The Australian and New Zealand Pulmonary Rehabilitation Guidelines present an evaluation of the evidence for nine PICO questions, with recommendations to provide guidance for clinicians and policymakers

Quantitative Analysis and Diagnostic Significance of Methylated SLC19A3 DNA in the Plasma of Breast and Gastric Cancer Patients

Background: Previously, we have examined the methylation status of SLC19A3 (solute carrier family 19, member 3) promoter and found that SLC19A3 was epigenetically down-regulated in gastric cancer. Here, we aim to develop a new biomarker for cancer diagnosis using methylated SLC19A3 DNA in plasma. Methodology/Principal Findings: SLC19A3 gene expression was examined by RT-qPCR. Methylation status of SLC19A3 promoter was evaluated by methylation-specific qPCR. SLC19A3 expression was significantly down-regulated in 80% (12/15) of breast tumors (P<0.005). Breast tumors had significant increase in methylation percentage when compared to adjacent non-tumor tissues (P<0.005). A robust and simple methylation-sensitive restriction enzyme digestion and real-time quantitative PCR (MSRED-qPCR) was developed to quantify SLC19A3 DNA methylation in plasma. We validated this biomarker in an independent validation cohort of 165 case-control plasma including 60 breast cancer, 45 gastric cancer patients and 60 healthy subjects. Plasma SLC19A3 methylated DNA level was effective in differentiating both breast and gastric cancer from healthy subjects. We further validated this biomarker in another independent blinded cohort of 78 plasma including 38 breast cancer, 20 gastric cancer patients and 20 healthy subjects. The positive predictive values for breast and gastric cancer were 90% and 85%, respectively. The negative predictive value of this biomarker was 85%. Elevated level in plasma has been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 100%. Conclusions: These results suggested that aberrant SLC19A3 promoter hypermethylation in plasma may be a novel biomarker for breast and gastric cancer diagnosis. © 2011 Ng et al.published_or_final_versio

A novel de novo BRCA1 mutation in a Chinese woman with early onset breast cancer

Germline mutations in the two breast cancer susceptibility genes, BRCA1 and BRCA2 account for a significant portion of hereditary breast/ovarian cancer. De novo mutations such as multiple exon deletion are rarely occurred in BRCA1 and BRCA2. During our mutation screening for BRCA1/2 genes to Chinese women with risk factors for hereditary breast/ovarian cancer, we identified a novel germline mutation, consisting of a deletion from exons 1 to 12 in BRCA1 gene, in a patient diagnosed with early onset triple negative breast cancer with no family history of cancer. None of her parents carried the mutation and molecular analysis showed that this novel de novo germline mutation resulted in down-regulation of BRCA1 gene expression

Patient characteristics.

<p>*DCIS, Ductal carcinoma in situ.</p

Down-regulated SLC19A3 gene expression in primary breast cancer tissues.

<p>Relative SLC19A3 mRNA expression between tumor tissues and their paired adjacent non-tumor breast from breast cancer patients (n = 15) by real-time qPCR. Expression of SLC19A3 mRNA (Log₁₀ scale at Y-axis) was normalized to GAPDH. The lines inside the boxes denote the medians. The boxes mark the interval between the 25^th and 75^th percentiles. The whiskers denote the interval between the 10^th and 90^th percentiles. Statistical difference was analyzed by Wilcoxon test, <i>P</i><0.005.</p

Quantitative analysis of plasma methylated SLC19A3 DNA on a group (n = 165) of plasma samples by MSRED-qPCR.

<p>Scatter plots of plasma levels of methylated SLC19A3 DNA in 60 healthy normal subjects, 45 gastric cancer (GC) and 60 breast cancer (BC) patients. Plasma level of methylated SLC19A3 DNA is expressed as 2^{ΔCt(undigest-digest)}. ΔCt_{(undigest-digest)} is calculated by subtracting the Ct values of digested plasma DNA from the Ct values of undigested plasma DNA. Since Ct of undigest should be ≤Ct of digest, the expression level is ranging from 1 to 0. The horizontal black lines denote the means. The blue errors bars denote the ± standard deviations (SD). Statistically significant differences were determined using Mann-Whitney <i>U</i> tests, <i>P</i><0.0001.</p

Increased percentage of SLC19A3 DNA methylation in primary breast cancer tissues.

<p>Percentage of SLC19A3 promoter methylation between tumor tissues and their paired adjacent non-tumor breast tissues from the 15 breast cancer patients by MS-qPCR. Percentage of methylation in tissue samples was calculated by the following equation: % meth = 100/[1+2^{ΔCt(meth-unmeth)}]%. ΔCt_{(meth-unmeth)} was calculated by subtracting the Ct values of methylated SLC19A3 signal from the Ct values of umnethylated SLC19A3 signal. Statistical difference was analyzed by Wilcoxon test, <i>P</i><0.005.</p

SLC19A3 is frequently down-regulated through promoter hypermethylation in breast cancer.

<p>(A) Fold change relationship between SLC19A3 mRNA expression and methylation percentage in those 15 breast cancer patients (mean ± SD). (B) Correlation analysis of the fold changes between SLC19A3 mRNA expression and methylation percentage (Spearman rank correlation, R² = −0.77, <i>P</i><0.0005).</p

Plasma methylated SLC19A3 DNA levels across tumor stages from both Phase I and II validation.

<p>(A) Box plot of plasma methylated SLC19A3 DNA level in 80 NC, 27 ductal carcinoma in situ (DCIS) plus 71 BC patients across various TNM stages. (B) Box plot of plasma methylated SLC19A3 DNA level in 80 NC and 65 GC patients across various TNM stages. The box represents the interquartile range and the line across the box indicates the median value. Relative plasma level of methylated SLC19A3 DNA is expressed as 2^{−ΔCt(Dig-Undig)}. ΔCt_(Dig-Undig) is calculated by subtracting the Ct values of digested plasma DNA from the Ct values of undigested plasma DNA. Statistically significant differences were determined using Mann-Whitney tests.</p