ABSTRACT
BACKGROUND: When suspicious lesions are observed on computer-tomography (CT), invasive tests are needed to confirm lung cancer. Compared with other procedures, bronchoscopy has fewer complications. However, the sensitivity of peripheral lesion through bronchoscopy including washing cytology is low. A new test with higher sensitivity through bronchoscopy is needed. In our previous study, DNA methylation of PCDHGA12 in bronchial washing cytology has a diagnostic value for lung cancer. In this study, combination of PCDHGA12 and CDO1 methylation obtained through bronchial washing cytology was evaluated as a diagnostic tool for lung cancer. METHODS: A total of 187 patients who had suspicious lesions in CT were enrolled. PCDHGA12 methylation test, CDO1 methylation test, and cytological examination were performed using 3-plex LTE-qMSP test. RESULTS: Sixty-two patients were diagnosed with benign diseases and 125 patients were diagnosed with lung cancer. The sensitivity of PCDHGA12 was 74.4% and the specificity of PCDHGA12 was 91.9% respectively. CDO1 methylation test had a sensitivity of 57.6% and a specificity of 96.8%. The combination of both PCDHGA12 methylation test and CDO1 methylation test showed a sensitivity of 77.6% and a specificity of 90.3%. The sensitivity of lung cancer diagnosis was increased by combining both PCDHGA12 and CDO1 methylation tests. CONCLUSION: Checking DNA methylation of both PCDHGA12 and CDO1 genes using bronchial washing fluid can reduce the invasive procedure to diagnose lung cancer.
Subject(s)
Lung Neoplasms , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Lung Neoplasms/pathology , DNA Methylation , Sensitivity and Specificity , Lung/pathology , Bronchoalveolar Lavage , Bronchoscopy/methodsABSTRACT
BACKGROUND: Brn3a/Pou4f1 is a class IV POU domain-containing transcription factor and has been found to be expressed in a variety of cancers. However, the mechanism and action of Brn3a in thyroid cancer has not been investigated. PURPOSE: To investigate the role of Brn3a in thyroid cancer progression and its clinical implication. METHODS: We examined Brn3a expression status in patients with thyroid cancer and analyzed relationships between Brn3a expression and clinicopathological findings using The Cancer Genome Atlas (TCGA) database. For functional in vitro analysis, proliferation, migration, invasion assay, and Western blotting were performed after overexpression or suppression of Brn3a. RESULTS: The promoter hypermethylation of Brn3a was found in patients with aggressive thyroid cancer and Brn3a was downregulated in tissues of patients with thyroid cancer. In TCGA database, the low-Brn3a-expression group revealed a more aggressive phenotype, including T stage and extrathyroid extension when compared with the high-Brn3a-expression group. Overexpression of Brn3a suppressed cell migration and invasion via regulation of epithelial-mesenchymal transition (EMT)-associated proteins in thyroid cancer cell lines. Brn3a overexpression also downregulated signal transducer and activator of transcription 3 (STAT3) signaling through suppression of tyrosine-protein kinase Met (c-MET). In contrast, knockdown of Brn3a by small interfering ribonucleic acid (siRNA) significantly increased cell migration and invasion through upregulation of c-MET/STAT3. These results imply that Brn3a suppresses tumor metastasis via c-MET/STAT3 inhibition and EMT suppression in thyroid cancer. CONCLUSIONS: Our findings show that Brn3a is a potential tumor suppressor that leads to reduced cancer cell migration and invasion in thyroid cancer. Elucidation of the Brn3a-regulated cancer pathways may therefore provide novel therapeutic strategies to control thyroid cancer metastasis.
Subject(s)
Receptor Protein-Tyrosine Kinases/genetics , STAT3 Transcription Factor/genetics , Thyroid Neoplasms/genetics , Transcription Factor Brn-3A/physiology , Cell Line, Tumor , Cell Movement/genetics , Epithelial-Mesenchymal Transition/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor/physiology , Humans , Microarray Analysis , Receptor Protein-Tyrosine Kinases/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/genetics , Thyroid Neoplasms/pathologyABSTRACT
OBJECTIVE: There are many conflicting reports about the clinical implications of BRAFV600E in papillary thyroid cancer (PTC). We investigated the associations between the extent of BRAFV600E alleles and both clinico-pathological features and recurrence of PTC. MATERIALS AND METHODS: Carcinoma tissues from 60 patients with PTC were genotyped for BRAFV600E using pyrosequencing, and the clinico-pathological factors and disease outcomes of the patients were examined. The associations between the extent of mutant BRAF alleles and both clinico-pathological parameters and recurrence-free survival (RFS) were analyzed. RESULTS: The BRAFV600E mutation was detected in 66.7% (40/60) of our PTC patients. When we defined four groups on the basis of the extent of BRAFV600E alleles by pyrosequencing-negative (less than 5%), low (5 - less than 15%), intermediate (15 - less than 25%), and high (25% or greater)- the four groups showed statistically significant differences regarding lymph node (LN) metastasis and recurrence (P<0.05). However, age, gender, tumor size, multicentricity, capsular invasion, and lymphovascular invasion were not significantly different among the groups. The 10-year RFS rates in PTC patients with greater than 25% and less than 25% mutated BRAF alleles were 74% and 100%, respectively. This difference was significant (P=0.043). CONCLUSIONS: A high extent more than 25% of BRAFV600E alleles may be associated with disease outcome in PTC patients. We need more data to verify a hypothesis that the extent of BRAF mutations may be clinically informative in the management of PTC, such as by tailoring proper surgical and radioactive iodine treatments and determining appropriate management during follow-up.
Subject(s)
Alleles , Carcinoma, Papillary/genetics , Proto-Oncogene Proteins B-raf/genetics , Thyroid Neoplasms/genetics , Adult , Aged , Female , Humans , Male , Middle Aged , Mutation , Neoplasm Recurrence, Local/genetics , Thyroid Cancer, Papillary , Young AdultABSTRACT
Secretory clusterin (sCLU), an anti-apoptotic protein, is overexpressed in many tumors and enhances tumorigenesis and chemo-resistance. However, the regulation mechanism controlling the sCLU maturation process or activity remains undetermined. In this study, we found PACAP as a negative regulator of CLU. Overexpression of the PACAP gene in cervical cancer cell lines lacking PACAP expression significantly inhibited cell growth and induced apoptosis. We further demonstrated that interaction of PACAP with CLU significantly downregulated CLU expression and secretion, inhibited the Akt-Raf-ERK pathway, and suppressed the growth of human tumor xenografts in nude mice. This novel inhibitory function of PACAP may be applicable for developing novel molecular therapies for tumors with increased sCLU expression.
Subject(s)
Clusterin/metabolism , Pituitary Adenylate Cyclase-Activating Polypeptide/metabolism , Uterine Cervical Neoplasms/pathology , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Cell Transformation, Neoplastic , Clusterin/chemistry , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Mice , Protein Structure, Tertiary , Protein Transport , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , raf Kinases/metabolismABSTRACT
Aberrant DNA methylation has shown promise as a biomarker for the early detection of cancer. To discover novel genes frequently methylated at an early stage in colorectal cancer (CRC), DNA microarray analysis coupled with enriched methylated DNA was performed in primary tumors and compared with adjacent nontumor tissues of 12 patients with CRC at stages I to IV. Stepwise filtering for candidate selection in microarray data analysis yielded a set of genes that are highly methylated across all CRC tumors and that can be used as a composite biomarker for CRC detection. Verification assay identified the SDC2 gene as a potential methylation biomarker for early CRC detection. In clinical validation in tissues from 139 CRC patients, a much higher level of aberrant SDC2 methylation was measured in most primary tumors (97.8%), compared with corresponding nontumor tissue of CRC patients, irrespective of clinical stage. Clinical validation of SDC2 methylation in serum DNA from CRC patients (n = 131) at stages I to IV and from healthy individuals (n = 125) by quantitative methylation-specific PCR demonstrated a high sensitivity of 87.0% (95% CI, 80.0% to 92.3%) in detecting cancers, with a specificity of 95.2% (95% CI, 89.8% to 98.2%). Importantly, sensitivity at stage I was 92.3%, indicating the potential of SDC2 methylation as a blood-based DNA test for early detection of CRC.
Subject(s)
Biomarkers, Tumor/blood , Colorectal Neoplasms/diagnosis , Syndecan-2/blood , Adult , Aged, 80 and over , Colorectal Neoplasms/blood , Colorectal Neoplasms/genetics , DNA Methylation , Early Detection of Cancer , Female , Gene Expression Regulation, Neoplastic , Genome, Human , Humans , Male , Middle Aged , Neoplasm Staging , Syndecan-2/biosynthesisABSTRACT
Herein, we describe a novel multiplex genotyping method, GTPlex-PyroSeq. This method consists of two phases: multiplex PCR followed by a single reaction of pyrosequencing. This study demonstrates how GTPlex-PyroSeq can be adapted for the determination of multiple human papillomavirus (HPV) genotypes. A biotinylated consensus primer, GP6+, and 15 high-risk HPV type-specific primers are used for multiplex PCR. Each type-specific primer has a 5'-tag unique ID sequence connected to a pyrosequencing primer binding region. The unique ID sequence is composed of three parts: i) a single nucleotide ID representing a specific genotype; ii) a sign post; and iii) an end mark. This design allows multiple genotype determination under an ID sequence-dependent nucleotide dispensation order during pyrosequencing. Following initial studies using HPV plasmids and cell lines, we evaluated the clinical utility and effectiveness by comparing our assay with direct sequencing and HPV DNA chip analysis of 80 samples from high-risk, HPV-positive patients. We found in single-type infections, 100% concordance with direct sequencing (70 of 80 perfect matches) and 97.5% concordance with HPV DNA chip data (50 of 80 perfect matches). Additionally, our system was superior to direct sequencing in detection of multiple infections (12 of 80), with a limit of detection of 100 copies. The scalability of this multiplex system, with its open-platform design and ability to use various sample types, makes the GTPlex applicable for use in multiple settings.
Subject(s)
Multiplex Polymerase Chain Reaction/methods , Papillomaviridae/genetics , Sequence Analysis, DNA/methods , Genotype , Humans , Papillomaviridae/classificationABSTRACT
Aspirin-exacerbated respiratory disease (AERD) is associated with severe asthma and aspirin can cause asthma to worsen, often in the form of a severe and sudden attack. The oral aspirin challenge is the gold standard to confirm the diagnosis of AERD, but it is time consuming and produces serious complications in some cases. Therefore, more efficient and practical method is needed to predict AERD patients. The aim of the present study was to identify AERD-related gene expression in peripheral blood mononuclear cells (PBMCs) and examine the diagnostic potential of these candidate gene(s) for predicting AERD. To do this, RNAs from 24 subjects with AERD and 18 subjects with aspirin-tolerant asthma (ATA) were subjected to microarray analysis of ~34,560 genes. In total, 10 genes were selected as candidate gene markers by applying p ≤ 0.001(t test) and ≥8-fold change, and to correct for multiple comparisons, the false discovery rate analyses were performed. By applying multiple logistic regression analysis, among possible 1,023 models (2(10)-1), a model consisting of CNKSR3, SPTBN2, and IMPACT was selected as candidate set, because this set showed the best AUC (0.98) with 88 % sensitivity and 89 % specificity. For validation, mRNA levels by real-time PCR on PBMCs from two population sets in a gene-chip study and another replication sample, 20 AERD, 20 ATA, and 8 normal controls, were significantly different between groups with 100 % sensitivity and 100 % specificity in each of the two population sets. However, IMPACT gene did not differentiate between AERD and normal controls. The set of the two genes (CNKSR3 and SPTBN2) showed the best AUC (0.96) with 88 % sensitivity and 94 % specificity in a gene-chip study sample. In addition, this set showed perfect discriminative power with AUC (1.0, 100 % sensitivity and 100 % specificity) in each of the two population sets: the gene-chip samples and the replication samples. It also showed perfect discrimination for AERD from NC (AUC: 1.0) and ATA from NC (AUC: 1.0). In conclusion, we developed the two gene markers (CNKSR3 and SPTBN2) of PBMC which differentiate between AERD and ATA with a perfect discriminative power. These gene markers may be an efficient and practical method for predicting AERD.
Subject(s)
Aspirin/adverse effects , Asthma, Aspirin-Induced/blood , Asthma, Aspirin-Induced/genetics , Asthma/blood , Asthma/genetics , Membrane Proteins/genetics , Spectrin/genetics , Adolescent , Adult , Aged , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Asthma/diagnosis , Asthma, Aspirin-Induced/diagnosis , Bleeding Time , Child , Child, Preschool , Diagnosis, Differential , Female , Gene Expression Profiling , Genetic Markers , Humans , Infant , Intracellular Signaling Peptides and Proteins , Leukocytes, Mononuclear/metabolism , Logistic Models , Male , Membrane Proteins/blood , Middle Aged , Proteins/genetics , Proteins/metabolism , RNA, Messenger/blood , RNA, Messenger/genetics , Spectrin/metabolism , Young AdultABSTRACT
Aberrant DNA methylation occurs early and frequently in tumorigenesis. Identification of DNA methylation biomarkers is a field that provides potential for improving the clinical process of breast cancer diagnosis. We utilized a genome-wide technique, methylated DNA isolation assay (MeDIA), in combination with high-resolution CpG microarray analysis to identify hypermethylated genes in breast cancer. Among differentially methylated genes between tumor and adjacent normal tissues, 3 candidate genes (LHX2, WT1 and OTP) were finally selected through a step-wise filtering process and examined for methylation status in normal tissues, primary tumor, and paired adjacent normal-appearing tissues from 39 breast cancer patients. Based on the calculated cut-off values, all genes showed significantly higher frequencies of aberrant hypermethylation in primary tumors (43.6% for LHX2, 89.7% for WT1 and 100% for OTP, p<0.05) while frequencies were intermediate in paired adjacent normal tissues and absent in normal tissues. On further analysis, the methylation level in primary tumors was not significantly correlated with clinicopathological features. Interestingly, DNA methylation of a novel gene OTP was detected in adjacent normal tissues even 6 cm away from primary tumors, suggesting that OTP methylation may qualify as a biomarker for the early detection of breast cancer. In conclusion, we successfully identified a novel gene OTP frequently methylated in breast cancer by genome-wide screening. Our results suggest that the OTP gene may play a crucial role in breast carcinogenesis, although further clinical validation will be needed to evaluate the potential application of OTP in the early detection of breast cancer.
Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , DNA Methylation , Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , Adult , Aged , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Gene Expression Profiling , Genome-Wide Association Study , Humans , LIM-Homeodomain Proteins/genetics , Middle Aged , Transcription Factors/genetics , WT1 Proteins/geneticsABSTRACT
Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29 genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/genetics , DNA Methylation , Laminin/genetics , Receptors, GABA-A/genetics , Caco-2 Cells , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Colorectal Neoplasms/pathology , CpG Islands , Down-Regulation , Epigenomics/methods , Female , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis/methodsABSTRACT
CONTEXT: Recently, tremendous efforts have been made towards the development of sensitive techniques to detect the BRAF(V600E) mutation in fine needle aspiration biopsy (FNAB) samples. However, newly developed quantitative and semi-quantitative methods, such as dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR), have the potential to generate false-positive (FP) results. OBJECTIVES: To eliminate the possibility of FP results, we generated a receiver operating characteristic (ROC) curve to investigate the diagnostic accuracy of pyrosequencing using quantitative data. DESIGN: Cytological diagnoses of 983 thyroid nodules were made according to the Bethesda System 2007. The BRAF(V600E) mutation was analysed by pyrosequencing, and statistical analyses were performed. RESULTS: Of the 983 nodules, 902 were adopted to evaluate the diagnostic value of pyrosequencing. The number of pathologically confirmed malignancies was 192, of which 182 were papillary thyroid cancer (PTC). By generating an ROC curve, we defined the optimal cut-off value of the mutant allele peak as 5·95% (area under the curve, 0·849; sensitivity, 0·55; 1-specificity, 0). When we applied this selective cut-off value, the number of PTCs positive for BRAF(V600E) was 99 (54·4% of the total number of PTCs). With cytology alone, the diagnostic sensitivity and specificity of detecting malignancy were 71·2% and 100%, respectively. Pyrosequencing improved the diagnostic sensitivity from 71·2% to 78·5% (McNemar's test, P < 0·001), without any change in the diagnostic specificity. When 'suspicious for malignancy' was considered a positive cytological outcome, pyrosequencing increased the diagnostic sensitivity of cytology from 95·8% to 96·9%; however, this improvement did not show statistical significance (McNemar's test, P > 0·05). CONCLUSIONS: Pyrosequencing is an effective method for detecting the BRAF(V600E) mutation in FNAB samples. By allowing the optimal cut-off value to be determined, pyrosequencing improves the diagnostic sensitivity while eliminating the possibility of FP results.
Subject(s)
Biopsy, Fine-Needle , Proto-Oncogene Proteins B-raf/genetics , Thyroid Nodule/genetics , Adult , DNA Mutational Analysis , Female , Humans , Male , Middle Aged , MutationABSTRACT
BACKGROUND: Lung cancer is a leading cause of cancer deaths. Unfortunately, no effective early screening modality exists for lung cancer. We aimed to evaluate the prevalence of HOXA9 promoter methylation in tissue and induced sputum samples from Korean patients with lung cancer. METHODS: Using pyrosequencing, HOXA9 methylation was analyzed for 40 pairs of primary lung cancer and normal tissues and 185 induced sputum specimens, including 76 patients with lung cancer. RESULTS: The methylation of HOXA9 in lung cancer tissue was significantly higher compared with normal tissues (67.4% ± 17.6% vs. 23.6% ± 10.3%, respectively; p<0.001). With a cut-off of >45.6% of HOXA9 gene methylation in tissues, the sensitivity was 90.5% and the specificity was 97.5%. In induced sputum specimens, the HOXA9 gene in lung cancer patients was significantly more hypermethylated compared with patients with benign lung diseases and the healthy group (23.4% ± 15.9%, 14.9% ± 7.9%, and 9.7% ± 5.0%, respectively; p<0.001). CONCLUSIONS: The HOXA9 gene was hypermethylated in 32 of 40 tumors (80%), especially in early stages of lung cancer. HOXA9 methylation could be a potential biomarker to aid early detection and prognosis.
Subject(s)
DNA Methylation/genetics , Homeodomain Proteins/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Sputum/metabolism , Aged , Base Sequence , Case-Control Studies , Female , Gene Expression Regulation, Neoplastic , Humans , Lung/cytology , Lung/metabolism , Lung/pathology , Male , Middle Aged , Promoter Regions, Genetic/geneticsABSTRACT
Multiple cytosine guanine dinucleotides (CpG island) are found in the VIM promoter region. The levels of VIM promoter methylation and VIM gene expression were investigated in 7 cervical cancer cell lines and 50 human tissue samples with a distinctive degree of malignant trans-formation. While multiple CpG sites in the VIM promoter were highly methylated in CIN III and invasive carcinoma cells, they were rarely methylated in normal cells. Our result shows that methylation in the VIM promoter appears to start from CIN I and CIN II, relatively early stages of multistep carcinogenesis. This epigenetic alteration in VIM promoter suggests the availability as a biomarker for the early diagnosis and prevention of cervical cancer. We also show that hypermethylation in the VIM promoter is responsible for transcriptional silencing of the VIM gene in cervical cancer cells. In addition, our result shows that exogenous overexpression of the VIM gene in SiHa cervical cancer cells slightly activated cell proliferation and migration as shown in soft agar colony formation and migration assays.
Subject(s)
Biomarkers, Tumor/genetics , DNA Methylation , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Vimentin/genetics , Cell Line, Tumor , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , Early Detection of Cancer , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Promoter Regions, Genetic , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Dysplasia/genetics , Uterine Cervical Dysplasia/pathologyABSTRACT
The ADCYAP1 gene encodes an adenylate cyclase activating polypeptide 1. ADCYAP1 has been known to be involved in various biological processes. Multiple cytosine guanine dinucleotides (CpG island) are found in the ADCYAP1 promoter region. Transcriptional silencing by promoter hypermethylation is an important regulatory mechanism in tumorigenesis in many cancers. Therefore, the methylation level of the ADCYAP1 promoter was investigated in eight cervical cancer cell lines and human tissue samples with a distinctive degree of malignant transformation. While multiple CpG sites in the ADCYAP1 promoter were highly methylated in CIN III and invasive carcinoma cells as well as seven cervical cancer cell lines, they were rarely methylated in normal cells. Importantly, methylation in the ADCYAP1 promoter seems to start from CIN I, relatively early stage of multistep carcinogenesis. This fact suggest that ADCYAP1 can be used as an effective and sensitive methylation biomarker for the early diagnosis of cervical cancer. Moreover, our data imply that the level of the ADCYAP1 promoter hypermethylation is correlated with cervical cancer development. We also show that ADCYAP1 gene expression was reactivated by the treatment of a DNA methyltransferase inhibitor of 5'-aza-2'deoxycytidine and/or a histone deacetylase inhibitor of trichostain A in cervical cancer cells suggesting that hypermethylation in the ADCYAP1 promoter is responsible for the transcriptional silencing of the ADCYAP1 gene in cervical cancer cells.
Subject(s)
Biomarkers, Tumor/genetics , DNA Methylation/genetics , Early Detection of Cancer , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Uterine Cervical Neoplasms/genetics , Biomarkers, Tumor/analysis , CpG Islands/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/genetics , Humans , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Uterine Cervical Neoplasms/diagnosisABSTRACT
CONTEXT: Dideoxy sequencing is the most commonly used method for detecting the BRAF(V600E) mutation in thyroid cancer and melanoma. However, this gold standard method often makes less definite results in detecting the BRAF(V600E) mutation when there are relatively low amounts of the mutant template in biopsy specimens, which are invariably contaminated with normal tissues. Pyrosequencing, which measures the incorporation of each of the four nucleotides at each template position and indicates the amounts of mutant template present, may be more useful in such situations. OBJECTIVE: To investigate the diagnostic efficiency of pyrosequencing for the mutant BRAF allele in ultrasound (US)-guided fine needle aspiration biopsies (FNABs) of thyroid incidentalomas. DESIGN, SETTING AND SUBJECTS: A total of 101 thyroid incidentaloma cases were included prospectively. Cytological diagnoses of the FNAB samples were made according to the American Thyroid Association (ATA) guidelines, 2006. The presence of the BRAF(V600E) mutation was investigated by pyrosequencing and dideoxy sequencing. RESULTS: On the basis of cytological analysis, the thyroid incidentalomas were classified into benign (n = 43), malignant (n = 30), indeterminate or suspicious neoplasm (n = 24), and nondiagnostic (n = 4) categories. Pyrosequencing detected the BRAF(V600E) mutation in 30 cases: 22 malignant cases, 7 indeterminate cases, and 1 nondiagnostic case. Dideoxy sequencing also detected the BRAF(V600E) mutation in 28 of the same cases but failed to clearly distinguish the mutant allele from the wild-type allele in one indeterminate case and one nondiagnostic case. Histopathological analysis ascertained that all BRAF(V600E)-positive cases were papillary thyroid carcinomas. CONCLUSIONS: Pyrosequencing may be suitable for detecting the BRAF(V600E) mutation in thyroid incidentaloma and may be superior to dideoxy sequencing when low amounts of the mutant template are present in the biopsy.
Subject(s)
DNA Mutational Analysis/methods , Proto-Oncogene Proteins B-raf/genetics , Thyroid Gland/pathology , Thyroid Neoplasms/genetics , Biopsy, Fine-Needle , Humans , Incidental Findings , Mutation , Thyroid Neoplasms/diagnostic imaging , Thyroid Neoplasms/pathology , UltrasonographyABSTRACT
The global pattern of growth-dependent gene expres-sion in Streptococcus pneumoniae strains was evalu-ated using a high-density DNA microarray. Total RNAs obtained from an avirulent S. pneumoniae strain R6 and a virulent strain AMC96-6 were used to compare the expression patterns at seven time points (2.5, 3.5, 4.5, 5.5, 6.0, 6.5, and 8.0 h). The expression profile of strain R6 changed between log and station-ary growth (the Log-Stat switch). There were clear differences between the growth-dependent gene ex-pression profiles of the virulent and avirulent pneumo-coccal strains in 367 of 1,112 genes. Transcripts of genes associated with bacterial competence and capsular polysaccharide formation, as well as clpP and cbpA, were higher in the virulent strain. Our data suggest that late log or early stationary phase may be the most virulent phase of S. pneumoniae.