BRAF Mutation Testing of Thyroid Fine-Needle Aspiration Specimens Enhances the Predictability of Malignancy in Thyroid Follicular Lesions of Undetermined Significance

Background/Objective: The Bethesda 2007 Thyroid Cytology Classification defines atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) as a heterogeneous category of cases that are neither convincingly benign nor sufficiently atypical for a diagnosis of follicular neoplasm or suspicious for malignancy. At our institution, we refer to these cases as 'indeterminate' and they are further subclassified into two categories. BRAF mutation occurs in 40-60% of papillary thyroid carcinoma (PTC). In this study, we examined cases in the AUS/FLUS category in correlation with BRAF mutation analysis and surgical pathology outcome. Study Design: Thyroid fine-needle aspiration (FNA) cytology specimens interpreted as 'indeterminate' were selected from our files, and available remnants of thin-layer processed specimens were used for BRAF mutation analysis. Surgical pathology reports were reviewed for the final outcomes in these patients. Results: Of the 84 indeterminate cases with BRAF mutation analysis, only 49 had follow-up with surgical intervention. Sixteen cases had BRAF mutation. All of the BRAF-positive cases had a final diagnosis of PTC. Conclusions: The sensitivity and specificity of BRAF mutation in detecting PTC in FNA specimens with indeterminate diagnosis was 59.3 and 100%, respectively, while the positive and negative predictive values were 100 and 65.6%, respectively. The limited data supports the use of BRAF mutation analysis to predict the risk of malignancy in patients with indeterminate thyroid FNAs.     

BRAF Mutation Analysis of Fine-Needle Aspiration Biopsies of Papillary Thyroid Carcinoma: Impact on Diagnosis and Prognosis

Objective: The BRAF V600E mutation has been associated with aggressive disease in papillary thyroid carcinoma (PTC). Molecular testing has been proposed as a useful adjunct to cytology in the diagnosis of malignancy and for tailoring clinical management. The aims of our study were to evaluate the BRAF mutational status using archived fine-needle aspiration biopsy (FNAB) material from patients with long-term follow-up and to correlate it with the original cytology diagnosis, clinicopathological stage at surgery, and prognosis. Study Design: FNAB material from 52 cases of PTC, with a mean follow-up of 8.4 years, was used in this study. DNA was extracted from archival cytology slides. Mutation analysis was performed by standard sequencing and locked nucleic acid-PCR/sequencing. Results: The BRAF V600E mutation was present in 46% of cases, but it was absent in all FNABs diagnosed originally as atypical and in 14 of 17 suspicious cases. Recurrence was significantly more frequent (p = 0.006) in cases with BRAF mutations and 54% of these cases presented with stage 2 or higher. Conclusion: The BRAF V600E mutation is associated with a higher pathological stage at surgery and a higher rate of recurrence. BRAF mutation analysis did not provide a significant increase in the accuracy of thyroid FNABs diagnosed as suspicious or atypical in our institution.     

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.     

Prediction of Nondiagnostic Results in Fine-Needle Aspiration of Thyroid Nodules: utility of On-Site Gross Visual Assessment of Specimens for Liquid-Based Cytology

Objective: To explore a comprehensive approach for on-site gross visual assessments of liquid-based cytology (LBC) specimens of thyroid nodules and determine morphologic criteria that help predict nondiagnostic rates.Methods: Two-hundred nodules from 165 patients who underwent fine-needle aspiration (FNA) at our hospital were included in this prospectively designed, retrospective analysis. Specimens were visually assessed on-site for three morphologic categories (specimen color, specimen volume, and particle count) using a 5-point grading.Results: Twenty-two nodules (11%) showed nondiagnostic results. Regarding specimen color, nondiagnostic rates tended to be higher in grades 1 (75%) and 5 (100%) than in grades 2 (18%), 3 (8%), or 4 (17%), with a significant difference between grade 1 and grade 3 (P = .003). For specimen volume, nondiagnostic results were significantly more common in grade 1 (33%) and 5 (33%) than in grades 3 (5%) or 4 (1%) (P<.005). There was a significant negative correlation between the grading of the particle count and the nondiagnostic rate (Spearman ρ = -1.000; P<.001). The sensitivity and specificity in the prediction of nondiagnostic results were 77% and 76%, respectively, at the optimal cutoff value of 2 (grade 2 or lower).Conclusion: Particle count was an important morphologic criterion that helped predict nondiagnostic rates in LBC specimens of thyroid nodules, and the specimen color and volume were also useful adjuncts. In routine practice, on-site gross visual assessment followed by resampling (if necessary) may potentially help reduce the rates of nondiagnostic results, repeat FNAs, and the number of unnecessary needle passes.Abbreviations: FNA = fine-needle aspiration; LBC = liquid-based cytology; ROC = receiver operating characteristic; US = ultrasonography     

Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection

Background

For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA), sequence variation in particular mt haplogroups (hgs) and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements.

Methodology/Principal Findings

A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples) or the derived 7028C allele (hg H samples) in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA). The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed ∼2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification.

Conclusions

Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the analysis of mixtures. Homogeneous ARMS-DCA is a valuable tool for large-volume studies targeting small numbers of single nucleotide polymorphisms (SNPs).     

Comparison of Ultrasound-Guided Fine-Needle Aspiration Biopsy with Core-Needle Biopsy in the Evaluation of Thyroid Nodules

ObjectiveTo compare the diagnostic rate of ultrasound-guided fine-needle aspiration biopsy (FNAB) with the diagnostic rate of combined FNAB and core-needle biopsy in the evaluation of nodular thyroid disease.MethodsWe performed a retrospective case-control study by reviewing charts of patients who underwent ultrasound-guided FNAB and core-needle biopsy of the thyroid at a tertiary referral center from January 1999 to December 2001. Results were classified as diagnostic (negative, suspicious, or positive for malignancy) or nondiagnostic. These findings were compared with an age- and sex-matched control group who underwent only FNAB. Complications between the groups were reviewed.ResultsThe patient group consisted of 320 patients who underwent 340 ultrasound-guided fine-needle aspiration and core-needle biopsies of the thyroid; the control group consisted of 311 patients who underwent 340 FNABs. There was no significant difference in the nondiagnostic rates between groups—12.9% in patients who had FNAB–only compared with 10.9% in patients who had both procedures (proportion difference, -2.1%; 95% confidence interval, -7.0% to 2.9%; P = .41). There was a trend towards an increased incidence of hematoma and infection in the core biopsy group. In the group that underwent FNAB and core-needle biopsies, 10 patients (3.1%) developed biopsy-specific complications (hematomas in 8 patients, biopsy site infections in 2 patients). In the FNAB only group, 3 patients (1.0%) developed hematomas; there was no incidence of infection.ConclusionsIn the evaluation of thyroid nodules, the addition of core-needle biopsies to FNAB confers little benefit in decreasing the nondiagnostic rates and may be associated with increased complications. Core-needle biopsies should not be routinely performed in the evaluation of thyroid nodules, but rather, patient selection for the more invasive core biopsy should be done judiciously. (Endocr Pract. 2008;14:426-431)     

Single-Tubed Wild-Type Blocking Quantitative PCR Detection Assay for the Sensitive Detection of Codon 12 and 13 KRAS Mutations

The high degree of intra-tumor heterogeneity has meant that it is important to develop sensitive and selective assays to detect low-abundance KRAS mutations in metastatic colorectal carcinoma (mCRC) patients. As a major potential source of tumor DNA in the aforementioned genotyping assays, it was necessary to conduct an analysis on both the quality and quantity of DNA extracted from formalin-fixed paraffin-embedded (FFPE). Therefore, four commercial FFPE DNA extraction kits were initially compared with respect to their ability to facilitate extraction of amplifiable DNA. The results showed that TrimGen kits showed the greatest performance in relation to the quality and quantity of extracted FFPE DNA solutions. Using DNA extracted by TrimGen kits as a template for tumor genotyping, a real-time wild-type blocking PCR (WTB-PCR) assay was subsequently developed to detect the aforementioned KRAS mutations in mCRC patients. The results showed that WTB-PCR facilitated the detection of mutated alleles at a ratio of 1:10,000 (i.e. 0.01%) wild-type alleles. When the assay was subsequently used to test 49 mCRC patients, the results showed that the mutation detection levels of the WTB-PCR assay (61.8%; 30/49) were significantly higher than that of traditional PCR (38.8%; 19/49). Following the use of the real-time WTB-PCR assay, the ΔC _q method was used to quantitatively analyze the mutation levels associated with KRAS in each FFPE sample. The results showed that the mutant levels ranged from 53.74 to 0.12% in the patients analyzed. In conclusion, the current real-time WTB-PCR is a rapid, simple, and low-cost method that permits the detection of trace amounts of the mutated KRAS gene.     

BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications

Pleomorphic xanthoastrocytoma (PXA) is low-grade glial neoplasm principally affecting children and young adults. Approximately 40% of PXA are reported to recur within 10 years of primary resection. Upon recurrence, patients receive radiation therapy and conventional chemotherapeutics designed for high-grade gliomas. Genetic changes that can be targeted by selective therapeutics have not been extensively evaluated in PXA and ancillary diagnostic tests to help discriminate PXA from other pleomorphic and often more aggressive astrocytic malignancies are limited. In this study, we apply the SNaPshot multiplexed targeted sequencing platform in the analysis of brain tumors to interrogate 60 genetic loci that are frequently mutated in 15 cancer genes. In our analysis we detect BRAF V600E mutations in 12 of 20 (60%) WHO grade II PXA, in 1 of 6 (17%) PXA with anaplasia and in 1 glioblastoma arising in a PXA. Phospho-ERK was detected in all tumors independent of the BRAF mutation status. BRAF duplication was not detected in any of the PXA cases. BRAF V600E mutations were identified in only 2 of 71 (2.8%) glioblastoma (GBM) analyzed, including 1 of 9 (11.1%) giant cell GBM (gcGBM). The finding that BRAF V600E mutations are common in the majority of PXA has important therapeutic implications and may help in differentiating less aggressive PXAs from lethal gcGBMs and GBMs.     

High-frequency microsatellite instability and BRAF mutation (V600E) in unselected Serbian patients with colorectal cancer

Microsatellite instability (MSI) is a genetic consequence of a MisMatch Repair defect in colorectal cancer (CRC). We compared clinicopathohistological features with MSI status of CRC and evaluated prognostic significance of MSI status and BRAF mutation in the group of MSI-H tumors. 155 primary CRCs were excised surgically, 2006–2008. MSI analysis was carried out using a fluorescence-based pentaplex polymerase chain reaction technique. BRAF mutation (V600E) was analyzed by direct sequencing in MSI-H tumors. For all patients were evaluated: age, gender, localization, tumor cell type, tumor differentiation, mucin production, lymphocytic infiltration (TILs) and TNM stage. Patients’ disease-free survival (DFS) was compared according to MSI and BRAF status using Kaplan–Meier test. Of the 155 CRCs, 19 (12.3%) were MSI-H, and 136 (87.7%) were MSS/L. BRAF mutations were found in 4 of the MSI-H tumors. Patients with MSI-H CRC had lower recurrence rate (log rank test; P = 0.04) than MSS/L group. Patients with MSI-H tumor and BRAF mutation had worse DFS than MSI-H tumors without this mutation (log rank test; P = 0.01). Most of the clinicopathologic characteristics of MSI-H CRC in Serbian patients are similar to those reported in previous studies. Patients with MSI tumor phenotype had favourable prognosis, but in those with BRAF mutation higher recurrence rate was observed.     

Fine-Needle Aspiration Biopsy as a Preoperative Procedure in Patients with Malignancy in Solitary and Multiple Thyroid Nodules

Background

Fine-needle aspiration biopsy (FNAB) is a recognized technique for the basic, preoperative cytological diagnosis of thyroid nodules.

Aim of the Study

To analyze the accuracy of FNAB in the diagnosis of thyroid cancer in patients with solitary and multiple thyroid nodules and to compare the demographic, clinical and pathological characteristics of patients with thyroid carcinoma in solitary and multiple tumors.

Materials and Methods

The case records of 2,403 patients with solitary and multiple thyroid tumors treated consecutively between 2008 and 2013 were analyzed retrospectively. We selected 1,645 for further analysis. A solitary thyroid nodule was observed in 493 patients, and multiple nodules were detected in 1,152 patients. Further classification of the patients in these two groups was performed on the basis of the FNAB results, type of surgery performed and histopathology. TC was histopathologically confirmed in 166 patients, and benign disease was found in 1,479. The TC patients were assigned to the study group, and those with benign thyroid disease were placed into the control group. The study group was divided into two subgroups according to the presence of cancer in a single thyroid nodule or in multiple nodules. Malignancy in a solitary thyroid nodule was diagnosed in 98 (59.0%) patients, and cancer in multiple nodules was diagnosed in 68 (41.0%). Comparative analyses of the demographic, clinical and histopathological characteristics were performed for both subgroups. The following statistical analyses were performed: comparative characteristic of subgroups, ROC analysis for study group and subgroup of patients, and multivariable logistic regression analysis for study group.

Results

The rate of prediction of TC by FNAB was three times higher in the patients with a solitary thyroid nodule compared with those with multiple thyroid nodules and it was statistically significant (p<0.001). The rate of total thyroid resection and lack of necessity for reoperation were also significantly higher in the TC patients with a solitary nodule. The histopathological results showed that significantly more patients with a solitary nodule had advanced-stage TC (stage III or IV) and tumor progression (pT3 or pT4) (p = 0.002 for both). ROC analysis demonstrated that the overall accuracy of FNAB as a predictor of thyroid cancer presence was high, especially for the subgroup of patients with a solitary thyroid nodule (AUC = 0.958, p<0.0001). Multivariable logistic regression analysis confirmed that a positive FNAB result was the sole predictor of the performance of total resection in the TC study group (p<0.0001), while a negative FNAB result and the presence of a papillary cancer type were independent predictors of the risk of reoperation (p<0.0001 and p = 0.002, respectively).

Conclusions

FNAB often produces false-negative results in patients with multiple malignant thyroid tumors, which results in reoperation in many cases. False-negative FNAB results are rare in patients with a solitary tumor. Because of the low predictive capacity of FNAB for thyroid cancer in patients with multiple thyroid tumors, total thyroid excision should be considered in most cases despite a "negative" (no malignant) FNAB result.     

Correlation of Findings from Iodine 123 Scan and Ultrasonography in the Recommendation for Thyroid Fine-Needle Aspiration Biopsy

ObjectiveTo assess the impact of correlating findings from iodine I 123 (¹²³I) radionuclide scans and thyroid ultrasonography on the decision to perform fine-needle aspiration (FNA) biopsy of thyroid nodules.MethodsIodine 123 scans and sonographic images of adult patients who had both examinations performed within 6 months of each other at our institution were retrospectively reviewed. The presence of 1 or more nodules satisfying imaging-specific criteria for recommending FNA biopsy was recorded. Iodine 123 scan and sonographic images were then directly compared to determine how frequently the FNA recommendation would be affected by discordant findings.ResultsThe study included 97 adult patients, with a total of 291 thyroid lobes (right thyroid lobe, left thyroid lobe, and isthmus). Recommendations for FNA biopsy were concordant in 231 of 291 lobes (79.4%), with both modalities recommending FNA biopsy in 55 lobes and not recommending FNA biopsy in 176 lobes. A discordant recommendation occurred in 60 of 291 lobes (20.6%). Using only ultrasonography findings, a recommendation for FNA biopsy was not indicated for 11 of the 291 lobes (3.8%) with functioning nodules. Using only ¹²³I findings, a recommendation for FNA biopsy was not indicated for 23 of the 291 lobes (7.9%); 13 had nodules, but none that fulfilled sonographic criteria, and 10 had no identifiable nodule on ultrasonography. Iodine 123 scan did not identify 26 lobes with nodules (8.9%) for which FNA biopsy was recommended based on ultrasonography findings.ConclusionRecommendations for FNA biopsy should not be based on the presence of hypofunctioning regions on ¹²³I scan without sonographic confirmation. (Endocr Pract. 2011;17:699-706)     

Intra- and inter-tumor heterogeneity of BRAF(V600E))mutations in primary and metastatic melanoma

The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene. With the emergence of BRAF^V600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAF^V600E mutation as a marker of clonality. BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18). Nineteen patients had tissues available from multiple metastatic sites. Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR. The better sensitivity of the MS-PCR to detect the mutant BRAF^V600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes. To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAF^V600E-specific SNaPshot analysis in 9 cases. Six of these cases demonstrated differing proportions of BRAF^V600Eand BRAF^wild-type cells in distinct microdissected regions within individual tumors. Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAF^V600E mutation. In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAF^V600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients. Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.     

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

Background

Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.

Methodology

In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.

Results

Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.

Conclusions

Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.     

The Role of Core-Needle Biopsy for Thyroid Nodules with Initially Nondiagnostic Fine-Needle Aspiration Results: A Systematic Review and Meta-Analysis

Objective: This study evaluated the prevalence of nondiagnostic results, diagnostic performance, and complications of core-needle biopsy (CNB) compared with repeat fine-needle aspiration (FNA) for thyroid nodules with previous nondiagnostic FNA findings.Methods: The Ovid-MEDLINE and EMBASE databases were thoroughly searched for studies evaluating CNB or repeat FNA for thyroid nodules with initially nondiagnostic FNA results. Pooled proportions of nondiagnostic results of CNB and repeat FNA were calculated. A meta-analysis was performed to evaluate the diagnostic accuracy of CNB and repeat FNA for a diagnosis of malignancy using a bivariate random-effects model. Complication rates were also evaluated.Results: A review of 52 articles identified 4 eligible articles, involving 1,028 patients with 1,028 thyroid nodules, which were included in the meta-analysis. CNB demonstrated significantly lower rates of nondiagnostic results (6.4%) than repeat FNA (36.5%) (P<.0001). In the 3 studies that analyzed the diagnostic accuracy of CNB in diagnosing malignancy, CNB demonstrated significantly higher summary estimates of sensitivity (89.8%) than repeat FNA (60.6%) (P = .022), but summary specificity did not differ between CNB (99.2%) and repeat FNA (99.0%) (P = .576). None of the patients who underwent CNB or repeat FNA experienced any major complications.Conclusion: CNB demonstrates lower rates of nondiagnostic results and higher diagnostic accuracy than repeat FNA. CNB, rather than repeat FNA, can be utilized to diagnose thyroid nodules previously nondiagnostic on FNA.Abbreviations:CI = confidence intervalCNB = core-needle biopsyFNA = fine-needle aspirationSROC = summary receiver operating characteristicUS = ultrasound     

A Multiplexed Microfluidic PCR Assay for Sensitive and Specific Point-of-Care Detection of Chlamydia trachomatis

Background

Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes.

Methodology and Principal Findings

Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively.

Conclusions

This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.     

Detection of the G17V RHOA Mutation in Angioimmunoblastic T-Cell Lymphoma and Related Lymphomas Using Quantitative Allele-Specific PCR

Angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) are subtypes of T-cell lymphoma. Due to low tumor cell content and substantial reactive cell infiltration, these lymphomas are sometimes mistaken for other types of lymphomas or even non-neoplastic diseases. In addition, a significant proportion of PTCL-NOS cases reportedly exhibit features of AITL (AITL-like PTCL-NOS). Thus disagreement is common in distinguishing between AITL and PTCL-NOS. Using whole-exome and subsequent targeted sequencing, we recently identified G17V RHOA mutations in 60–70% of AITL and AITL-like PTCL-NOS cases but not in other hematologic cancers, including other T-cell malignancies. Here, we establish a sensitive detection method for the G17V RHOA mutation using a quantitative allele-specific polymerase chain reaction (qAS-PCR) assay. Mutated allele frequencies deduced from this approach were highly correlated with those determined by deep sequencing. This method could serve as a novel diagnostic tool for 60–70% of AITL and AITL-like PTCL-NOS.     

5-FU preferably induces apoptosis in BRAF V600E colorectal cancer cells via downregulation of Bcl-xL

Molecular and Cellular Biochemistry - Diallyl trisulfide (DATS) is distinguished as the most potent polysulfide isolated from garlic. The aim of our study was to investigate effects of oral...