Detection Of BRAF V600E Mutation Research Articles

Detection of BRAF V600E in Fine-Needle Aspiration Samples of Thyroid Nodules by Droplet Digital PCR.

Background BRAF exon 15 p.V600E (BRAF V600E) mutation has been established as an important molecular marker for papillary thyroid carcinoma diagnosis by ultrasound-guided fine-needle aspiration biopsy (FNAB). Sanger sequencing is the gold standard for detecting BRAF V600E mutations but fails to identify low-frequency mutations. However, droplet digital PCR (ddPCR) is a popular new method for detecting low-frequency mutations. Here, we compare the efficiency of droplet digital PCR (ddPCR) and Sanger sequencing for detection of the BRAF V600E mutation in thyroid fine-needle aspiration (FNA) samples. Methods Thyroid fine-needle aspiration samples from 278 patients with 310 thyroid nodules were collected. Sanger sequencing and ddPCR were conducted to detect the BRAF V600E mutation. Results The BRAF V600E mutation was found in 94 nodules (30.32%) by ddPCR and 40 nodules (12.90%) by Sanger sequencing in 310 FNA samples. A total of 119 nodules were confirmed PTC by postsurgical pathology. Among which the BRAF mutation was found in 80 (67.23%) nodules by ddPCR and 31 (26.05%) by Sanger sequencing. All nodules carrying the mutation detected by Sanger sequencing (SS+) were verified by ddPCR (ddPCR+). Also, all nodules with no mutation detected by ddPCR were interpreted as wild-type by Sanger sequencing (SS−). In addition. Almost all SS+/ddPCR + nodules (95.00%; 38/40) and SS−/ddPCR + nodules (100.00%; 54/54) displayed a BRAF mutation rate of >5% and <15%, respectively, indicating easy misdetection by Sanger sequencing when the mutation rate is between 5 and 15%. Conclusion ddPCR has higher sensitivity than Sanger sequencing and we propose ddPCR as a supplement to Sanger sequencing in molecular testing of BRAF using FNAB samples.

Comparison of Two Rapid Assays for the Detection of BRAF V600 Mutations in Metastatic Melanoma including Positive Sentinel Lymph Nodes

Testing for the BRAF mutation is mandatory for the management of patients with locally advanced or metastatic melanoma. Molecular analysis based on DNA sequencing remains the gold-standard method for the screening of the different BRAF mutations. These methods must be rapid, sensitive, and specific enough to allow optimal therapeutic management in daily practice and also to include patients in clinical trials. Here, we compared the Idylla BRAF Mutation Test and the anti-BRAF V600E (clone VE1) immunohistochemistry (IHC) in 90 melanoma samples, with a focus on a challenging cohort of 32 positive sentinel lymph nodes. The BRAF status was assessed with both methods independently of the percentage of tumor cells. The concordance rate was calculated excluding both non-contributory analyses and BRAF V600K/R/M mutants due to the specific V600E-IHC test design. The incidence of the BRAF V600E mutation was 33% with both BRAF Idylla and BRAF IHC. The agreement rate was 91% (72/79). Although the agreement rate was high, we suggest that the use of IHC is more suitable for rapid BRAF testing on sentinel lymph node biopsies when associated with a low percentage and scattered tumor cells, which gave a high risk of non-contributory analysis and/or false negative results with the IdyllaTM BRAF Mutation Test.

Diagnostic accuracy of immunohistochemistry compared with molecular tests for detection of BRAF V600E mutation in ameloblastomas: Systematic review and meta-analysis.

The objective of this systematic review with meta-analysis was to critically evaluate the available data on sensitivity and specificity of IHC compared with molecular tests in the detection of BRAF V600E mutation in ameloblastomas. This systematic review was performed based on the PRISMA statement and registered in Prospero (CRD42021259117). PubMed, Web of Science, Scopus, and Cochrane Library databases were searched for observational studies to answer the question "What is the diagnostic accuracy of immunohistochemistry compared with molecular tests for the diagnosis of BRAF V600E mutation in ameloblastomas?". Methodological quality and risk of bias assessment of the selected studies were based on the QUADAS-2. Meta-analysis based on hierarchical SROC curve model and summary measures for sensitivity and specificity were computed. A total of 226 records were found, but only 05 articles met the inclusion criteria, with 277 FFPE specimens of ameloblastoma included in the quantitative analysis. The sensitivity of the IHC compared to molecular tests ranged from 0.71 to 1.00, while all of the included studies showed perfect specificity (1.00). Pooled measures for sensitivity and specificity were 0.95 [95% CI 0.89, 1.00] and 1.00 [95% CI 0.95, 1.00], respectively. The diagnostic odds ratio was 4.05, and the AUC for SROC curve was calculated as 0.979. BRAF V600E-specific IHC using VE1 antibody showed extremely high sensitivity and specificity when compared with molecular tests in the detection of the mutation in ameloblastomas.

BRAF V600E mutations in right-side colon cancer: Heterogeneity detected by liquid biopsy

IntroductionThe prognosis for metastatic colorectal cancer patients (mCRC) with the BRAFV600E mutation is poor. BRAFV600E mutation frequency is reportedly low among Asians; however, the frequency of the BRAFV600E mutation in right-side colon cancer may not be low, even among Asians. In addition, spatial heterogeneity of BRAFV600E mutations also exists, as for RAS mutations. In this prospective observational study, we evaluated BRAFV600E mutations in cancer tissue and plasma of Japanese right-side colon cancer patients. Method215 patients with right-side colon cancer were included. BRAFV600E mutations of cancer tissue and plasma were detected using droplet digital PCR. Blood plasma of patients with BRAFV600E mutations in cancer tissue or plasma was drawn at intervals throughout chemotherapy, and BRAFV600E mutations were evaluated. ResultsBRAFV600E mutations were detected in tissue samples from 35 of 215 patients (16.3%, cecum; 22.4%, ascending colon; 17.8%, and transverse colon; 9.0%). BRAFV600E mutations were detected in plasma of 10 of 215 (4.7%) patients. Eight of the ten patients had BRAFV600E mutations in their primary tumours, but two (both were Stage IV) patients did not. Sensitivity of liquid biopsy to detect BRAFV600E mutations was 10.3% (3/29) in Stage I-III patients and 83.3% (5/6) in Stage IV patients. ConclusionBRAFV600E mutations are observed in right-side colon cancer at high frequency, especially in the cecum. BRAFV600E mutations can be detected in plasma and the detection rate is high in patients with advanced cancer. Spatial heterogeneity was observed using liquid biopsy.

BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

A dual identification strategy based on padlock ligation and CRISPR/Cas14a for highly specific detection of BRAF V600E mutation in clinical samples.

BRAF V600E mutation is a single-nucleotide variation (SNV) that is widely found in various cancers and has been demonstrated to have a strong association with the prognosis and development of some diseases. Thus, we developed a strategy based on rolling circle amplification (RCA) and CRISPR/Cas14a to meet the great need for detecting highly specific BRAF V600E mutation in fine-needle biopsy samples. In this study, a padlock probe was designed to recognize and trigger subsequent ligase chain reactions (LCR). And due to the Taq DNA ligase, a great number of ligated annular padlock probes were generated in the presence of BRAF V600E mutation, subsequently generating long repeated single-strand DNA by RCA. The obtained amplicons were activators triggering the trans-cleavage of CRISPR/Cas14a. CRISPR/Cas14a shows outstanding performance in identifying ssDNA with single base mutation, which significantly increases the specificity of mutation discrimination. Under the optimal conditions, our strategy can identify BRAF V600E mutation down to 0.307 fM with a wide linear range from 1 fM to 10 pM. On the other hand, the dual identification strategy endows the method with terrific specificity for the detection of SNV. Furthermore, our method has been successfully employed to identify BRAF V600E mutation in clinical fine-needle aspiration samples, proving great potential for ultra-specific identification of low abundance BRAF V600E mutation and providing a novel method for diagnosis and treatment of cancer.

Four Variations Detected in 3 Genes Related to Thrombophilia by Multiplex ARMS – PCR

Thrombophilia is a condition that may cause venous obstruction, leading to serious illnesses such as stroke, respiratory failure, and even death. This condition is hereditary or related to the immune system. Inherited thrombophilia is screened by analysing genetic variants (allele). In this study, we analyse 4 variations in 3 genes, which occur at high variable frequency, namely Methytetrahydrofolate Reductase (MTHFR C677T, MTHFR A1298C), Factor V Leiden (FVL C1691T) and Plasminogen Activator Inhibitor 1 (PAI-1 5G/4G), using allele-specific polymerase chain reaction (ARMS-PCR). The primers were designed using FastPCR program and PCR conditions were optimized to identify 8 alleles of 4 variants through 3 multiplex ARMS - PCR reactions. The accuracy of the ARMS - PCR results was confirmed by Sanger sequencing and the real time PCR commercial kit (SNP). Mastering the allele specific PCR technique contributes to the survey of genetic variations in the Vietnamese population in order to screen and treat thrombophilia-related diseases.&#x0D; Keywords&#x0D; Thrombophilia, allele-specific polymerase chain reaction (ARMS-PCR), variants MTHFR C677T, MTHFR A1298C, FVL C1691T, PAI-1 4G/5G.&#x0D; References&#x0D; [1] A. Dautaj, G. Krasi, V. Bushati et al., Hereditary Thrombophilia, Acta Biomedica, Vol. 90, No. 10S, 2019, pp. 44-46, https://doi.org/10.23750/abm.v90i10-S.8758.[2] M. G. Beckman, W. C. Hooper, S. E. Critchley, T. L. Ortel, Venous Thromboembolism: A Public Health Concern, American Journal of Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S495-S501, https://doi.org/10.1016/j.amepre.2009.12.017.[3] G. E. Raskob, R. Silverstein, D. W. Bratzler et al., Surveillance for Deep Vein Thrombosis and Pulmonary Embolism: Recommendations from A National Workshop, American Journal Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S502-S509, https://doi.org/10.1016/j.amepre.2010.01.010.[4] J. A. Heit, M. D. Silverstein, D. N. Mohr et al., The Epidemiology of Venous Thromboembolism in the Community, Thrombosis and Haemostasis, Vol. 86, No. 1, 2001, pp. 452-463, https://doi.org/10.1055/s-0037-1616243.[5] M. Cushman, A. W. Tsai, R. H. White et al., Deep Vein Thrombosis and Pulmonary Embolism in Two Cohorts: The Longitudinal Investigation of Thromboembolism Etiology, The American Journal of Medicine, Vol. 117, No. 1, 2004, pp. 19-25, https://doi.org/10.1016/j.amjmed.2004.01.018.[6] J. A. Heit, The Epidemiology of Venous Thromboembolism in the Community: Implications for Prevention and Management, Journal of Thrombosis and Thrombolysis, Vol. 21, No. 1, 2006, pp. 23-29, https://doi.org/10.1007/s11239-006-5572-y.[7] R. H. White, The Epidemiology of Venous Thromboembolism, Circulation, Vol. 107, No. 23 S1, 2003, pp. I4-I8, https://doi.org/10.1161/01.cir. 0000078468.11849.66.[8] A. Wahed, A. Dasgupta, Thrombophilias and Their Detection, Hematology and Coagulation: A Comprehensive Review for Board Preparation, Certification and Clinical Practice, Elsevier, United States of America, 2015, pp. 263-275.[9] SNP Biotechnology, SNP Detection Real-Time PCR Kits, http://www.snp.com.tr/EN,37/snp-detection-real-time-pcr-kits.html/, 2021 (accessed on: May 1st, 2021).[10] V. S. Le, K. T. Tran, H. T. P. Bui et al., A Vietnamese Human Genetic Variation Database, Human Mutation, Vol. 40, No. 10, 2019,pp. 1664-1675, https://doi.org/10.1002/humu.23835.[11] dbSNP – NCBI, Reference SNP (rs) Report (rs6025, rs1799762), https://www.ncbi.nlm.nih.gov/snp/,02021 2021 (accessed on: May 1st, 2021).[12] D. C. Rees, M. Cox, J. B. Clegg, World Distribution of Factor V Leiden, The Lancet, Vol. 346, No. 8983, 1995, pp. 1133-1134, https://doi.org/10.1016/s0140-6736(95)91803-5. A. Fateh, M. Aghasadeghi, S. D. Siadat et al., Comparison of Three Different Methods for Detection of IL28 rs12979860 Polymorphisms as A Predictor of Treatment Outcome in Patients with Hepatitis C Virus, Osong Public Health and Research Perspectives, Vol. 7, No. 2, 2016, pp. 83-89, https://doi.org/10.1016/j.phrp.2015.11.004. [13] T. Huang, J. Zhuge, W. W. Zhang, Sensitive Detection of BRAF V600E Mutation by Amplification Refractory Mutation System (ARMS)-PCR, Biomarker Research, Vol. 1, No. 3, 2013, https://doi.org/10.1186/2050-7771-1-3.[14] R. F. V. Medrano, C. A. D. Oliveira, Guidelines for the Tetra-primer ARMS-PCR Technique Development, Molecular Biotechnology, Vol. 56, 2014, pp. 599-608, https://doi.org/10.1007/s12033-014-9734-4.[15] C. Runcharoen, K. Fukunaga, I. Sensorn et al., Prevalence of Pharmacogenomic Variants in 100 Pharmacogenes Among Southeast Asian Populations under Tarmacogenomics Research Network (SEAPharm), Human Genome Variation, Vol. 8, No. 7, 2021, https://doi.org/10.1038/s41439-021-00135-z.

Detection of BRAF V600E mutation in metastatic colorectal carcinoma : AQuIP round robin test

Ringversuche sind ein wichtiges Instrument zur Qualitätssicherung. Dies betrifft in zunehmendem Maße auch die molekulare Diagnostik in der Pathologie, von deren Ergebnissen Therapieentscheidungen in der Präzisionsonkologie direkt abhängen. Beim metastasierten kolorektalen Karzinom (mKRK) stand bisher der Nachweis von KRAS-und NRAS-Mutationen im Vordergrund, deren Abwesenheit eine Therapie mit EGFR-blockierenden Antikörpern ermöglicht. Nun ist BRAF als weiterer prädiktiver Marker hinzugekommen, da mKRK Patienten mit einer BRAF-V600E-Mutation nach systemischer Vortherapie von einer Behandlung mit Encorafenib (einem BRAF-Inhibitor) in Kombination mit Cetuximab (Anti-EGFR-Antikörper) profitieren. Aufgrund der 2020 erfolgten Zulassung für diese Behandlung ist es wichtig, dass der diagnostische Nachweis einer BRAF-V600E-Mutation zuverlässig in den Pathologien durchgeführt werden kann. Daher wurde dieser Ringversuch durchgeführt, bei dem der Nachweis der BRAF-V600E-Mutation entweder mittels Immunhistochemie oder molekularer Verfahren erfolgen konnte. Die Ergebnisse des Ringversuchs belegen eindeutig, dass derzeit die molekulare BRAF-V600E-Bestimmung dem immunhistologischen Nachweis überlegen ist.

Glucometer‐based Ultra‐sensitive BRAF V600E Mutation Detection Facilitated by Magnetic Nanochains and a Self‐made Point‐of‐Care (POC) Device

AbstractWe realized the user‐friendly and field‐based point‐of‐care detection on a self‐made portable device with the aid of personal glucose meter (PGM) for single nucleotide polymorphisms (SNPs) of melanoma and colon cancer marker BRAF V600E with good sensitivity and selectivity by integrating the loop‐mediated isothermal amplification (LAMP) reaction and the strand exchange signal transduction. The device is equipped with a rotating magnetic field as well as a heating element, under which the pre‐added magnetic nanochains (MNCs) help to homogenize the reaction mixture and overcome the low diffusion efficacy of the reactant and the required temperature for different reactions can be met.

Predicting BRAF V600E mutation in glioblastoma: utility of radiographic features

Detection of BRAF V600E mutation in glioblastomas (GBMs) is important because of potential therapeutic implications. Still, the relative paucity of these mutations makes molecular detection in all GBMs controversial. In the present study, we analyzed clinical, radiographic and pathologic features of 12 BRAF V600E-mutant GBMs and 12 matched controls from 2 institutions. We found that a majority of BRAF V600E-mutant GBMs displayed a combination of well-circumscribed lesions, large cystic components with thin walls and solid cortical component on MRI, but with some overlap with matched BRAF wildtype controls (p = 0.069). BRAF V600E-mutant GBMs were also apt to gross total resection (83% vs 17%, p = 0.016) and morphologically displayed epithelioid features (83% vs 0%, p < 0.0001). Identification of these clinical, radiographic, and pathologic characteristics should prompt testing for BRAF V600E in IDH-wildtype GBM.

Correlation between US-FNAC with BRAF V600E Mutation Analysis and Central Neck Lymph Node Metastasis in cN0 Papillary Thyroid Cancer.

The aim of this study was to explore the correlation between ultrasound-guided fine-needle aspiration cytology(US-FNAC) combined with BRAF V600E mutation analysis and central neck lymph node metastasis in cN0 papillary thyroid cancer, so as to provide reliable molecular evidence to use it for preoperative evaluation, operation procedure design, and postoperative follow-up planning in clinic. Specimens were obtained from 250 patients with cN0 thyroid cancer (TI-RADS≥4a, highly suspected of PTC by US-FNAC) after bilateral thyroidectomy and central neck lymph node dissection with accessible postoperative pathologic results of PTC and central neck lymph nodes and used for cytological diagnosis by H&E stain and BRAF V600E mutation detection. Single-factor analysis showed that differences between the central neck lymph node metastasis and nonmetastasis groups were statistically significant in gender, BRAF V600E mutation, and extracapsular extension. Logistic multivariate regression analysis showed significant differences in gender, BRAF V600E mutation, and extracapsular extension. Positive BRAF V600E mutations by US-FNAC, extracapsular extension, and male gender are risk factors of central neck lymph node metastasis in cN0 PTC metastatic PTC to central neck lymph node. Patients with those factors should undergo prophylactic central neck lymph node dissection.

Predictive Evaluation on Cytological Sample of Metastatic Melanoma: The Role of BRAF Immunocytochemistry in the Molecular Era.

Background: Cutaneous malignant melanoma is an aggressive neoplasm. In advanced cases, the therapeutic choice depends on the mutational status of BRAF. Fine needle aspiration cytology (FNA) is often applied to the management of patients affected by melanoma, mainly for the diagnosis of metastases. The evaluation of BRAF mutational status by sequencing technique on cytological samples may be inconvenient, as it is a time and biomaterial-consuming technique. Recently, BRAF immunocytochemistry (ICC) was applied for the evaluation of BRAF V600E mutational status. Although it may be useful mainly in cytological samples, data about BRAF ICC on cytological samples are missing. Methods: We performed BRAF ICC on a series of 50 FNA samples of metastatic melanoma. BRAF molecular analysis was performed on the same cytological samples or on the corresponding histological samples. Molecular analysis was considered the gold standard. Results: BRAF ICC results were adequate in 49 out of 50 (98%) cases, positive in 15 out of 50 (30%) cases and negative in 34 out of 50 (68%) of cases. Overall, BRAF ICC sensitivity, specificity, positive predictive value and negative predictive value results were 88.2%, 100%, 100% and 94.1%, respectively. The diagnostic performance of BRAF ICC results was perfect when molecular evaluation was performed on the same cytological samples. Hyperpigmentation represents the main limitation of the technique. Conclusions: BRAF ICC is a rapid, cost-effective method for detecting BRAF V600E mutation in melanoma metastases, applicable with high diagnostic performance to cytological samples. It could represent the first step to evaluate BRAF mutational status in cytological samples, mainly in poorly cellular cases.

Machine learning algorithm improved automated droplet classification of ddPCR for detection of BRAF V600E in paraffin-embedded samples

Somatic mutations in cancer driver genes can help diagnosis, prognosis and treatment decisions. Formalin-fixed paraffin-embedded (FFPE) specimen is the main source of DNA for somatic mutation detection. To overcome constraints of DNA isolated from FFPE, we compared pyrosequencing and ddPCR analysis for absolute quantification of BRAF V600E mutation in the DNA extracted from FFPE specimens and compared the results to the qualitative detection information obtained by Sanger Sequencing. Sanger sequencing was able to detect BRAF V600E mutation only when it was present in more than 15% total alleles. Although the sensitivity of ddPCR is higher than that observed for Sanger, it was less consistent than pyrosequencing, likely due to droplet classification bias of FFPE-derived DNA. To address the droplet allocation bias in ddPCR analysis, we have compared different algorithms for automated droplet classification and next correlated these findings with those obtained from pyrosequencing. By examining the addition of non-classifiable droplets (rain) in ddPCR, it was possible to obtain better qualitative classification of droplets and better quantitative classification compared to no rain droplets, when considering pyrosequencing results. Notable, only the Machine learning k-NN algorithm was able to automatically classify the samples, surpassing manual classification based on no-template controls, which shows promise in clinical practice.

Frequency detection of BRAF V600E mutation in a cohort of pediatric langerhans cell histiocytosis patients by next-generation sequencing

BackgroundLangerhans cell histiocytosis (LCH) is a rare neoplastic disease that occurs in both children and adults, and BRAF V600E is detected in up to 64% of the patients. Several studies have discussed the associations between BRAF V600E mutation and clinicopathological manifestations, but no clear conclusions have been drawn regarding the clinical significance of the mutation in pediatric patients.ResultsWe retrieved the clinical information for 148 pediatric LCH patients and investigated the BRAF V600E mutation using next-generation sequencing alone or with droplet digital PCR. The overall positive rate of BRAF V600E was 60/148 (41%). The type of sample (peripheral blood and formalin-fixed paraffin-embedded tissue) used for testing was significantly associated with the BRAF V600E mutation status (p-value = 0.000 and 0.000). The risk of recurrence declined in patients who received targeted therapy (p-value = 0.006; hazard ratio 0.164, 95%CI: 0.046 to 0.583). However, no correlation was found between the BRAF V600E status and gender, age, stage, specific organ affected, TP53 mutation status, masses close to the lesion or recurrence.ConclusionsThis is the largest pediatric LCH study conducted with a Chinese population to date. BRAF V600E in LCH may occur less in East Asian populations than in other ethnic groups, regardless of age. Biopsy tissue is a more sensitive sample for BRAF mutation screening because not all of circulating DNA is tumoral. Approaches with low limit of detection or high sensitivity are recommended for mutation screening to avoid type I and II errors.

Diagnostic value of puncture feeling combined with BRAF V600E mutation in repeat US-FNA biopsy of Bethesda III thyroid nodules.

The diagnosis and treatment of Bethesda III thyroid nodules has always been controversial. Our aim is to study the value of puncture feeling combined with BRAF V600E mutation detection in the diagnosis of Bethesda III thyroid nodules in repeat fine needle aspiration (FNA). From January 1, 2017 to December 31, 2021, a total of 1,114 thyroid nodules were included, of which Bethesda III thyroid nodules accounted for 12.1%. We analyzed the correlation between puncture feeling and postoperative pathology. Then, the diagnostic value of puncture feeling in Bethesda III thyroid nodules and the mutation rate of BRAF V600E in repeated FNA and its correlation with puncture feeling were analyzed. The results showed that there was a significant correlation between puncture feeling and postoperative pathology in the 1114 thyroid nodules (P<0.001). Additionally, 93.4% (299/320) of the thyroid nodules with soft puncture feeling were benign, 89.0% (460/517) of the thyroid nodules with hard puncture feeling were malignant, and 93.1% (258/277) of the thyroid nodules with a puncture feeling of hard with grittiness were malignant. There was high consistency of puncture feelings among different operators (P<0.001). The distribution of puncture feeling between benign and malignant thyroid nodules was significantly different (P<0.001) in Bethesda III thyroid nodules. In the puncture feeling ROC curve of Bethesda III thyroid nodules based on pathology, the AUC was 0.895 (95% CI: 0.817-0.973, P<0.001). In repeat FNA, BRAF V600E was significantly correlated with puncture feeling (P<0.001). BRAF V600E mutation was found in 22 (68.8%) malignant nodules. Puncture feelings of hard and hard with grittiness were malignant risk factors. Repeated FNA and BRAF V600 mutation detection should be performed for Bethesda III thyroid nodules with a hard or hard with grittiness puncture feeling.

Highly Sensitive Detection of Low-Abundance BRAF V600E Mutation in Fine-Needle Aspiration Samples by Zip Recombinase Polymerase Amplification.

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

Blood-Based Detection of BRAF V600E in Gliomas and Brain Tumor Metastasis.

Simple SummaryThe BRAF V600E mutation has been identified as a key driver in brain tumors and brain tumor metastasis. The ability to detect this mutation in a minimally invasive plasma assay offers advantages over traditional tissue-based biopsy for the disease diagnosis and monitoring. The aim of this study was to develop an assay for the detection of BRAF V600E in the plasma of patients with brain tumors and brain tumor metastasis. We demonstrate BRAF V600E detection using a novel plasma-based ddPCR assay. We detect the mutation in circulating nucleic acids in 4/5 patients with mutant gliomas and metastatic melanoma. We also show correlation between plasma BRAF V600E and clinical status. This proof of principle study is important in the context of application of liquid biopsy in plasma to the neuro-oncologic field. The assay may be useful as a diagnostic adjunct, prognostication tool, and method for monitoring of disease and treatment response.Liquid biopsy provides a minimally invasive platform for the detection of tumor-derived information, including hotspot mutations, such as BRAF V600E. In this study, we provide evidence of the technical development of a ddPCR assay for the detection of BRAF V600E mutations in the plasma of patients with glioma or brain metastasis. In a small patient cohort (n = 9, n = 5 BRAF V600E, n = 4 BRAF WT, n = 4 healthy control), we were able to detect the BRAF V600E mutation in the plasma of 4/5 patients with BRAF V600E-tissue confirmed mutant tumors, and none of the BRAF WT tumors. We also provide evidence in two metastatic patients with longitudinal monitoring, where the plasma-based BRAF V600E mutation correlated with clinical disease status. This proof of principle study demonstrates the potential of this assay to serve as an adjunctive tool for the detection, monitoring, and molecular characterization of BRAF mutant gliomas and brain metastasis.

Clinical, radiological and genomic features and targeted therapy in BRAF V600E mutant adult glioblastoma.

Although uncommon, detection of BRAF V600E mutations in adult patients with glioblastoma has become increasingly relevant given the widespread application of molecular diagnostics and encouraging therapeutic activity of BRAF/MEK inhibitors. We performed a retrospective study of adult glioblastoma patients treated at Dana-Farber Cancer Institute/Brigham and Women's Hospital or Massachusetts General Hospital from January 2011 to July 2019 with an identified BRAF V600E mutation by either immunohistochemistry or molecular testing. Patient characteristics, molecular genomics, and preoperative MRI were analyzed. Nineteen glioblastoma patients were included, with median age at diagnosis of 41-years-old (range 22-69). Only 1/18 was IDH1/2-mutant; 10/17 had MGMT unmethylated tumors. The most common additional molecular alterations were CDKN2A/2B biallelic loss/loss-of-function (10/13, 76.9%), polysomy 7 (8/12, 66.7%), monosomy 10 (5/12, 41.7%), PTEN biallelic loss/loss-of-function (5/13, 38.5%) and TERT promoter mutations (5/15, 33.3%). Most tumors were well-circumscribed (11/14) and all were contrast-enhancing on MRI. Twelve patients eventually developed subependymal or leptomeningeal dissemination. Six patients were treated with BRAF/MEK inhibition following disease progression after standard of care therapy, with 4/6 patients showing partial response or stable disease as best response. Median time to progression after BRAF/MEK inhibition was 6.0months (95% CI 1.2-11.8). Grade 1 skin rash was present in 2 patients, but no other adverse events were reported. Median OS for the entire cohort was 24.1months (95% CI 15.7-38.9). Understanding the natural history and features of BRAF V600E glioblastoma may help better identify patients for BRAF/MEK inhibition and select therapeutic strategies.

Paediatric Gliomas: BRAF and Histone H3 as Biomarkers, Therapy and Perspective of Liquid Biopsies.

Simple SummaryGliomas are major causes of worldwide cancer-associated deaths in children. Generally, paediatric gliomas can be classified into low-grade and high-grade gliomas. They differ significantly from adult gliomas in terms of prevalence, molecular alterations, molecular mechanisms and predominant histological types. The aims of this review article are: (i) to discuss the current updates of biomarkers in paediatric low-grade and high-grade gliomas including their diagnostic and prognostic values, and (ii) to discuss potential targeted therapies in treating paediatric low-grade and high-grade gliomas. Our findings revealed that liquid biopsy is less invasive than tissue biopsy in obtaining the samples for biomarker detections in children. In addition, future clinical trials should consider blood-brain barrier (BBB) penetration of therapeutic drugs in paediatric population.Paediatric gliomas categorised as low- or high-grade vary markedly from their adult counterparts, and denoted as the second most prevalent childhood cancers after leukaemia. As compared to adult gliomas, the studies of diagnostic and prognostic biomarkers, as well as the development of therapy in paediatric gliomas, are still in their infancy. A body of evidence demonstrates that B-Raf Proto-Oncogene or V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF) and histone H3 mutations are valuable biomarkers for paediatric low-grade gliomas (pLGGs) and high-grade gliomas (pHGGs). Various diagnostic methods involving fluorescence in situ hybridisation, whole-genomic sequencing, PCR, next-generation sequencing and NanoString are currently used for detecting BRAF and histone H3 mutations. Additionally, liquid biopsies are gaining popularity as an alternative to tumour materials in detecting these biomarkers, but still, they cannot fully replace solid biopsies due to several limitations. Although histone H3 mutations are reliable prognosis biomarkers in pHGGs, children with these mutations have a dismal prognosis. Conversely, the role of BRAF alterations as prognostic biomarkers in pLGGs is still in doubt due to contradictory findings. The BRAF V600E mutation is seen in the majority of pLGGs (as seen in pleomorphic xanthoastrocytoma and gangliomas). By contrast, the H3K27M mutation is found in the majority of paediatric diffuse intrinsic pontine glioma and other midline gliomas in pHGGs. pLGG patients with a BRAF V600E mutation often have a lower progression-free survival rate in comparison to wild-type pLGGs when treated with conventional therapies. BRAF inhibitors (Dabrafenib and Vemurafenib), however, show higher overall survival and tumour response in BRAF V600E mutated pLGGs than conventional therapies in some studies. To date, targeted therapy and precision medicine are promising avenues for paediatric gliomas with BRAF V600E and diffuse intrinsic pontine glioma with the H3K27M mutations. Given these shortcomings in the current treatments of paediatric gliomas, there is a dire need for novel therapies that yield a better therapeutic response. The present review discusses the diagnostic tools and the perspective of liquid biopsies in the detection of BRAF V600E and H3K27M mutations. An in-depth understanding of these biomarkers and the therapeutics associated with the respective challenges will bridge the gap between paediatric glioma patients and the development of effective therapies.

A study of pathological characteristics and BRAF V600E status in Langerhans cell histiocytosis of Vietnamese children.

BackgroundLangerhans cell histiocytosis (LCH) is more common in children than adults and involves many organs. In children, the BRAF V600E mutation is associated with recurrent and high-risk LCH.MethodsWe collected paraffin blocks of 94 pediatric LCH patients to detect BRAF_V600E mutation by sequencing. The relationship between BRAF V600E status and clinicopathological parameters were also critically analyzed.ResultsBRAF V600E mutation exon 15 was detected in 45 cases (47.9%). Multiple systems LCH showed a significantly higher BRAF_V600E mutation rate than a single system (p=.001). No statistical significance was evident for other clinical characteristics such as age, sex, location, risk organs involvement, and CD1a expression.ConclusionsIn Vietnamese LCH children, the proportion of BRAF V600E mutational status was relatively high and related to multiple systems.