BRAF T1799A Research Articles

Increasing the Capture Rate of Circulating Tumor DNA in Unaltered Plasma Using Passive Microfluidic Mixer Flow Cells.

A limiting factor in using blood-based liquid biopsies for cancer detection is the volume of extracted blood required to capture a measurable number of circulating tumor DNA (ctDNA). To overcome this limitation, we developed a technology named the dCas9 capture system to capture ctDNA from unaltered flowing plasma, removing the need to extract the plasma from the body. This technology has provided the first opportunity to investigate whether microfluidic flow cell design can affect the capture of ctDNA in unaltered plasma. With inspiration from microfluidic mixer flow cells designed to capture circulating tumor cells and exosomes, we constructed four microfluidic mixer flow cells. Next, we investigated the effects of these flow cell designs and the flow rate on the rate of captured spiked-in BRAF T1799A (BRAFMut) ctDNA in unaltered flowing plasma using surface-immobilized dCas9. Once the optimal mass transfer rate of ctDNA, identified by the optimal ctDNA capture rate, was determined, we investigated whether the design of the microfluidic device, flow rate, flow time, and the number of spiked-in mutant DNA copies affected the rate of capture by the dCas9 capture system. We found that size modifications to the flow channel had no effect on the flow rate required to achieve the optimal capture rate of ctDNA. However, decreasing the size of the capture chamber decreased the flow rate required to achieve the optimal capture rate. Finally, we showed that, at the optimal capture rate, different microfluidic designs using different flow rates could capture DNA copies at a similar rate over time. In this study, the optimal capture rate of ctDNA in unaltered plasma was identified by adjusting the flow rate in each of the passive microfluidic mixer flow cells. However, further validation and optimization of the dCas9 capture system are required before it is ready to be used clinically.

BRAF V600E protect from cell death via inhibition of the mitochondrial permeability transition in papillary and anaplastic thyroid cancers.

BRAF T1799A mutation is the most common genetic variation in thyroid cancer, resulting in the production of BRAF V600E mutant protein reported to make cells resistant to apoptosis. However, the mechanism by which BRAF V600E regulates cell death remains unknown. We constructed BRAF V600E overexpression and knockdown 8505C and BCPAP papillary and anaplastic thyroid cancer cell to investigate regulatory mechanism of BRAF V600E in cell death induced by staurosporine (STS). Induced BRAF V600E expression attenuated STS‐induced papillary and anaplastic thyroid cancer death, while BRAF V600E knockdown aggravated it. TMRM and calcein‐AM staining showed that opening of the mitochondrial permeability transition pore (mPTP) during STS‐induced cell death could be significantly inhibited by BRAF V600E. Moreover, our study demonstrated that BRAF V600E constitutively activates mitochondrial ERK (mERK) to inhibit GSK‐3‐dependent CypD phosphorylation, thereby making BRAF V600E mutant tumour cells more resistant to mPTP opening. In the mitochondria of BRAF V600E mutant cells, there was an interaction between ERK1/2 and GSKa/ß, while upon BRAF V600E knockdown, interaction of GSKa/ß to ERK was decreased significantly. These results show that in thyroid cancer, BRAF V600E regulates the mitochondrial permeability transition through the pERK‐pGSK‐CypD pathway to resist death, providing new intervention targets for BRAF V600E mutant tumours.

Capturing ctDNA from Unaltered Stationary and Flowing Plasma with dCas9.

Many studies have established that blood-based liquid biopsies can be used to detect cancer in its early stages. However, the limiting factor for early cancer detection is the volume of blood required to capture the small amount of circulating tumor DNA (ctDNA). An apheresis machine is a device that can draw whole blood, separate the blood components, and infuse the blood components back into the individual. This device provides the opportunity to screen large volumes of plasma without extracting it from the body. However, current DNA capture technologies require the plasma to be altered before the ctDNA can be captured. Our goal was to develop the first technology that can capture ctDNA from flowing unaltered plasma. To simulate cancer patient plasma, we spiked BRAF T1799A (BRAFMut) DNA into plasma from healthy individuals. We used catalytically dead Cas9 (dCas9), guide RNA, and allele-specific quantitative polymerase chain reaction (qPCR) to capture and measure the number of captured BRAFMut DNA copies. We found that dCas9 captured BRAFMut alleles with equal efficiency at room temperature (25 °C) and body temperature (37 °C). Next, we showed that, in stationary unaltered plasma, dCas9 was as efficient in capturing BRAFMut as a commercial cell-free DNA (cfDNA) capture kit. However, in contrast to the cfDNA capture kit, dCas9 enriched BRAFMut by 1.8-3.3-fold. We then characterized the dCas9 capture system in laminar and turbulent flowing plasma. We showed that the capture rate using turbulent flow was greater than that in laminar flow and stationary plasma. With turbulent flow, the number of captured BRAFMut copies doubles with time (slope = -1.035 Ct) and is highly linear (R2 = 0.874). While we showed that the dCas9 capture system can capture ctDNA from unaltered flowing plasma, further optimization and validation of this technology is required before its clinical utility can be determined.

Prospective Study of Bethesda Categories III and IV Thyroid Nodules: Outcomes and Predictive Value of BRAFV600E Mutation.

Introduction:Atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) of The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) has emerged as the most controversial category because of its heterogeneity and inconsistent reporting. There is a definite paucity in data available from the Indian subcontinent about the outcome of nodules carrying Bethesda category III or Bethesda category IV diagnoses.Aims and Objectives:The primary objective of our study was to determine the malignancy rate in Bethesda categories III and IV nodules. The secondary objectives were to determine predictive value of BRAFV600E mutation analysis on indeterminate thyroid nodules, predictive value of Thyroid image reporting and data system (TI-RADS) in malignancy prediction and to study the common histological variants in indeterminate nodules.Materials and Methods:This prospective study included 176 consecutive nodules of Bethesda categories III and IV, diagnosed over a period of 2 years from August 2015 to August 2017. A part of the fine needle aspirate was used to perform the BRAFV600E mutation analysis. The malignancy risk associated with these Fine needle aspiration Cytology categories were discussed with the patients. Those with Bethesda category IV diagnosis was advised surgery, whereas those with Bethesda category III were given the options of close follow-up with repeat FNA in 3 months or immediate surgery.Results:In our prospective study, there were 176 consecutive samples of categories III (140/79.5%) and IV (36/20.5%). Seventy-five (53.6%) category III nodules and 29 (80.6%) category IV nodules underwent immediate surgical excision. Fifty-five consecutive indeterminate cytology nodules were subjected for BRAFV600E. One of the samples was found to be positive for BRAF T1799A (V600E) mutation. The second sample harboured a missense mutation at position 1819 (TCC--GCC), wherein the codon 607 (TCC) coding for serine was substituted by alanine (GCC) which is a variant of unknown significance. In our study, the malignancy rate of Bethesda categories III and IV, which were triaged for immediate surgery were 54.6% and 72.4%, respectively.Conclusion:Malignancy rate in Category III at our center was much higher than that described by ATA and by other studies published from centers around the world, including the only two studies from India. In view of the strikingly high malignancy rate in these indeterminate nodules, strong consideration to surgery should be given to patients with FNA results suggesting these two categories. BRAFV600E mutation analysis in FNA specimen has limited utility in improving the preoperative diagnostic rate for malignancy.

Low Prevalence of TERT Promoter, BRAF and RAS Mutations in Papillary Thyroid Cancer in the Greek Population.

Thyroid cancer is a common endocrine malignancy and displays a variety of histological patterns ranging from adenoma to malignant tumors. Molecular diagnostics have given significant insights into the genetic basis of thyroid tumorigenesis, known to be linked with signaling pathways affected by oxidative stress. We report for the first time a genotype study of TERT promoter combined with BRAF and RAS mutations in Papillary Thyroid Cancer (PTC) cases in the Greek population. Polymerase Chain Reaction and sequencing were used to identify TERT promoter (C228T, C250T, CC243-243TT) mutations, the BRAF (T1799A) mutation and mutations in codons 12, 13, 61 of the HRAS, KRAS and NRAS genes. The most common C228T TERT promoter mutation was identified in 2 PTC cases co-existing with the BRAF mutation. The BRAF T1799A mutation was detected in 10 PTC cases, while two different NRAS mutations in codon 61 (C181A and A182G) were found in 2 PTC cases. These mutations occur in a mutually exclusive manner. Our results indicate that despite the low frequencies, the study of the specific mutations should be encouraged because they are indicative of aggressive forms of thyroid cancer of the papillary histotype in this patient cohort, thus providing insights towards their therapeutic management.

Simultaneous Presentation of Cerebellopontine Angle Pleomorphic Xanthoastrocytoma and Malignant Melanoma in a Known Case of Neurofibromatosis 1; Probable Role of BRAF Gene: A Case Report and Review of Literature

Introduction: Pleomorphic Xanthoastrocytoma (PXA) is a rare astrocytic neoplasm which mostly affects supratentorial region. The vast majority are sporadic, with only rare examples described in association with neurofibromatosis 1 (NF1), a genetic disorder with neurocutaneous manifestations. Co-occurrence of these 2 rare entities with neoplasm of melanocytes, Cutaneous Malignant Melanoma (CMM), is described in this report for the first time. Case Presentation: A 34-year-old female with established diagnosis of NF1 and left optic nerve glioma from childhood was presented with visual field impairment, decreased level of consciousness and right VI and VII cranial nerves paresis. An irregular hyperpigmented skin lesion was also noted on thoracic area, which histopathological and immunohistochemistry (IHC) study with S100 confirmed CMM. Brain MRI demonstrated high signal intensity mass in right cerebellopontine. Right retro-mastoid craniectomy and total resection of tumor was performed and histopathological investigation of excised lesion along with IHC study with glial fibrillary acidic protein (GFAP), confirmed PXA. Sanger sequencing showed positive BRAF T1799A mutation in both PXA and melanoma’s tissue. Conclusions: The association between BRAF gene mutations and human cancers such as PXA and CMM was discovered. This first ever co-occurrence of mentioned rare disorders emphasizes on probable role of BRAF gene in pathogenesis and whether its inhibitors could serve as an adjuvant agent to facilitate surgical approaches to complex lesions.

PTPS-21MAPK PATHWAY INHIBITOR THERAPY FOR PROGRESSIVE PEDIATRIC ASTROCYTOMAS

BACKGROUND: Brain tumors are the most common solid tumor in childhood and astrocytomas account for the largest proportion of these tumors. Increasing sophistication in genetic testing has allowed for the detection of specific mutations within tumor subtypes that may represent targets for individualized tumor treatment. The MAPK pathway, and more specifically, BRAF mutations have been shown to be prevalent in pediatric astrocytomas and may represent one such area to target. METHODS: We describe the clinical course of four cases of non-operable pediatric astrocytomas (Grade I or II) whose tumor progressed despite chemotherapy. Testing of each tumor showed BRAF mutations and each patient was started on either a BRAF inhibitor or MEK inhibitor. A review of each patient's medical record and imaging studies was completed. Tumor volumetrics were calculated prior to and after treatment. RESULTS: Three patients had optic pathway gliomas; two pilocytic astrocytomas (WHO Grade I) and one pilomyxoid astrocytoma (WHO Grade II). The fourth tumor was a thalamic infiltrating astrocytoma (WHO Grade II). Genetic testing showed a duplication of BRAF (7q34) in 3 tumors and treatment with a MEK inhibitor, trametinib, was initiated. One tumor was found to have a BRAF T1799A (V600E) point mutation and the patient was started on vemurafenib, a BRAF inhibitor. Duration of treatment to date ranged from 105 to 805 days. Pretreatment tumor volumes varied from 15.1cm3-451.8cm3. All four tumors had a decrease in volume (2.1cm3-227.2cm3, 13.5-80.6% decrease) on these inhibitors. CONCLUSION: We have shown in this small subgroup of patients that MEK inhibitors and BRAF inhibitors in tumors with BRAF mutations are effective in decreasing tumor volume in otherwise progressive pediatric astrocytomas. This would suggest these may be ideal targets for treatment of tumors with these mutations and that tumor volume measurement is a reasonable method for documenting response in this patient group.

Frequency assessment of BRAF mutation, KRas mutation, and RASSF1A methylation in nodular goitre based on fine-needle aspiration cytology specimens Ocena częstości występowania mutacji genów BRAF, KRas oraz.

Standard pre-operative diagnosis of nodular goitre is not always conclusive. The decision about nodular goitre surgery is increasingly based on molecular methods. The aim of the study was to determine BRAF T1799A mutation and KRas proto-oncogene mutation, and the analysis of RASSF1A promoter methylation level in cytological material obtained from FNAB specimens of thyroid nodules. The study population consisted of 85 women and 12 men. The study material was genomic DNA isolated from peripheral blood and thyroid bioptates. Pyrosequencing was used for the evaluation of RASSF1 methylation level. KRas mutation was investigated with Sanger sequencing. BRAF mutation was analysed by standard methods of real-time amplification detection (real-time PCR) with the use of specific starters surrounding the mutated site. A significant positive correlation was demonstrated between mean methylation of four CpG islands of RASSF1A gene and thyroid tumour volume and its largest diameter (p < 0.05). KRas mutation was not detected in any of the 97 patients. In 7/85 subjects (8.2%) BRAF mutation was observed. In 6/7 patients with BRAF mutation, FNAB of thyroid nodules confirmed a benign nature of the lesions; the material was non-diagnostic in one patient, and papillary thyroid cancer was diagnosed on the basis of postoperative histopathology assessment. The results of genetic tests reported in our study indicate that the presence of BRAF mutation or higher RASSF1A methylation levels in FNAB cytology specimens of benign lesions may be useful in the assessment of oncological risk, while the evaluation of KRas proto-oncogene mutation is not a valuable test in pre-operative diagnosis of nodular goitre.

Surgical Perspective of T1799A BRAF Mutation Diagnostic Value in Papillary Thyroid Carcinoma

Throughout Indonesia, thyroid cancer is one of the ten commonest malignancies, with papillary thyroid carcinoma (PTC) in our hospital accounting for about 60% of all thyroid nodules. Although fine needle aspiration biopsy (FNAB) is the most reliable diagnostic tool, some nodules are diagnosed as indeterminate and second surgery is common for PTC. The aim of this study was to establish the diagnostic value and feasibility of testing the BRAF T1799A mutation on FNA specimens for improving PTC diagnosis. This prospective study enrolled 95 patients with thyroid nodules and future surgery planned. Results of mutational status were compared with surgical pathology diagnosis. Of the 70 cases included in the final analysis, 62.8% were PTC and the prevalence of BRAF mutation was 38.6%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for BRAF mutation analysis were 36%, 100%, 100% and 48%, respectively. With other data findings, nodules with "onset less than 5 year" and "hard consistency" were proven as diagnostic determinants for BRAF mutation with a probability of 62.5%. This mutation was also a significant risk factor for extra-capsular extension. Molecular analysis of the BRAF T1799A mutation in FNAB specimens has high specificity and positive predictive value for PTC. It could be used in the selective patients with clinical characteristics to facilitate PTC diagnosis and for guidance regarding extent of thyroidectomy.

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

Presencia de la mutación BRAF T1799A en el tumor primario como indicador de riesgo, recidiva o persistencia de carcinoma papilar de tiroides

Background and objective The BRAF T1799A mutation is reported to be associated to aggressive, persistent, and recurrent tumor in papillary thyroid carcinoma (PTC) patients. Association of the BRAF T1799A mutation in the primary tumor with the clinicopathological characteristics of PTC patients was analyzed. Patients, material and methods Ninety-seven PTC patients were followed up for a median of 64.1 months. The BRAF T1799A mutation was analyzed in DNA from initial thyroidectomy biopsies by PCR amplification and restriction fragment length polymorphism using TspRI enzyme. Positive cases were confirmed by DNA sequencing. Statistical association between BRAF T1799A mutation and clinicopathological characteristics was analyzed by the relevant hypothesis tests and logistic regression. Results 46.4% of patients were positive for the BRAF T1799A mutation. Bivariate and multivariate analysis showed the BRAF T1799A mutation to be only associated to age over 60 years (odds ratio [OR] = 5.5; 95% confidence interval [CI],1.4-21.9; p = 0.019) and to tumor size of 1 cm or greater (OR = 3.6, 95% CI, 1.2-10.3; p = 0.016). The BRAF T1799A mutation was not associated to histological subtype, metastasis, recurrence, more aggressive treatments (ablative I 131 therapy or surgery), or PTC persistence at the end of follow-up. Conclusions The BRAFT1799A mutation is associated to age over 60 and a tumor size of 1 cm or greater, but not to other clinicopathological characteristics, tumor recurrence or PTC persistence.

Factors influencing the detection of the BRAF T1799A mutation in papillary thyroid carcinoma

The BRAF T1799A mutation is a heterozygous point mutation and its reported prevalence in papillary thyroid carcinoma (PTC) has varied from 29 to 83%, with an overall mean of 44%. In Korea, the reported mutation rate reached 83% in PTC and 52% in micropapillary carcinoma. We hypothesized that the differences in prevalence may be influenced by the methods of mutation analysis, the sizes of tumor and ethnic differences. Three types of DNA samples from the same PTC mass (0.4-1.6 cm, mean 0.83 cm sized) per each patient (n=17) were isolated. The first type was obtained from frozen PTC tissues using laser-captured microdissection (Frozen-laser, n=17), the second was obtained from frozen tissue by manual tumor margin dissection using a blade (Frozen-blade, n=17) and the third was obtained from formalin-fixed, paraffin-embedded tissue by manual margin dissection (Paraffin-blade, n=15, 2 failed). The mutation rates of the three-matched DNA samples were compared by the SNP mode and AQ mode of pyrosequencing, and direct DNA sequencing. Both the AQ mode of pyrosequencing and the direct DNA sequencing detected the BRAF T1799A mutation in 100% of the 'Frozen-laser' samples, but the mutation was omitted in 1/17 of the 'Frozen-blade' samples and in 5/15 of the 'Paraffin-blade' samples, while the former was more rapid and objective than the latter. The SNP mode of pyrosequencing variably detected the mutation from 40 to 100%, and it showed the lowest sensitivity. Our results indicate that the reported prevalence of the BRAF T1799A mutation in PTC can be underestimated due the mutation analysis methods, and especially in small PTCs. The BRAF T1799A mutation may be an early and essential carcinogenic event in nearly all Korean PTCs, and even in micro-PTCs. For the accurate detection of the BRAF T1799A mutation from small PTCs, fresh or frozen tissues and more cautious microdissection are required, and the AQ mode of pyrosequencing assay is preferred.

The BRAFT1799A mutation confers sensitivity of thyroid cancer cells to the BRAF V600E inhibitor PLX4032 (RG7204)

Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF V600E, as a result of the BRAF T1799A mutation, plays a fundamental role in thyroid tumorigenesis. This study investigated the therapeutic potential of a BRAF V600E-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF T1799A mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF T1799A mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC 50 values (0.115–1.156 μM) in BRAF V600E mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC 50 values (56.674–1349.788 μM). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF T1799A mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF T1799A mutation-selective therapeutic agent for thyroid cancer.

A valuable adjunct to FNA diagnosis of papillary thyroid carcinoma: In-house PCR assay for BRAF T1799A (V600E)

The diagnostic approach to thyroid nodules generally starts with FNA cytology. However, approximately one-fifth of cytologic evaluations yield indeterminate cytological findings but only 20% of cases with indeterminate thyroid nodule cytology have a cancer diagnosis, emphasizing the need for an effective ancillary test based on FNA material to help prevent unnecessary surgery. Detection of BRAFV600E mutation, the genetic signature of papillary thyroid carcinoma (PTC) in FNA material provides an invaluable diagnostic adjunct to overcome the limitations of FNA cytology. There are many ways to detect V600E, such as direct DNA sequencing, allele-specific PCR and hybridization-based colorimetric methods. In this study, a newer simple PCR method is presented that removes requirements for sequencing special equipment and commercial kits. Two forward primers including the mutant sequence specific (F2), and one common reverse (R) primer were optimized to generate a 241 bp fragment (F1R), an internal PCR control, and a 141 bp fragment (F2R) denoting the presence of V600E. Sensitivity studies revealed that the assay is capable of detecting V600E even in 1 ng of DNA. Direct sequencing data of 241 bp F1R fragment proved the specificity of the assay. For validation studies of the sequence specific multiplex PCR assay, archival FNA slides were used in a group of thyroid lesions including PTC, follicular carcinoma, follicular adenoma, Hashimoto thyroiditis, and benign thyroid nodules. The newer PCR-based method presented in this study is a practical, inexpensive one-step assay to detect the BRAF T1796A mutation on FNA samples.

RET/PTC Rearrangement Occurring in Primary Peritoneal Carcinoma

RET/PTC rearrangements are initiating events in the development of a significant proportion of papillary thyroid carcinomas. Activated RET/PTC mutations are thought to be restricted to thyroid disease, but this study proposes that these events may also occur in nonthyroid tumors. A total of 57 nonthyroid papillary tumors were examined for RET/PTC rearrangements using interphase fluorescence in situ hybridization, Taqman reverse transcriptase polymerase chain reaction, and immunohistochemistry. Taqman single nucleotide polymorphism detection was used to analyze for expression of mutated BRAF T1799A. In all, 20% (3/15) of primary peritoneal carcinoma had detectable RET/PTC1 rearrangements by all 3 methodologies. A further case of similar histotype had an alternate RET/ PTC rearrangement. No RET/PTC1 rearrangements were detected in the remaining tumor cohort. All 57 tumors were homozygous for wild-type BRAF. The results indicate that RET/PTC rearrangements occur in a small subset of nonthyroid papillary tumors. These rearrangements may not be directly implicated in tumor growth; rather representing "passenger" mutations reflecting RET instability in secondary tumor subclones.

Association of the T1799A BRAF mutation with tumor extrathyroidal invasion, higher peripheral platelet counts, and over-expression of platelet-derived growth factor-B in papillary thyroid cancer

The relationship among BRAF mutation, platelet counts, and platelet-derived growth factor (PDGF) with respect to clinicopathological outcomes of papillary thyroid cancer (PTC) may play a role in PTC pathogenesis but remains undefined. We examined the T1799A BRAF mutation by direct genomic DNA sequencing in 108 primary PTC samples from a Chinese cohort and analyzed its relationship with clinicopathological, hematological, and other laboratory results as well as the levels of expression of PDGF in tumors. We found that the BRAF mutation was significantly associated with extrathyroidal invasion and advanced tumor stages III and IV. Specifically, extrathyroidal invasion was seen in 30/54 (56%) PTC with BRAF mutation versus 18/54 (33%) PTC without the mutation (P=0.02). Tumor stages III and IV were seen in 16/54 (30%) PTC with BRAF mutation versus 7/54 (13%) PTC without the mutation (P=0.04). The BRAF mutation was also significantly associated with a higher platelet count, with 249.28+/-53.76 x 10(9)/l in the group of patients with BRAF mutation versus 207.79+/-58.98 x 10(9)/l in the group without the mutation (P=0.001). An association of higher platelet accounts with extrathyroidal invasion was also seen, with 242.66+/-51.85 x 10(9)/l in patients with extrathyroidal invasion versus 218.49+/-59.10 x 10(9)/l in patients without extrathyroidal invasion (P=0.03). The BRAF T1799A-positive PTC tissues harbored a significantly higher level of PDGF-B than BRAF T1799A-negative PTC tissues. The data suggest that the BRAF T1799A mutation is associated with aggressive pathological outcomes of PTC in which high platelet counts and increased PDGF production may play a role.

BRAF T1799A Mutation Occurring in a Case of Malignant Struma Ovarii

Struma ovarii is an extremely rare tumor that occasionally undergoes malignant transformation. Because struma ovarii is composed of thyroid tissue, it is conceivable that the pathogenetic events involved in thyroid follicular transformation may take place also in struma ovarii. The authors describe a case of a classical variant of papillary thyroid carcinoma arising in a struma ovarii of a 22-year-old female. The tumor was heterozygous for BRAF T1799A mutation. No ret/ PTC-1 or ret/PTC-3 rearrangements were detected. This finding would suggest that malignant struma ovarii is similar histologically and genetically to primary papillary thyroid carcinoma.

Joint research project of genetic diagnosis of papillary thyroid cancer between Semipalatinsk Medical Academy and Nagasaki University

The activating point mutation of BRAF gene, BRAF T1799A , is the most common and specific genetic alteration in adult papillary thyroid carcinoma. BRAF T1799A seems to be associated with clinical aggressiveness of papillary thyroid carcinoma. We have established that PCR-RFLP method enables us to detect the BRAF T1799A mutation using DNA extracted from fine-needle aspiration biopsy samples and then applied this proprietary method not only to our clinical practice but also to international medical assistance around the Semipalatinsk Nuclear Testing Site in Kazakhstan. Seventy cases from Japan and one hundred cases from Kazakhstan were examined. There were 14 Japanese and 50 Kazakhstani cases of cytological malignant tumors in all of the examined samples, among which we detected 12 and 14 cases with BRAF T1799A in Japanese and Kazakhstan cases, respectively. Of these cases, we found mutations in two cytological “suspicious” cases and even two pathological “benign” cases (after surgery in Kazakhstan). All of the BRAF mutation-positive cases, including those four, were confirmed as papillary thyroid carcinoma by careful pathological examination including immunohistochemical analysis. In conclusion, our PCR-RFLP method for BRAF T1799A detection using fine-needle aspiration biopsy samples is useful not only for preoperative diagnosis but also for accurate pathological decision of difficult cases as a complementary diagnostic method even after surgery. The introduction of appropriate genetic evaluation together with cytological cancer diagnosis into Kazakhstan makes a novel possibility for our future joint project.

Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA)

The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis. BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.

T1799ABRAFMutations in Conjunctival Melanocytic Lesions

To gain a better understanding of the molecular events leading to the development of conjunctival melanocytic lesions and conjunctival melanoma, this study was conducted to investigate the presence of T1799A BRAF oncogenic mutation in these lesions. Forty-eight surgically excised conjunctival melanocytic lesions from 48 patients were examined for the presence of the BRAF T1799A mutation. Twenty-eight lesions were conjunctival nevi, of which 20 were excised from children younger than 18 years. Fifteen lesions were conjunctival primary acquired melanosis (PAM; 11 without atypia and 4 with atypia) and five were conjunctival melanomas. To detect the BRAF T1799A mutation, both a newly developed kit (Mutector; TrimGen, Sparks, MD) and direct DNA sequence analysis of exon 15 after PCR amplification were used. The T1799A BRAF mutation was identified in 14 of 28 (50%) conjunctival nevi analyzed, but in none of the 15 conjunctival PAMs, with and without atypia. The T1799A BRAF mutation was identified in two of the five (40%) conjunctival melanomas. There was no difference in the BRAF mutation detected in conjunctival nevi in children or adults, as the BRAF mutation was detected in 50%. The results showed that conjunctival nevi, similar to skin nevi, have a high frequency of oncogenic BRAF mutations. Furthermore, the results suggest that the oncogenic event leading to BRAF mutations affect only conjunctival nevi and not conjunctival PAM. The clinical significance of these observations remains to be determined.