No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
BRAF mutations in papillary thyroid carcinoma
Abstract
Papillary thyroid carcinoma (PTC) is the most frequent malignant neoplasm of the thyroid originating from the thyroid follicular cell (TFC). Although the formation of PTC is believed to result from rearrangements of RET or TRK oncogenes or MET point mutations, these structural aberrations or point mutations do not correlate with the clinicopathological features of PTC and do not seem to be a useful prognostic marker of the disease. Therefore, further experiments should be carried out in order to find new practical clinical markers. Recently, oncogene BRAF has become a subject of great interest. The mutation of BRAF gene is characteristic for PTC and poorly differentiated and/or undifferentiated cancers derived from PTC. The occurrence of BRAF mutation has often been observed in various human tumours. The presence of mutation was confirmed in melanoma, colon cancer, gliomas and lung cancer. In the majority of cases, there is only one type of point mutation - V600E. The RAS/RAF/MEK/MAPK kinase pathway mediates the cellular response to mitogenic signals. BRAF gene mutation results in increased kinase activity, leading to excessive activation of the above mitogenic pathway and to uncontrolled proliferation of cancer cells. Some correlation was noticed between BRAF gene mutation and the clinical stage of the neoplastic disease in question. Preliminary investigations indicate that the presence of BRAF mutation might be a valuable diagnostic and prognostic marker of the disease. Further investigations could also bring further improvements into the therapeutic management of thyroid cancer. There are reports emphasizing the possibility of using the inhibitors of BRAF proteins in the treatment of PTC. Certainly, in order to confirm the diagnostic usefulness of this marker, further studies should be carried out.
... Thus, we created a prototype antigen array for differential diagnostics of thyroid tumors which can be regarded as a platform for design of more complicated panel, highly sensitive in thyroid cancer detection, which can significantly improve the accuracy of preoperative diagnosis of thyroid cancer. [2, 3, 7, 8, 13, 29, 38]. По сравнению с классическими циркулируу ющими биомаркерами, представляющими собой " прямые " репортеры канцерогенеза (биологические субстанции, напрямую секк ретируемые опухолевыми клетками), аутоанн титела являются своего рода " непрямыми " репортерами, отражающими реакцию имм мунной системы на происходящие в опухоли молекулярные изменения [10, 11, 20, 25]. ...
... К преимуществам аутоантительных биомарр керов по сравнению с классическими секрее тируемыми опухолями гликопротеинами следует отнести высокую стабильность имм муноглобулинов G в пробах сыворотки и плазмы крови, высокое время полужизни, минимальные концентрационные флуктуаа ции в зависимости от времени суток, приема лекарственных препаратов и других фактоо ров и отсутствие специфических требований к забору материала и пробоподготовке [4]. Также важнейшим преимуществом аутоантии тельных биомаркеров является высокая (в типичных случаях – 90–100%) диагностии ческая специфичность в отношении очага злокачественного роста [7, 37, 38] * Наличие аутоантител хотя бы к одному из трех антигенов (наличие – 1, отсутствие – 0). ** Наличие на цитограмме пунктата изменений, подозрительных в отношении малигнизации (наличие – 1, отсутствие – 0). ...
Currently the “gold standard” in diagnostics of thyroid tumors is a fine-needle aspiration cytology (FNAC). However, FNAC cannot discriminate between benign and malignant thyroid tumors in 15 to 30% of observa tions. In order to develop an additional tool for differential diagnostics of thyroid tumors we evaluated the diagnostic performance of 3-antigen serum autoantibody signature in groups of benign (n = 22) and malignant (n = 26) thyroid tumors using a dot-blot ELISA-based analysis The sensitivity and specificity of resultant array were estimated to be 55–60% and 95–100%, respectively (p < 0.001 according to one-sided Fisher Exact Test). Thus, we created a prototype antigen array for differential diagnostics of thyroid tumors which can be regarded as a platform for design of more complicated panel, highly sensitive in thyroid cancer detection, which can significantly improve the accuracy of preoperative diagnosis of thyroid cancer.
... BRAF mutations are the most common genetic alteration occurring in thyroid carcinomas especially in PTC, accounting for 28-83%, with an overall rate of 44% (46)(47)(48). The most common type of BRAF mutations found in PTC is a T to A substitution at nucleotide 1,799 in exon 15, which results in the conversion of a valine to glutamic acid at codon 600 (V600E) of the BRAF protein (49,50). All of the relevant studies mainly demonstrate the influence of BRAF V600E on thyroid cancer. ...
Driver oncogene alterations have always been one of leading causes in the process of occurrence and development of tumors. And the effects of driver oncogene alterations on tumorigenesis and progression in different kinds of tumors have been studied heatedly. And the roles that the driver oncogenes alterations play have been elucidated clearly in previous studies. The phenomenon of concomitant driver oncogenes mutations and driver genes fusions has gained much concentration in the past two decades. And a growing number of studies reported this phenomenon, either coexistence or mutually exclusivity. Here we reviewed on the phenomenon of concomitant mutations in three common types of carcinomas—lung cancer, thyroid cancer, and leukemia, which have been studied relatively more detailed and more general compared with others.
... It comprises a T1799A nucleotide transversion within exon 15, and is detected in over 50% of PTCs (Sipos and Mazafferri, 2010). The T1799A mutation leads to the substitution of a valine residue at position 600 of the protein for a glutamate residue (V600E), with consequent gain of function and constitutive activation of the mitogen activated protein kinase (MAPK) pathway (Wojciechowska and Lewinski, 2006). The MAPK pathway is a signal transduction cascade driven by phosphorylation that connects growth factors and their respective cell surface receptors to intracellular responses such as cell proliferation (Vidwans et al., 2011). ...
A common mutation in the BRAF gene, comprising the T1799A nucleotide transversion, which leads to the V600E amino acid substitution in the BRAF protein, has been observed in about 50% of papillary thyroid carcinomas (PTCs). However, BRAF protein expression has been rarely examined in such tumors. Clinical studies have shown important associations between BRAF mutation and clinical parameters in PTC, such as progression, invasion, and recurrence. The aim of this study was to evaluate the association between BRAF protein overexpression and the BRAF V600E mutation in a group of PTC patients. The study group included 116 patients with PTC from Araújo Jorge Hospital, Goiânia, Goiás, Brazil. Immunohistochemistry was utilized to analyze BRAF protein expression. Presence of the BRAF V600E mutation was determined by polymerase chain reaction amplification and restriction fragment length polymorphism, and confirmed by direct sequencing. The chi-square test with Yates correction and the Fisher exact test were used for statistical analysis. BRAF overexpression was detected in 55 patients with PTC (47.4%) and the BRAF V600E mutation was observed in 74 patients (63.8%). In the studied group, significant associations were observed between the BRAF V600E mutation and BRAF protein overexpression (P = 0.0115), and also between BRAF overexpression and extra-thyroid extension of the tumor (P = 0.0111). This study demonstrated a significant association between BRAF overexpression and the BRAF V600E mutation in PTC, highlighting the importance of these molecular events in the process of PTC carcinogenesis.
... However, 25% are due to autoso- Well-differentiated thyroid carcinomas include PTC and FTC and are sporadic in most cases. PTC can be associated with the V600E BRAF activating point mutation on chromosome 7. Rearrangements of two transmembrane tyrosine kinase receptor genes (RET and NTRK1) have also been shown to be associated with PTC [7] . FTC is associated with several oncogenes -somatic mutations in MET (tyrosine kinase receptor gene), RAS (G protein signal transduction), and a translocation in PPARγ1 (tumor suppressor gene). ...
Papillary thyroid carcinoma is the most common thyroid cancer (85%). Follicular thyroid carcinoma is the second most common type of thyroid cancer, accounting for up to 10% of all thyroid cancers. Medullary thyroid carcinoma accounts for only 5-8% of thyroid cancers. Concurrent medullary, follicular, and papillary carcinomas of the thyroid gland are extremely rare and reported scarcely.
A 72-year-old male presented with nonspecific neck pain. The workup revealed a nodular thyroid gland with a follicular lesion on fine-needle aspiration. Total thyroidectomy was performed and pathological examination identified a 25-mm follicular carcinoma, two papillary microcarcinomas, and two medullary microcarcinomas. The genetic workup was negative and no other family members were diagnosed with any endocrinopathy. Two months after surgery, the patient was diagnosed with Cushing's syndrome that was treated with laparoscopic left adrenalectomy. On 3-year follow-up, the patient is asymptomatic with no evidence of recurrent disease.
We present a rare case of a patient with follicular, papillary, and medullary thyroid carcinoma, and Cushing's syndrome. To date, no known genetic mutation or syndrome can account for this combination of neoplastic thyroid and adrenal pathologies, although future research may prove differently.
... Mutation of the BRAF gene is most prominent in PTCs with an appearance up to 50% (Kondo et al., 2006;Pallante et al., 2010). The BRAF mutation in primary PTC is a T-A substitution at nucleotide 1799 in exon 15, which results in a change from valine acid to glutamic acid at codon 600 (V600E) of the BRAF protein (Wellbrock et al., 2004;Wojciechowska and Lewinski, 2006;Mitsiades et al., 2007), resulting in activation of the mitogen-activated protein kinase (MAPK) pathway (Wan et al., 2004;Knauf et al., 2005). The presence of a BRAF mutation has been associated with the poorer prognosis in PTC patients, such as an advanced stage at diagnosis, extrathyroidal extension, and lymph node metastasis (Xing, 2007;Elisei et al., 2008a;Yip et al., 2009). ...
MicroRNAs (miRNAs or miRs) are endogenous non-coding RNAs that negatively regulate gene expression by binding to the 3' non-coding regions of target mRNAs, resulting in their cleavage or blocking their translation. miRNAs may have an impact on cell differentiation, proliferation, and survival, and their deregulation can be inclined to diseases and cancers, including thyroid tumors. The purpose of this review is to summarize the existing findings of deregulated miRNAs in different types of thyroid tumors and to exhibit their potential target genes, especially to demonstrate those involved in tumor invasion and metastasis. In addition, new findings of circulating miRNA expression profiles, single nucleotide polymorphism (SNP) in thyroid tumors, and the correlation of somatic mutations with deregulated miRNA expression in thyroid tumors were all included in this review.
... Differences were taken to be significant at P<0.05. A high incidence of B-Raf mutations has also been detected in melanoma and thyroid tumors [23][24][25] . A series of melanoma and thyroid cancer cell lines was therefore characterized for presence of B-Raf mutations and Rac1b over-expression but revealed no association (Supplementary Table 2). ...
BACKGROUND & AIMS: In colorectal tumors, activating BRAF mutations occur alternative to KRAS oncogenic mutations, but in cell culture possess a much lower transforming capacity. Rac1b, a hyperactive Rac1 spliced variant, is over expressed in some colorectal tumors and activates the transcription factor nuclear factor-kappaB, which initiates a transcriptional response that promotes cell cycle progression and inhibits apoptosis. The aim of this study was to determine whether Rac1b overexpression is associated with B-Raf(V600E) in primary colorectal tumors and whether a functional cooperation between these 2 proteins exists in colorectal cells with a wild-type KRAS genotype.
METHODS: Screening of BRAF and KRAS mutations by direct sequencing and Rac1b mRNA expression analysis by quantitative real-time polymerase chain reaction were conducted in 74 samples (13 normal colonic mucosa, 45 primary colorectal tumors, and 16 colorectal cancer [CRC] cell lines). RNA interference and focus formation assays were used to assess the cooperation between Rac1b and B-Raf(V600E) in cancer cell viability.
RESULTS: Rac1b overexpression and B-Raf(V600E) are significantly associated in primary colorectal tumors (P = .008) and colorectal cell lines. The simultaneous suppression of both proteins dramatically decreased CRC cell viability through impaired cell-cycle progression and increased apoptosis.
CONCLUSIONS: Our data demonstrate that Rac1b and B-Raf(V600E) functionally cooperate to sustain colorectal cell viability and suggest they constitute an alternative survival pathway to oncogenic K-Ras. These results reveal a novel molecular characteristic of colon tumors containing B-Raf mutations and should help in defining novel targets for cancer therapy.
... from the thyroid follicular cells (TFCs) include a variety of histotypes ranging from well-differentiated (papillary and follicular) to undifferentiated (anaplastic) cancers. Altogether , they represent approximately 1% (3% in women) of all human cancers [154, 155]. Well-differentiated thyroid carcinomas account for approximately 90% of all thyroid cancers. ...
Endocrine cancers are a heterogeneous group of diseases that may arise from endocrine cells in any gland of the endocrine system. These malignancies may show an aggressive behavior and resistance to the common anticancer therapies. The etiopathogenesis of these tumors remains mostly unknown. The normal embryological development and differentiation of several endocrine glands are regulated by specific pituitary tropins, which, in adult life, control the function and trophism of the endocrine gland. Pituitary tropins act in concert with peptide growth factors, including the insulin-like growth factors (IGFs), which are considered key regulators of cell growth, proliferation, and apoptosis. While pituitary TSH is regarded as tumor-promoting factor for metastatic thyroid cancer, the role of other pituitary hormones in endocrine cancers is uncertain. However, multiple molecular abnormalities of the IGF system frequently occur in endocrine cancers and may have a role in tumorigenesis as well as in tumor progression and resistance to therapies. Herein, we will review studies indicating a role of IGF system dysregulation in endocrine cancers and will discuss the possible implications of these findings for tumor prevention and treatment, with a major focus on cancers from the thyroid, adrenal, and ovary, which are the most extensively studied.
... The Braf V600E mutation has been detected in greater than 60% of patients with metastatic melanoma. It has also been observed in patients with colon [1,2,3], thyroid [4,5,6,7,8] and other cancers [9,10,11]. The present technology for determining Braf V600E status either utilizes mass spectrometry [12,13], real time PCR [14,15], allelic specific PCR [7,16,17], PCR using locked oligonucleotides to suppress wild type sequences [18,19,20] or direct sequencing of RNA or DNA [8,21]. ...
The Braf(V600E) mutation has been detected in patients with metastatic melanoma, colon, thyroid and other cancers. Recent studies suggested that tumors with this mutation are especially sensitive to Braf inhibitors, hence the need to reliably determine the Braf status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type Braf alleles and thus may fail to detect the presence of mutant Braf(V600E) tumors. We have developed a rapid, inexpensive method that reduces the contamination of wild-type Braf sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild type and mutant Braf RNA at position 600. Using this protocol, mutant Braf can be detected in RNA from mixed populations with as few as 0.1% Braf(V600E) mutant cells.
... Most PTC carry one of two mutations, a BRAF mutation and RET/PTC rearrangement. The most common BRAF mutation is a T to A substitution at nucleotide 1799 in exon 15 that results in the conversion of a valine to glutamic acid at codon 600 (V600E) of the BRAF protein (2,3). The negative charge introduced by glutamic acid mimics the effect of phosphorylation at an adjacent site when BRAF is activated and results in constitutive activation of BRAF. ...
Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy. Most PTC carry one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling transduction pathway leading to cellular proliferation, differentiation, and apoptosis. PD0325901 is a specific MEK1/2 inhibitor and therefore is a promising drug to treat thyroid cancers with either RET/PTC or BRAF mutation. In this study we tested the effects of PD0325901 on PTC cells harboring either mutation in vitro by growth curves and Western blots and in vivo using a murine orthotopic xenograft model. We found that 50% growth inhibition (GI(50)) by PD0325901 was 11 nmol/L for the PTC cells with the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells with a BRAF mutation, with both concentrations readily achievable in serum. After 1 week of oral administration of PD0325901 (20-25 mg/kg/day) in mice, no tumor growth was detected in mice inoculated with PTC cells bearing a BRAF mutation. For PTC with the RET/PTC1 rearrangement, the average tumor volume of the orthotopic tumor was reduced by 58% as compared with controls. In conclusion, our data suggested that PTC cells carrying a BRAF mutation were more sensitive to PD0325901 than were PTC cells carrying the RET/PTC1 rearrangement. Our findings support the clinical evaluation of PD0325901 for patients with PTC and potentially other carcinomas with BRAF mutations.
... Although a6b4 integrin signals stimulate NFAT activity in breast cancer (Jauliac et al, 2002), we now identify that NFAT is activated by oncogenic BRAF signalling, which is the most frequently mutated gene in melanoma and a gene frequently mutated in many other cancers. Activating BRAF mutations are present in 29 -69% of papillary thyroid carcinomas (Wojciechowska and Lewinski, 2006), are also common in colorectal cancers (Ogino et al, 2009) and present in 4% of small cell lung cancers (Halilovic and Solit, 2008). Identifying that NFAT functions downstream of mutant BRAF in melanoma, raises the possibility that NFAT may be similarly activated in other BRAF-mutated cancers. ...
Background:
Metastatic melanoma is the most deadly form of skin cancer and with an overall 5-year survival rate of <11%, there is an acute need for novel therapeutic strategies. Activating mutations in the BRAF oncogene are present in 50-70% of cases and contribute to tumourigenesis, thus, defining downstream targets of oncogenic BRAF may help define novel targets for therapeutic intervention. The Ca(2+)/calcineurin-regulated transcription factor, Nuclear factor of activated T-cells (NFAT), is important in the pathogenesis of several human cancers, target genes of which are also known to contribute to melanoma progression. One such NFAT target gene is COX-2, increased expression of which correlates with poor prognosis; however, upstream regulators of COX-2 in melanoma remain undefined. Therefore, the aim of this study was to evaluate NFAT expression and activity in metastatic melanoma and establish whether or not oncogenic BRAF signalling modulates NFAT activity and determine if NFAT is a key upstream regulator of COX-2 in melanoma.
Methods:
Nuclear factor of activated T-cells transcriptional activity and protein expression were determined in three human metastatic melanoma cell lines with differing B-RAF mutational status. NFAT activation by oncogenic BRAF(V600E) was explored by BRAF(V600E) overexpression and application of the specific MEK inhibitor PD98059. Regulation of COX-2 expression by NFAT was investigated using NFAT-targeted siRNA, calcineurin inhibitors cyclosporin A and FK506, in addition to COX-2 luciferase reporter vectors that selectively lacked NFAT binding sites.
Results:
NFAT transcriptional activity was increased in BRAF-mutated melanoma cells compared with wild-type cells. Furthermore, in wild-type cells, overexpression of BRAF(V600E) increased NFAT activity, which was blocked by the MEK inhibitor PD98059. Using calcineurin inhibitors and siRNA-mediated knockdown of NFAT2 and 4, we show NFAT is required for COX-2 promoter activation and protein induction in metastatic melanoma cells.
Conclusion:
NFAT2 and 4 are expressed in human metastatic melanoma cell lines and are activated by oncogenic BRAF(V600E) via MEK/ERK signalling. NFAT is an important upstream regulator of COX-2 in metastatic melanoma. Furthermore, as the BRAF/MEK/ERK pathway is hyperactive in other malignancies and MEK/ERK are also activated by oncogenic RAS in 30% of all human cancers, the potential to exploit NFAT signalling for therapeutic benefit warrants further investigation.
Papillary thyroid cancer (PTC) is a kind of thyroid cancer and frequently presents with epithelial–mesenchymal transition (EMT). MicroRNAs (miRNAs) were previously reported to be associated with PTC. Thus, this study aims to define the role of microRNA‐520a‐3p (miR‐520a‐3p) in PTC through the JAK/STAT signaling pathway by targeting JAK1. The PTC and normal thyroid tissues of 137 PTC patients were collected. First of all, the expression pattern of miR‐520a‐3p, JAK1, JAK2, STAT3, E‐cadherin, and vimentin in PTC was identified. The relationship between miR‐520a‐3p and JAK1 was predicted and analyzed. And a series of miR‐520a‐3p mimic or inhibitor, or siRNA JAK1 introduced into PTC cells were applied to examine the effect of miR‐520a‐3p on PTC cell viability, migration, invasion, cell cycle, apoptosis, and EMT. Meanwhile, the regulatory effect of miR‐520a‐3p and JAK1 on the JAK/STAT signaling pathway was also determined. The expression of JAK1, JAK2, STAT3, and vimentin increased yet miR‐520a‐3p and E‐cadherin decreased in PTC tissue. JAK1 was negatively regulated by miR‐520a‐3p. Functionally, EMT induction was prevented by miR‐520a‐3p upregulation through downregulating JAK1. When upregulating miR‐520a‐3p or silencing JAK1 in PTC cells, PTC cell viability, migration, and invasion were inhibited yet cell apoptosis promoted with cells arrested at G1 phase, indicating that miR‐520a‐3p prevented PTC progression by downregulating JAK1. Moreover, miR‐520a‐3p elevation or JAK1 inhibition inactivated the JAK/STAT signaling pathway. Collectively, miR‐520a‐3p prevents cancer progression through inactivating the JAK/STAT signaling pathway by downregulating JAK1 in PTC.
Testing of tumor tissue remains the recommended method for detecting the presence of somatic mutations in human malignancies. V600E is the most frequent somatic point mutation in metastatic melanoma, providing a unique molecular marker for this malignancy. In addition, tumors carrying this mutation are primary candidates for BRAF-targeted therapy. Although metastatic melanoma patients usually have sufficient tumor tissue available for genetic analyses, the detection of V600E in blood can have prognostic and predictive value. In addition, patients are rarely re-biopsied and genetic testing in blood can be useful for monitoring response to therapy. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA), RNA associated to platelets and circulating tumor cells (CTCs) are some of the materials that can be derived from the blood of cancer patients. cfDNA can be easily purified from serum and plasma and contains DNA fragments of tumor origin. For this reason, it is the most widely used material for the detection of somatic mutations in blood. Several methodologies have been used to determine V600E status in the cfDNA of metastatic melanoma and some studies have demonstrated that the identification and follow-up of V600E in cfDNA can have prognostic and predictive value.
Tumor necrosis factor -related apoptosis-inducing ligand (TRAIL) represents an effective agent for the treatment of many cancers, though the majority of thyroid cancers are found to be resistant. Therefore it would be necessary to identify agents capable of increasing the sensitivity of these cancers to TRAIL-mediated cell death. Here, we examined the therapeutic effect and its underlying mechanism of combination treatment of TRAIL and histone deacetylase inhibitor, Valproic acid (VPA) in vitro using human papillary thyroid cancer (PTC) cells and in vivo using an orthotopic mouse model of PTC. TRAIL-VPA combination therapy synergistically induced apoptotic cell death in TRAIL-resistant PTC through caspase activation. In addition, downregulation of antioxidant transcription factor, Nrf2 by co-treatment of TRAIL-VPA induces cell death via suppression of Bcl-xL in vitro and in vivo; these effects were further enhanced following siRNA inhibition of these proteins in combination with TRAIL or TRAIL-VPA. Taken together, VPA sensitized TRAIL-resistant PTC cells to apoptotic cell death through involvement of Nrf2 and Bcl-xL. Thus, the combination of VPA and TRAIL may be a promising therapy for TRAIL-resistant PTC.
The Braf(V600E) mutation has been detected in patients with metastatic melanoma, colon, thyroid, and other cancers. Studies suggested that tumors with this mutation are especially sensitive to BRAF inhibitors-hence the need to reliably determine the BRAF status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type BRAF alleles and thus may fail to detect the presence of mutant BRAF(V600E) tumors. We have developed a rapid, inexpensive method of BRAF analysis that reduces the contamination of wild-type BRAF sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild-type and mutant BRAF RNA at codon 600. Using this protocol, mutant BRAF can be detected in RNA from mixed populations with as few as 0.1 % BRAF(V600E) mutant containing cells.
Objective:
To investigate the aggressive properties of papillary thyroid cancer (PTC) with concurrent BRAF(V600E) mutation and rearranged during transfection (RET) proto-oncogene protein expression.
Methods:
Fifty pathologically confirmed PTC patients who had received thyroidectomy were enrolled in this study. BRAF(V600E) mutation was detected by real time polymerase chain reaction (RT-PCR), while RET protein expression was measured by immunohistochemical SP method. Clinical and pathological features were compared between the concurrent BRAF(V600E) mutation and RET protein expression group (n=24) and BRAF(V600E) mutation or RET protein expression alone group (n=19). Seven patients were ruled out from the final analysis due to the absence of either BRAF(V600E) mutation or RET protein expression.
Results:
Of these 50 patients, BRAF(V600E) mutation and RET protein expression were detected in 38 patients (76%) and 28 patients (56%), respectively. Concurrent BRAF(V600E) mutation and RET expression was detected in 24 patients (48%). Compared with the concurrent BRAF(V600E) mutation and RET protein expression group, the BRAF(V600E) mutation or RET protein expression alone group had relatively poorer tissue differentiation and higher prognostic score (P=0.011, P=0.022).
Conclusion:
PTC patients with concurrent BRAF(V600E) mutation and RET expression present poorer differentiation, more highly aggressive variant in carcinoma tissues, and higher cancer-related mortality risk.
Objective:
To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR).
Study design:
Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe.
Results:
Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens.
Conclusion:
Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.
Pheochromocytomas vary in presentation, tumor size, and in catecholamine production. Whether pheochromocytoma size correlates with hormone levels, clinical presentation, and perioperative complications is not known. The goal of this study was to determine if tumor size and hormone level correlate according to the clinical presentation at diagnosis.
We retrospectively analyzed all patients who underwent an adrenalectomy with a diagnosis of a pheochromocytoma from February 1996 to October 2008. We grouped patients according to their clinical presentation at diagnosis (routine biochemical screening, incidentaloma, classic symptoms, pheochromocytoma crisis) and obtained preoperative radiographic tumor size and catecholamine hormone levels. ANOVA was used for the group effects and the Kruskal-Wallis rank test was used for pairwise comparison between groups with the Sidak/Bonferroni method for multiplicity adjustment according to age, tumor size, and hormone level. The Pearson correlation coefficient was then calculated to determine if hormone level correlated with tumor size.
Eighty-one of 107 patients had data available for complete analysis. The average age at diagnosis for all patients was 47.1 years, and the average tumor size was 4.9 cm. The average highest hormone ratio among all patients was 27.4. Tumor size and hormone ratio levels differed among all groups (p < or = 0.03). A direct correlation (p = 0.014) was apparent between tumor size and hormone level. Complication rates also differed among the four groups of patients (p < or = 0.02).
Our study showed that tumor size directly correlates with hormone level. Smaller tumors tend to secrete lower levels of catecholamines, but larger tumors have a wider variation in secretory potential. Larger tumors, however, produced the highest hormone ratios.
There has been considerable progress identifying biomarkers in thyroid tumors that improve the accuracy of fine-needle aspiration biopsy and also help predict tumor aggressiveness or behavior. In this review we address both the clinical potential of molecular biomarkers and their usefulness, based on the most recent literature. We describe the current best clinical staging systems and the common somatic mutations in thyroid cancer. The BRAF mutation is the most common mutation in papillary thyroid cancer and has recently been reported to be associated with disease aggressiveness; it is also an independent predictor of tumor behavior. Combined testing of RET/PTC, NTRK, RAS and PAX8-PPARgamma, which are mutually exclusive mutations, helps improve the accuracy of fine-needle aspiration biopsy. Gene-expression profiling studies have identified a variety of potential molecular markers to help distinguish benign from malignant thyroid neoplasms. Expression analysis of differentially expressed microRNAs also appears to be a promising diagnostic approach for distinguishing benign from malignant thyroid neoplasm. It is especially useful for indeterminate nodules by fine-needle aspiration biopsy.
Papillary thyroid carcinoma (PTC), the most common thyroid malignancy, usually possesses BRAF mutation or rearranged in translation (RET)/PTC rearrangements. PTC usually possesses BRAF mutation or RET/PTC rearrangements. The mutation status of patients with recurrent PTC has never been characterized in a large population.
Mutation status was determined in a cohort of 54 patients with recurrent PTC and analyzed for clinicopathologic relationships. BRAF and ras mutations were determined by PCR and sequencing of genomic DNA. RET/PTC rearrangements were analyzed by reverse transcription-PCR.
BRAF mutation in exon 15 (V600E) was found in 42/54 (77.8%) recurrent PTC patients. The RET/PTC rearrangements were detected in 9 of 54 (16.7%) patients. In addition, 5 of 54 (9.3%) recurrent PTC patients had both a BRAF mutation and a RET/PTC rearrangement. The prevalence of tumors with dual mutations found in the recurrent population far exceeds the frequency historically reported for patients with primary PTC. Patients with dual mutations were significantly older (80% older than 45 years) than patients with a BRAF mutation alone (38% older than 45 years).
Recurrent PTC is significantly associated with a predominant BRAF mutation. RET/PTC rearrangements, although commonly associated with primary PTCs in younger patients, are uncommonly found in recurrent PTC patients. In addition, the incidence of dual mutations was higher in patients with recurrent PTC than in those primary PTC, as reported by others.
To evaluate BRAF(V600E) mutation on consecutive fine-needle aspiration biopsy (FNAB) specimens in order to assess FNAB's usefulness in preoperative papillary thyroid carcinoma (PTC) diagnosis with the contemporaneous analysis of RET/PTC1 and RET/PTC3 rearrangements obtained from ex vivo thyroid nodules.
Thyroid FNABs from 156 subjects with nodules and 49 corresponding surgical samples were examined for the presence of BRAF mutation by real-time allele-specific polymerase chain reaction, confirmed with the use of a laser pressure catapulting system. Samples were also examined for RET/PTC rearrangements. The results were compared with the cytological diagnosis and histopathology.
13/156 cytological examinations were diagnostic for PTC and 19/156 showed suspicious/indeterminate FNAB (12.2%). FNAB-BRAF(V600E) mutation was detected in 11/16 (69%) cases with histological confirmation of PTC. In our series, RET/PTC rearrangement was detected in only one case of PTC, whereas it was not present in any case of adenoma, goiter, or Hashimoto's thyroiditis. No PTC case was found positive at the same time for BRAF mutation and RET/PTC rearrangements.
BRAF(V600E) mutation detected on FNAB specimens, more than RET/PTC rearrangements, is highly specific for PTC and its routine research might well be an adjunctive and integrative diagnostic tool for the preoperative diagnostic iter.
In recent years, the T1799A B-type Raf kinase (BRAF) mutation in thyroid cancer has received enthusiastic investigation, and significant progress has been made toward understanding its tumorigenic role and clinical significance. Among various thyroid tumors, this mutation occurs uniquely in papillary thyroid cancer (PTC), the most common endocrine malignancy, and some apparently PTC-derived anaplastic thyroid cancers. Many studies have found this mutation to be associated with those clinicopathological characteristics of PTC that are conventionally known to predict tumor progression and recurrence, including, for example, old patient age, extrathyroidal invasion, lymph node metastasis, and advanced tumor stages. Direct association of BRAF mutation with the clinical progression, recurrence, and treatment failure of PTC has also been demonstrated. The BRAF mutation has even been correlated with PTC recurrence in patients with conventionally low-risk clinicopathological factors. Some molecular mechanisms determining BRAF mutation-promoted progression and the aggressiveness of PTC have recently been uncovered. These include the down-regulation of major tumor suppressor genes and thyroid iodide-metabolizing genes and the up-regulation of cancer-promoting molecules, such as vascular endothelial growth factor, matrix metalloproteinases, nuclear transcription factor kappaB, and c-Met. Thus, BRAF mutation represents a novel indicator of the progression and aggressiveness of PTC. Significant advances have also occurred in the preclinical testing of new therapeutic strategies targeting the MAPK pathway aberrantly activated by BRAF mutation and other related mutations. New mitogen extracellular kinase (MEK) inhibitors developed recently are particularly promising therapeutic agents for thyroid cancer. With these advances, it has become clearer that BRAF mutation will likely have significant impact on the clinical management of PTC.
The aim of this study was hypermethylation of multiple genes for papillary thyroid carcinomas (PTCs).
We examined 39 lesions using methylation-specific PCR to assess hypermethylation in genes, including p16(INK4a), p14(ARF), RB1, p27(Kip1)and 0(6)-MGMT. Homozygous deletions of p16(INK4a) and p14(ARF) were investigated by differential PCR, all with reference to clinicopathological factors.
We found methylation of p16(INK4a) in 35.9% (14/39); p14(ARF) in 2.6% (1/39); RB1 in 23.1% (9/39); p27(Kip1) in 15.4% (6/39),and 0(6)-MGMT in 15.4% (6/39). Hypermethylation of at least one of these genes was apparent in 66.7% (26/39). Homozygous deletions of p14(ARF) and p16(INK4a) were detected in 7.7 (3/39) and 2.6% (1/39), respectively. In cases with p16(INK4a) alterations, tumors were significantly increased. A history of chronic thyroid disease and lymphocytic infiltration was significantly associated with p14(ARF) alterations, without regional lymph node metastases.
Our data suggest that alterations in p16(INK4a), mainly hypermethylation, may be linked to tumor growth but not tumor development, while alterations in p14(ARF) may contribute to the induction of chronic inflammation-related PTCs.
The detection of papillary microcarcinomas of the thyroid is increasing due to frequent use of ultrasound and fine-needle aspiration biopsy. Although most of the papillary microcarcinomas remain quiescent and follow an indolent clinical course, some behave aggressively and metastasize early, giving rise to clinically significant disease. There have been few studies concerning factors predictive of lymph node metastasis in papillary microcarcinomas. We analyzed the expression of S100A4, cyclin D1, p27 and MUC1, the presence of the BRAF V600E mutation and the clinicopathological features of the tumors, including patient age, tumor size (>or=5 vs <5 mm), extrathyroidal extension, multifocality, histological subtype, sclerosis and encapsulation, in a series of 198 papillary microcarcinomas in relation to lymph node metastasis to determine the predictive factors of lymph node metastasis. On univariate analysis, tumor size of 5 mm or more, extrathyroidal extension, multifocality, sclerosis and the expression of S100A4 and cyclin D1 predicted lymph node metastasis, whereas patient age, expression of p27 and MUC1 and the BRAF V600E mutation did not. Moreover, tumor size 5 mm or more, multifocality and expression of S100A4, especially its strong expression in the invasive fronts, were significantly associated with macrometastasis and lateral node metastasis. On multivariate analysis, multifocality and expression of S100A4 were found to be common independent predictive factors of lymph node metastasis, macrometastases, and lateral node metastasis. In conclusion, S100A4 expression in papillary microcarcinomas may indicate the presence of nodal metastasis. Thus, S100A4 immunohistochemistry may be valuable for predicting metastatic potential in papillary microcarcinomas.
Genes of the RAF family, which mediate cellular responses to growth signals, encode kinases that are regulated by RAS and participate in the RAS, RAF, mitogen/extracellular signal-regulated kinase, extracellular signal-regulated kinase and mitogen-activated protein kinase pathway. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation, it may provide possible diagnostic and therapeutic targets in human malignant tumors. We analyzed exon 15 of the BRAF gene for mutations in 58 lung, 12 breast, six kidney, 14 cervical, four endometrial and 10 ovarian carcinoma cell lines by PCR-SSCP and direct sequencing. The T1796A transversion was found in one (2.9%) of 34 small cell lung carcinoma and one (8.3%) of 12 breast carcinoma cell lines, resulting in a valine-to-glutamate substitution at residue 599 (V599E). One (4.2%) of 24 non-small cell lung carcinoma cell line showed the C1786G transversion, leading to a leucine-to-valine substitution at residue 596 (L596V). No BRAF point mutations were found in any of the other cell lines examined. Our present results suggest that BRAF may not be a frequent target of mutations involved in the pathogenesis of human lung, breast, kidney, cervical, endometrial and ovarian carcinomas.
In colorectal tumors, activating BRAF mutations occur alternative to KRAS oncogenic mutations, but in cell culture possess a much lower transforming capacity. Rac1b, a hyperactive Rac1 spliced variant, is over expressed in some colorectal tumors and activates the transcription factor nuclear factor-kappaB, which initiates a transcriptional response that promotes cell cycle progression and inhibits apoptosis. The aim of this study was to determine whether Rac1b overexpression is associated with B-Raf(V600E) in primary colorectal tumors and whether a functional cooperation between these 2 proteins exists in colorectal cells with a wild-type KRAS genotype.
Screening of BRAF and KRAS mutations by direct sequencing and Rac1b mRNA expression analysis by quantitative real-time polymerase chain reaction were conducted in 74 samples (13 normal colonic mucosa, 45 primary colorectal tumors, and 16 colorectal cancer [CRC] cell lines). RNA interference and focus formation assays were used to assess the cooperation between Rac1b and B-Raf(V600E) in cancer cell viability.
Rac1b overexpression and B-Raf(V600E) are significantly associated in primary colorectal tumors (P = .008) and colorectal cell lines. The simultaneous suppression of both proteins dramatically decreased CRC cell viability through impaired cell-cycle progression and increased apoptosis.
Our data demonstrate that Rac1b and B-Raf(V600E) functionally cooperate to sustain colorectal cell viability and suggest they constitute an alternative survival pathway to oncogenic K-Ras. These results reveal a novel molecular characteristic of colon tumors containing B-Raf mutations and should help in defining novel targets for cancer therapy.
The B-raf gene is the human homolog of the avian c-Rmil proto-oncogene encoding a 94-kDa serine/threonine kinase detected in avian cells. We have previously shown that this protein contains amino-terminal sequences not found in other proteins of the mil/raf gene family. These sequences are encoded by three exons in the avian genome. We report that these three exons are conserved in the human B-raf gene and that they encode an amino acid sequence similar to that of the avian c-Rmil gene, indicating that in both avian and mammalian species the product of the B-raf/c-Rmil gene is a 94-kDa protein. We also identified two human B-raf loci: B-raf-1, located on chromosome 7q34, which encodes the functional B-raf/Rmil gene product, and B-raf-2, an inactive processed pseudogene located on chromosome Xq13.
The BRAF gene has been found to be activated by mutation in human cancers, predominantly in malignant melanoma. We tested
476 primary tumors, including 214 lung, 126 head and neck, 54 thyroid, 27 bladder, 38 cervical, and 17 prostate cancers, for
the BRAF T1796A mutation by polymerase chain reaction (PCR)–restriction enzyme analysis of BRAF exon 15. In 24 (69%) of the
35 papillary thyroid carcinomas examined, we found a missense thymine (T)→adenine (A) transversion at nucleotide 1796 in the
BRAF gene (T1796A). The T1796A mutation was detected in four lung cancers and in six head and neck cancers but not in bladder,
cervical, or prostate cancers. Our data suggest that activating BRAF mutations may be an important event in the development
of papillary thyroid cancer.
Rearrangement of RET proto-oncogene is the major event in the etiopathogenesis of papillary thyroid carcinoma (PTC). We report a high prevalence of BRAF(V599E) mutation in sporadic PTC and in PTC-derived cell lines. The BRAF(V599E) mutation was detected in 23 of 50 PTC (46%) and in three of four PTC-derived cell lines. The prevalence of the BRAF(V599E) mutation in PTC is the highest reported to date in human carcinomas, being only exceeded by melanoma. PTC with RET/PTC rearrangement as well as the TPC-1 cell line (the only one harboring RET/PTC rearrangement) did not show the BRAF(V599E) mutation. BRAF(V599E) mutation was not detected in any of 23 nodular goiters, 51 follicular adenomas and 18 follicular carcinomas. A distinct mutation in BRAF (codon K600E) was detected in a follicular adenoma. Activating mutations in RAS genes were detected in 15% of FA, 33% of FTC and 7% of PTC. BRAF(V599E) mutation did not coexist with alterations in any of the RAS genes in any of the tumors. These results suggest that BRAF(V599E) mutation is frequent in the etiopathogenesis of PTC. The BRAF(V599E) mutation appears to be an alternative event to RET/PTC rearrangement rather than to RAS mutations, which are rare in PTC. BRAF(V599E) may represent an alternative pathway to oncogenic MAPK activation in PTCs without RET/PTC activation.
Numerous biomolecular markers have been studied to improve the accuracy of fine needle aspiration biopsy (FNAB) in the diagnostic and prognostic evaluation of thyroid tumors, but none of them has yet become clinically useful. The recently discovered BRAF mutation, which occurs specifically in papillary thyroid cancers (PTC) with a high prevalence and is associated with poor clinicopathological outcomes, has the potential to be a useful diagnostic and prognostic marker for PTC. In the present study, we investigated whether detection of BRAF mutation on FNAB specimens was technically possible and could be used as an adjunct diagnostic tool with routine FNAB. Evaluation of a new colorimetric mutation detection method demonstrated 100% sensitivity and 100% specificity in comparison with conventional DNA sequencing as the "gold standard" in a large pool of DNA samples from various primary thyroid tumor specimens and cell lines. We found this novel technique even more sensitive in detecting BRAF mutation on FNAB specimens than conventional sequencing. In a series of 48 patients undergoing thyroidectomy, mostly for thyroid cancer or for suspicion of cancer, we performed preoperative FNAB and, using the colorimetric mutation detection method, identified BRAF mutation on the cytological specimens. Prospective analysis showed that 50% of the nodules that proved to be PTC on surgical histopathology were correctly diagnosed by BRAF mutation analysis on FNAB specimens; there were no false positive findings. Thus, we have demonstrated the usefulness of BRAF mutation detection on FNAB specimens that can help diagnose and identify those PTC patients who may need more aggressive surgical treatment and vigilant clinical monitoring.
Papillary thyroid carcinoma (PTC) is associated with RET and NTRK1 rearrangements and BRAF mutations. A series of 60 PTCs collected in a single center from Italian patients were histologically re-examined and subclassified as well differentiated or tall cell variant. The sample collection was analysed for the presence of all the reported PTC-associated genetic alterations through DNA or cDNA amplification, followed by automated sequencing. The analysis of exons 11 and 15 of BRAF gene revealed the T1796A (V599E) mutation in 32% of cases, and this alteration is significantly associated with PTC tall cell variant. Oncogenic rearrangements of RET and NTRK1 receptors were found in 33 and 5% of cases, respectively. No Ras mutations were detected. Overall, genetic alterations were detected in two-thirds of samples, and in no single case more than one mutational event was found simultaneously. Gene expression profiling of a subset of 31 tumors performed using cDNA microarray chips showed no strong differences in global gene expression among the different cases. However, a supervised analysis of the obtained data identified a subset of genes differentially expressed in tumors carrying BRAF mutation or RTK rearrangement.
Point mutations in BRAF are genetic hallmarks of papillary thyroid carcinoma (PTC). In this retrospective study, we examined thyroid aspirates and corresponding paraffin-embedded surgical samples for the presence of BRAF mutations. Altogether, we examined 96 cases, including 69 PTCs, 19 follicular adenomas, and eight nontoxic nodular goiters for BRAF; 60 of these samples were also examined for RET/PTC rearrangements. The results were correlated with the cytological diagnosis and the final histopathology. The BRAF mutation (V599E) was detected in 38% of the samples that were PTC on histopathology; RET/PTC was found in 18% of the PTC cases. In all the cases, the presence of the genetic alteration was confirmed in the surgically resected tumor. The identification of BRAF mutation and RET/PTC refined the diagnosis of PTC in five of 15 samples that were considered either indeterminate or insufficient at cytology. No mutation was found in aspirates of follicular adenomas and nontoxic nodular goiters. These results indicate that BRAF mutation and RET/PTC rearrangements are molecular markers of PTC that can be applied to FNA in adjunct to traditional cytology.
Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.
Fine-needle aspiration biopsy (FNAB) samples from thyroid tumor tissues were analyzed for the presence of the BRAF(T1796A) mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. This assay utilized a specific mismatched primer and has proved to be a relatively simple, accurate, and highly sensitive method. The analysis of 130 aspirated samples from thyroid tumors (18 follicular adenomas, 72 papillary carcinomas [PTCs], 8 follicular carcinomas, 2 undifferentiated carcinomas, 1 medullary carcinoma, 2 malignant lymphomas, and 27 adenomatous goiters) revealed BRAF(T1796A) mutations in 37 (51.4%) of 72 PTCs, supporting the usefulness of this method. We examined BRAF(T1796A) in 21 patients with thyroid tumors using leftover cells in the needle at the preoperative FNAB. BRAF(T1796A) was detected in 4 patients, of which 3 cases were diagnosed as positive and 1 case as suspicious by cytologic examination. Furthermore, BRAF(T1796A) mutations were found to occur more often in tumors of 3 cm or larger in size. Our results indicate that the preoperative determination of the presence of a BRAF(T1796A) mutation by conventional PCR-RFLP may be potentially useful in the diagnosis of the most common thyroid malignancies.
A high prevalence of the BRAF(V600E) somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF(V600E) mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF(K601E) mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF(V600E) (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF(V600E) (29.5 years). The BRAF (BRAF(V600E)) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.
Genetic alteration is the driving force for thyroid tumorigenesis and progression, based upon which novel approaches to the management of thyroid cancer can be developed. A recent important genetic finding in thyroid cancer is the oncogenic T1799A transversion mutation of BRAF (the gene for the B-type Raf kinase, BRAF). Since the initial report of this mutation in thyroid cancer 2 years ago, rapid advancements have been made. BRAF mutation is the most common genetic alteration in thyroid cancer, occurring in about 45% of sporadic papillary thyroid cancers (PTCs), particularly in the relatively aggressive subtypes, such as the tall-cell PTC. This mutation is mutually exclusive with other common genetic alterations, supporting its independent oncogenic role, as demonstrated by transgenic mouse studies that showed BRAF mutation-initiated development of PTC and its transition to anaplastic thyroid cancer. BRAF mutation is mutually exclusive with RET/PTC rearrangement, and also displays a reciprocal age association with this common genetic alteration in thyroid cancer. The T1799A BRAF mutation occurs exclusively in PTC and PTC-derived anaplastic thyroid cancer and is a specific diagnostic marker for this cancer when identified in cytological and histological specimens. This mutation is associated with a poorer clinicopathological outcome and is a novel independent molecular prognostic marker in the risk evaluation of thyroid cancer. Moreover, preclinical and clinical evaluations of the therapeutic value of novel specific mitogen-activated protein kinase pathway inhibitors in thyroid cancer are anticipated. This newly discovered BRAF mutation may prove to have an important impact on thyroid cancer in the clinic.
Use of BRAF mutation in papillary thyroid cancer (PTC) has the potential to improve risk stratification of this cancer.
The objective of the study was to investigate the prognostic value of BRAF mutation in patients with PTC.
In a multicenter study of 219 PTC patients, data on their clinicopathological characteristics and clinical courses between 1990 and 2004 were retrospectively collected, and their tumor BRAF mutation status was determined. Associations of BRAF mutation with initial tumor characteristics and subsequent recurrence were analyzed.
Relationships between the BRAF mutation status and clinicopathological outcomes, including recurrence, were measured.
We found a significant association between BRAF mutation and extrathyroidal invasion (P < 0.001), lymph node metastasis (P < 0.001), and advanced tumor stage III/IV (P = 0.007) at initial surgery. This association remained significant on multivariate analysis, adjusting for conventional clinicopathological predictors of recurrence excluding the histological PTC subtype, but was lost when the tumor subtype was included in the model. BRAF mutation was also significantly associated with tumor recurrence, 25 vs. 9% with and without mutation, respectively (P = 0.004), during a median of 15 (interquartile range, 3-29) months of follow-up. This association remained significant on multivariate analysis adjusting for conventional clinicopathological predictors of recurrence, even including the PTC subtype (odds ratio, 4.0; 95% confidence interval, 1.1-14.1; P = 0.03). BRAF mutation was even an independent predictor of recurrence in patients with stage I/II disease, 22 vs. 5% with and without BRAF mutation, respectively (P = 0.002). BRAF mutation was also more frequently associated with absence of tumor I-131 avidity and treatment failure of recurrent disease.
In patients with PTC, BRAF mutation is associated with poorer clinicopathological outcomes and independently predicts recurrence. Therefore, BRAF mutation may be a useful molecular marker to assist in risk stratification for patients with PTC.
The aim of the study was to establish the LOH frequency of selected polymorphic markers in different histological types of thyroid tumors: 18 colloid goiters (CG), five follicular adenomas (FA), nine follicular carcinomas (FTC), 40 papillary carcinomas (PTC), and one anaplastic carcinoma (ATC). For PTC, tumors negative for RET/PTC rearrangements were preferred.
LOH studies were performed using 14 highly polymorphic markers previously described as frequently lost in thyroid tumors.
In 20 cases (27%) the loss of at least one marker was found. No difference between the frequency of the LOH in FTC and PTC tumors was revealed (33% v. 33%). No differences between histopathological subtypes of PTC in LOH were found. Papillary thyroid carcinomas showed a tendency to higher LOH frequency from patients older than 45 years of age compared to younger ones (9/23 v. 4/17) although it was not statistically significant.
We conclude that papillary thyroid cancers, particularly those diagnosed in patients older than 45 years of age, do exhibit LOH at least with the same frequency as follicular cancers. This increased number of LOH events may contribute to the clinical aggressiveness of cancer in older patients.
BRAF belongs to the RAF family of protein kinases that are important components of the MAPK signaling pathway mediating cell growth, differentiation and survival. Activating point mutation of the BRAF gene resulting in V600E (previously designated as V599E) is a common event in thyroid papillary carcinoma, being found in approx 40% of this tumor. It has strong association with classical papillary carcinoma and tall cell and possibly Warthin-like variants. This mutation also occurs in thyroid poorly differentiated and anaplastic carcinomas, usually those containing areas of papillary carcinoma. Alterations in the BRAF gene do not overlap with RAS mutations and RET/PTC rearrangement, indicating that activation of one of the effectors of the MAPK pathway is sufficient for papillary thyroid carcinogenesis. Recently, another mechanism of BRAF activation has been identified, which involves chromosome 7q inversion that results in the AKAP9-BRAF fusion. It is rare in sporadic papillary carcinomas and is more common in tumors associated with radiation exposure. Yet another mechanism of BRAF activation may involve copy number gain, which is seen in a significant portion of thyroid follicular tumors of both conventional and oncocytic (Hürthle cell) types.
Fine needle aspiration (FNA) is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC). In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC.
Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE) surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 mum FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR). Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing.
A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8%) FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4%) FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells.
When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.
Recently, a somatic point mutation of the B-RAF gene (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence variable among different series. Since discordant data on the clinico-pathologic features of B-RAF mutated PTC are present in the literature, the aim of the present co-operative study was to establish the prevalence of this genetic alteration and to perform a genotype-phenotype correlation in a large cohort of patients with PTC. To this purpose, a series of 260 sporadic PTCs with different histological variants were included in the study. The mutational analysis of the B-RAF gene was performed either by RT-PCR followed by single-stranded conformational polymorphism or by PCR and direct sequencing. Statistical analyses were obtained by means of chi2/Fisher's exact test and t-test. Overall, a heterozygous T > A transversion at nucleotide 1799 (V600E) was found in 99 out of 260 PTCs (38%). According to the histological type of the tumor, the B-RAF (V600E) mutation was present in 48.3% of cases of classic PTCs (85 out of 176), in 17.6% (nine out of 51) of follicular variants of PTCs, in 21.7% (five out of 23) in other PTC variants and in none of the ten poorly differentiated tumors. B-RAF (V600E) was significantly associated with the classic variant of PTC (P = 0.0001) and with an older age at diagnosis (P = 0.01). No statistically significant correlation was found among the presence of B-RAF (V600E) and gender, tumor node metastasis (TNM), multicentricity of the tumor, stage at diagnosis and outcome. In conclusion, the present study reports the prevalence of B-RAF (V600E) (38%) in the largest series of sporadic PTCs, including 260 cases from three different Italian referring centers. This prevalence is similar to that calculated by pooling together all data previously reported, 39.6% (759 out of 1914 cases), thus indicating that the prevalence of this genetic event lies around 38-40%. Furthermore, B-RAF (V600E) was confirmed to be associated with the papillary growth pattern, but not with poorer differentiated PTC variants. A significant association of B-RAF mutation was also found with an older age at diagnosis, the mutation being very rare in childhood and adolescent PTCs. Finally, no correlation was found with a poorer prognosis and a worse outcome after a median follow-up of 72 months.
Activating mutations in the gene encoding BRAF are the most commonly identified oncogenic abnormalities in papillary thyroid cancer. In vitro and in vivo models have demonstrated that overexpression of activated BRAF induces malignant transformation and aggressive tumour behaviour. BRAF and other RAF kinases are frequently activated by other thyroid oncogenes and are important mediators of their biological effects including dedifferentiation and proliferation. Because current therapeutic options for patients with thyroid cancers that are aggressive and/or do not respond to standard therapies are limited, BRAF and its downstream effectors represent attractive therapeutic targets. In this review, data supporting a role for BRAF activation in thyroid cancer development and establishing the potential therapeutic efficacy of BRAF-targeted agents in patients with thyroid cancer will be reviewed.
The activating point mutation of the BRAF gene, BRAF(T1799A), is the most common and specific genetic alteration in adult papillary thyroid carcinoma (PTC) and a possible marker of malignant potential of PTC. We have applied the PCR-RFLP method using fine-needle aspiration biopsy samples not only to our clinical practice but also to the international medical assistance effort around the Semipalatinsk Nuclear Testing Site in Kazakhstan. Seventy-seven cases (100 nodules) from Japan and 131 cases (137 nodules) from Kazakhstan were examined. There were 14 Japanese and 76 Kazakhstani cases of cytological malignant tumors from the examined samples. We detected 12 (85.7% of PTC) and 19 (25% of PTC) cases with BRAF(T1799A) among the Japanese and Kazakhstani cases, respectively. Of these cases, we found mutations in one cytologically "suspicious" case and even in two pathologically "benign" cases (after surgery in Kazakhstan). All of the BRAF mutation-positive cases, including those three, were confirmed as PTC by careful pathological examination, including immunohistochemical analysis. In summary, our PCR-RFLP method for BRAF(T1799A) detection using FNAB samples is useful not only for preoperative diagnosis of PTC but also as a complementary diagnostic tool for accurate pathological diagnosis, even after surgery.
To evaluate BRAF(V600E) mutation on consecutive fine-needle aspiration biopsy (FNAB) specimens in order to assess FNAB's usefulness in preoperative papillary thyroid carcinoma (PTC) diagnosis with the contemporaneous analysis of RET/PTC1 and RET/PTC3 rearrangements obtained from ex vivo thyroid nodules.
Thyroid FNABs from 156 subjects with nodules and 49 corresponding surgical samples were examined for the presence of BRAF mutation by real-time allele-specific polymerase chain reaction, confirmed with the use of a laser pressure catapulting system. Samples were also examined for RET/PTC rearrangements. The results were compared with the cytological diagnosis and histopathology.
13/156 cytological examinations were diagnostic for PTC and 19/156 showed suspicious/indeterminate FNAB (12.2%). FNAB-BRAF(V600E) mutation was detected in 11/16 (69%) cases with histological confirmation of PTC. In our series, RET/PTC rearrangement was detected in only one case of PTC, whereas it was not present in any case of adenoma, goiter, or Hashimoto's thyroiditis. No PTC case was found positive at the same time for BRAF mutation and RET/PTC rearrangements.
BRAF(V600E) mutation detected on FNAB specimens, more than RET/PTC rearrangements, is highly specific for PTC and its routine research might well be an adjunctive and integrative diagnostic tool for the preoperative diagnostic iter.
Because very few studies have examined the correlation between BRAF mutations and clinicopathological features of papillary thyroid carcinoma (PTC), we analyzed here a large and homogeneous cohort of patients with PTC for the presence of the BRAF mutation.
We examined BRAF mutations in a consecutive series of 500 PTC patients who underwent surgery in the Department of Surgery of the University of Pisa, and we correlated the presence of the mutation with clinicopathological parameters of the patients: age, gender, tumor size, presence of tumor capsule, extrathyroidal invasion, multicentricity, presence of node metastases, and tumor class.
BRAF (exon 15) mutation was examined by PCR-single strand conformational polymorphism followed by DNA sequencing in laser-capture microdissected tissue samples.
In this study, BRAF mutation was found in 219 of 500 cases (43.8%). In particular, we found the most common BRAF V600E mutation in 214 cases (42.8%), BRAF K601E mutation in three cases (0.6%), BRAF VK600-1E (0.2%) in one case, whereas in one case we found a new 14-bp deletion with concomitant 2-bp insertion, VKSR600-3del and T599I, respectively. BRAF V600E was associated with extrathyroidal invasion (P < 0.0001), multicentricity (P = 0.0026), presence of nodal metastases (P = 0.0009), class III vs. classes I and II (P < 0.00000006), and absence of tumor capsule (P < 0.0001), in particular in follicular- and micro-PTC variants. By multivariate analysis, the absence of tumor capsule remained the only parameter associated (P = 0.0005) with BRAF V600E mutation.
Our data suggest that BRAF V600E mutation is associated with high-risk PTC and in particular in follicular variant with invasive tumor growth.
Experiments were conducted in order to examine the expression of the IGF-I gene and of genes for IGF-binding protein (IGFBP)-1, -2, -3 and -4 in human thyroid cells from nodular goiter (NG), follicular adenoma (FTA) and follicular carcinoma (FTC), cultured in monolayers. The effect of exogenous IGF-I on the expression of the genes in question was also studied. Thyroid tissue specimens were collected from 55 patients. After approximately 2-3 weeks of culture, the cells were incubated for 24 hours with IGF-I (0, 1, 10 and 100 ng/ml). Following total mRNA isolation, mRNA encoding IGF-I, IGFBP 1-4 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was amplified by RT-PCR. The GAPDH gene served as a control housekeeping gene. PCR products were electrophoresed and then submitted to densitometric analysis. The essential differences in the expression of IGF-I and IGFBP 1-4 genes between benign (NG, FTA) and malignant (FTC) lesions may perhaps allow for differentiation between these two types of lesions by molecular biology techniques.
Fine needle aspiration (FNA) cytology represents a reliable diagnostic procedure for preoperative identification of thyroid carcinoma. For improving its diagnostic accuracy, a technique that allows the analysis of cancer-related gene abnormalities on thyroid FNA smears has been developed. Cells were collected by scraping from thyroid smears, DNA-extracted and ras proto-oncogene sequences amplified by the polymerase chain reaction (PCR). Preliminary destaining of cytologic smears was essential for efficient PCR amplification. Twelve thyroid FNA cytologic smears, characterized by the indeterminate pattern of follicular neoplasia, were analysed for the presence of ras mutations known to confer an oncogenic potential. The same nucleotide substitution at codon 12 of the H-ras proto-oncogene was detected in two different thyroid nodules among six cases that, at final histology, were identified as follicular carcinomas. No ras mutations were observed in the remaining six cases that were diagnosed as follicular adenoma at histologic examination. Molecular analysis of FNA smears may provide additional information on the nature of the lesion underlying thyroid neoplasia, thus improving diagnostic accuracy of conventional FNA cytology.
(C) Lippincott-Raven Publishers.
BACKGROUND
Fine-needle aspiration (FNA) of the thyroid is of limited value in discriminating between nonneoplastic and neoplastic lesions in approximately 5–29% of patients. Indeterminate lesions are due primarily to the overlapping cytologic features found in follicular lesions. In this report, the authors describe their experience with FNA biopsy of the thyroid, concentrating on the analysis of those aspirates placed in the follicular lesion category.METHODSA blinded, retrospective analysis of 92 patients who underwent FNA and were diagnosed with follicular lesions was performed by three of the authors (T.S.G., B.D.F., and M.O.) at a multihead microscope. A worksheet assessing a variety of cytologic and architectural features was filled out for each FNA patient. The reviewers then reached a consensus diagnosis.RESULTSThe reviewers agreed with the reported FNA diagnosis of follicular lesion in 63 of the 92 patients studied. No distinguishing cytologic features predictive of the histologic outcome were found in any of these 63 patients. Seven patients were judged by the reviewers to have insufficient cells for evaluation. In the remaining 22 patients, the reviewers' diagnoses were in agreement with the histologic diagnoses in 17 patients.CONCLUSIONS
The authors found that there is a gray area in the cytologic diagnosis of patients with thyroid lesions by FNA due to inherent similarities at the light microscopic level. However, increased specificity may be achieved by careful attention to cytologic features and morphologic detail. Skillful application of FNA techniques, with the recovery of an adequate sample, will further decrease both interpretive errors and the number of patients diagnosed with “follicular lesions.” Cancer (Cancer Cytopathol) 2000;90:335–341. © 2000 American Cancer Society.
High-frequency ultrasound examination of the thyroid was performed in 253 subjects (130 women and 123 men; age range, 19-50 years) that were randomly selected from the population in an area of Finland where goiter is not endemic. Thyroid echo abnormalities were detected in 69 subjects (27.3%). Prevalence of abnormalities increased with age, and women showed more lesions than did men in each of the 3 decades. The abnormality was solitary in 39 subjects (57%), multiple in 15 (22%), and diffuse in 15 (22%). Of the 68 individual nodules, 48 (70%) were smaller than 1 cm in diameter. Anechoic rounded nodules 1-5 mm in diameter were found in 28 subjects. Fine-needle aspiration biopsy was performed in 30 subjects. Cytologic examination revealed no unequivocal malignancies. In eight subjects (3.2%) with a diffuse echo abnormality, cytologic evaluation indicated lymphocytic thyroiditis. It is concluded that the prevalence of small thyroid echo abnormalities in a randomly selected adult population is rather high, a fact that supports use of a conservative approach to these types of findings.
Thyroid nodules are common. Most are benign lesions since clinically important thyroid carcinoma is a relatively rare disease. The most sensitive and specific test for the diagnosis of thyroid cancer is fine-needle aspiration biopsy, but its diagnostic accuracy depends upon whether or not one excises all suspicious nodules, thus including them as correctly diagnosed. Nevertheless, fine-needle aspiration biopsy is the most sensitive, specific, and cost-effective test for thyroid cancer. Therapy depends upon the cause of the thyroid nodule.
Very high frequency (13 MHz) ultrasonographic examination of the neck successfully visualized one or more normal lymph nodes (weakly echoic oval structures with an echoic central hilum) in 67.6% of 1000 healthy volunteers. Efficacy was not related to recent ear, nose, and throat infection, sex, or age. The longitudinal-transverse diameter ratio was greater than or equal to 2 in 86.2% of cases. No signal was detected with color Doppler imaging. One or more thyroid nodules were also identified in 34.7% of the subjects: the frequency was 44% in women and 42% in subjects over 50 years of age.
To determine the long-term impact of medical and surgical treatment of well differentiated papillary and follicular thyroid cancer.
Patients with papillary and follicular cancer (n = 1,355) treated either in U.S. Air Force or Ohio State University hospitals over the past 40 years were prospectively followed by questionnaire or personal examination to determine treatment outcomes. Outcomes were analyzed by Kaplan-Meier survival curves and Cox proportional-hazard regression model.
Median follow-up was 15.7 years; 42% (568) of the patients were followed for 20 years and 14% (185) for 30 years. After 30 years, the survival rate was 76%, the recurrence rate was 30%, and the cancer death rate was 8%. Recurrences were most frequent at the extremes of age (< 20 and > 59 years). Cancer mortality rates were lowest in patients younger than 40 years and increased with each subsequent decade of life. Thirty-year cancer mortality rates were greatest in follicular cancer patients, who were more likely to have adverse prognostic factors: older age, larger tumors, more mediastinal node involvement, and distant metastases. When patients with distant metastases at diagnosis were excluded, follicular and papillary cancer mortality rates were similar (10% versus 6%, P not significant [NS]). In a Cox regression model that excluded patients who presented with distant metastases, the likelihood of cancer death was (1) increased by age > or = 40 years, tumor size > or = 1.5 cm, local tumor invasion, regional lymph-node metastases, and delay in therapy > or = 12 months; (2) reduced by female sex, surgery more extensive than lobectomy, and 131I plus thyroid hormone therapy; and (3) unaffected by tumor histologic type. Following 131I therapy given only to ablate normal thyroid gland remnants, the recurrence rate was less than one third the rate after thyroid hormone therapy alone (P < 0.001). No patient treated in this way with 131I has died of thyroid cancer. Low 131I doses (29 to 50 mCi) were as effective as high doses (51 to 200 mCi) in controlling tumor recurrence (7% versus 9%, P = NS). Following 131I therapy, whether given for thyroid remnant ablation or cancer therapy, recurrence and the likelihood of cancer death were reduced by at least half, despite the existence of more adverse prognostic factors in patients given 131I. At 30 years, the cumulative cancer mortality rate following 131I therapy, regardless of the reason for its use, was one third that in patients not so treated (P = 0.03).
Over the long term, for tumors > or = 1.5 cm that are not initially metastatic to distant sites, near-total thyroidectomy followed by 131I plus thyroid hormone therapy confers a distinct outcome advantage. This therapy reduces tumor recurrence and mortality sufficiently to offset the augmented risks incurred by delayed therapy, age > or = 40 at the time of diagnosis, and tumors that are much larger than 1.5 cm, multicentric, locally invasive, or regionally metastatic.
The solitary thyroid nodule, defined as a palpably discrete swelling within an otherwise apparently normal gland, is usually a benign lesion. However, patient and physician alike are typically concerned about the possibility of thyroid cancer. This review describes a strategy for the treatment of clinically euthyroid patients who have a solitary thyroid nodule that prevents unnecessary testing while identifying the few patients who require therapy. Management has changed in recent years, but important differences of opinion remain over which nodules should be surgically excised. Several recent reviews address these issues comprehensively1–3. Prevalence of Thyroid Nodules and Cancer The . . .
The burden of thyroid disease in the general population is enormous. As many as 50% of people in the community have microscopic nodules, 3.5% have occult papillary carcinoma, 15% have palpable goiters, 10% demonstrate an abnormal thyroid-stimulating hormone level, and 5% of women have overt hypothyroidism or hyperthyroidism. Despite this high prevalence of thyroid disease, screening for these disorders is not recommended by any major health agency. This article explores the epidemiologic issues surrounding this complex problem by analyzing prevalence, incidence, and mortality data from a worldwide variety of sources.
The RET/PTC oncogenes, activated forms of the RET protooncogene, almost exclusively found in papillary thyroid carcinoma (PTC). What is more, the targeted expression of RET/PTC in mice leads to the development of thyroid tumors very similar to human PTCs. In all RET/PTC types the RET tyrosine kinase domain is fused to the N-terminus of ubiquitously expressed genes that is capable of ligand-independent dimerization. The majority of RET/PTC identified consists of two types which results from the inversion of chromosome 10: RET/PTC1 and RET/PTC3. The prevalence of RET/PTC in papillary thyroid carcinomas of thyroid varies widely from a few to about 80% with the highest frequency in tumors arising in children after ionizing radiation. In Polish population the frequency of RET rearrangements in papillary cancers is 27%, although, it was reported to be twice higher in young patients (50% in patients younger than 21 at operation). Correlation with clinical outcome as well as prognostic value of RET/PTC is controversial. Some authors suggest that it predicts metastases, others found rearranged RET in more favourable, slow growing tumors. RET/PTC3 seems to be associated with solid/follicular variant PTC and short latency period (it is found more frequently in children) whereas RET/PTC1--with classic PTC variant and long latency.
Mutations in the BRAF gene have recently been detected in a wide range of neoplastic lesions with a particularly high prevalence in melanoma and papillary thyroid carcinoma (PTC). The hot-spot mutation BRAF(V599E) is frequently detected in PTC (36-69%), in contrast to its absence in other benign or malignant thyroid lesions. In order to unravel whether there is any association between the occurrence of the BRAF mutation and the histological pattern of PTC, in this study a previous series of 50 PTCs was extended to 134 cases, including ten cases of PTC-related entities-hyalinizing trabecular tumour (HTT) and mucoepidermoid carcinoma (MEC). Using PCR/SSCP and sequencing, the BRAF(V599E) mutation was detected in 45 of the 124 PTCs (36%). No mutations were detected in any case of HTT and MEC. BRAF(V599E) was present in 75% of Warthin-like PTCs and 53% of conventional PTCs, whereas no BRAF(V599E) mutations were detected in any of the 32 cases of the follicular variant of PTC. BRAF(V599E) was also detected in 6 of 11 cases of the oncocytic variant of PTC that displayed a papillary or mixed follicular-papillary growth pattern and in none of the four oncocytic PTCs with a follicular growth pattern. A distinct mutation in BRAF (codon K600E) was detected in three cases of the follicular variant of PTC. This study has confirmed the high prevalence of BRAF(V599E) in PTC and has shown that the mutation is almost exclusively seen in PTC with a papillary or mixed follicular-papillary growth pattern, regardless of the cytological features of the neoplastic cells. The results support the existence of an oncocytic variant of PTC that should be separated from the oncocytic variant of follicular carcinoma and suggest that the follicular variant of PTC may be genetically different from conventional PTC.
Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.
Fine needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules, but subsequent patient management is often complicated by the inability to decisively recognize malignancy on cytologic grounds alone. Activating mutations of the BRAF oncogene commonly occur in papillary thyroid carcinomas (PTCs) but not in other types of benign and malignant thyroid lesions. Mutational analysis of FNAs could enhance selection of thyroid nodules for surgical removal.
Ninety-five excised PTCs along with 49 corresponding FNAs were evaluated for BRAF mutations by a newly developed assay that uses a novel primer extension method (MutectorR assay) and by direct sequencing. An additional 42 FNAs from thyroid nodules that were excised based on a suspicion of malignancy were also evaluated.
BRAF mutations were identified in 36 of the 95 (38%) excised PTCs. By histological subtype, BRAF mutations were more common in conventional PTCs than in the follicular variant (67% versus 12%; P < 0.0001; chi(2)). Analysis of the preoperative FNAs accurately reflected BRAF status of the resected PTC in 46 of the 49 paired samples (94% concordance). In FNA samples grouped according to the preoperative cytologic findings (malignant, n = 25; benign, n = 11; and indeterminate, n = 55), a BRAF mutation confirmed the diagnosis of PTC in 72% of carcinomas within the malignant group, and it established the diagnosis of PTC in 16% of carcinomas within the indeterminate group. BRAF mutations were not detected in FNAs from 32 benign thyroid lesions. Direct sequencing and the MutectorR assay yielded completely concordant results.
BRAF mutations are common in conventional PTCs, and they are specific for PTC. A BRAF mutation can be reliably detected in cells aspirated from a thyroid nodule suggesting a role for this marker in the preoperative evaluation of thyroid nodules.
Activation of the RAS/RAF/MEK/ERK/MAP kinase pathway is a known mediator of signaling that results in cellular proliferation. Moreover, this activation can lead to a growth advantage of tumor cells. Therefore, mitogenic mutations in the RAS family of oncogenes are detectable in a significant percentage in most tumors. Moreover, mutations in the BRAF gene have recently been suggested as an alternate predominant cause of colorectal and papillary thyroid cancers without ras mutations. Similar to neoplasms of other organs mutations of all three ras genes can be found in thyroid tumors. In our set of 40 cold thyroid adenoma and adenomatous nodules ras mutations were detected in only a single case. We therefore tested these hypofunctional tumors for BRAF mutations. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 11 and 15 of the BRAF gene were polymerase chain reaction (PCR) amplified and denaturing gradient gel electrophoresis was used to screen for mutations. No point mutation could be found in the two exons of the BRAF gene. Our study suggests that BRAF mutations are rather rare in solitary cold adenomas and adenomatous nodules and do not explain the molecular etiology of ras mutation-negative cold thyroid nodules.
To describe changes that have occurred between 1952 and 2002 in the evaluation and management of nodular thyroid disease.
A 30-year personal experience, institutional contributions, and the related published literature on evaluation of thyroid function and evolving strategies for management of thyroid nodules are reviewed.
Triiodothyronine (T(3)) was discovered in 1952, and measurement of plasma thyroxine by a competitive protein-binding technique became available in the 1960s. Late during that decade, the first radioimmunoassay for thyroid-stimulating hormone (TSH) was described, modified, and then used in clinical practice until the mid-1980s, when the more sensitive TSH assays became widely available. T(3) determination by radioimmunoassay was introduced early in the 1970s. Currently, sensitive thyroid function tests can detect early disease. In the general population, thyroid nodules have a prevalence higher than 50% after age 65 years, affecting more than 100 million people in the United States. Two important developments influenced thyroid nodule evaluation and management-- fine-needle aspiration (FNA) biopsy and ultrasonography. Because FNA biopsy has emerged as the most accurate test for nodule diagnosis, it has decreased the need for scanning and for thyroidectomy and thereby is likely to reduce health-care costs by more than $500 million annually in the United States. Thyroid ultrasonography is the imaging method of choice for evaluation of thyroid gland structure. Management of cytologically benign thyroid nodules remains controversial.
TSH seems to be only one of many factors in pathologic thyroid growth. FNA, because of its diagnostic accuracy, should be the initial procedure used in nodule evaluation.
In this issue of the JCI, Ciampi et al. report the identification of a novel oncogene in patients affected by radiation-associated thyroid papillary carcinomas. This oncogene derives from a paracentric inversion of the long arm of chromosome 7, which results in an in-frame fusion of the N-terminus of the A-kinase anchor protein 9 (AKAP9) gene with the C-terminal catalytic domain (exons 9-18) of the serine-threonine kinase BRAF. The resulting AKAP9-BRAF fusion protein shows constitutive kinase activity, and it is able to transmit mitogenic signals to the MAPK pathways and to promote malignant transformation of NIH3T3 cells.
In this study, the frequency of BRAF mutation was investigated in a series of 67 cases of papillary thyroid cancer (PTC) in patients from Ukraine. Thirty-two patients were aged 30 years or older at the time of diagnosis and 35 were under 16. Tumour was microdissected from paraffin wax-embedded sections, DNA extracted, and the presence of the BRAF T1796A mutation demonstrated by two different methods: PCR followed by restriction enzyme digestion or primer extension assay and detection using MALDI-TOF mass spectrometry. Eighteen (58%) of the adult cases, but only one of the 35 cases aged less than 16 harboured a BRAF T1796A mutation. There was complete agreement between the two methods used, suggesting that the MALDI-TOF assay is a robust alternative to conventional mutation analysis. RET rearrangement was also examined in the young cohort. The overall frequency of RET rearrangement was 45.7%. Eight of the younger group of patients were born after 1 December 1986 and were therefore not exposed to radioiodine in fallout from Chernobyl. None of the PTCs from these eight patients were positive for BRAF mutation. The frequency of RET rearrangement was 44% in the 27 cases exposed to radiation and 50% in the eight not exposed. These results suggest that the different molecular biological profiles observed are associated with the age of the patient at diagnosis with PTC, rather than being associated with radiation exposure.
Searching for multiple molecular markers in thyroid aspirates appears to be a promising approach for establishing a preoperative diagnosis of papillary thyroid carcinoma (PTC).
Based on this hypothesis, a total of 63 samples from 55 patients, were collected at random. RNA was extracted from the residue cells inside the needle used for fine needle aspiration cytology (FNAC) and thereafter molecular analysis was carried out both for RETrearrangements (type 1, 2, 3) and BRAF codon 599 mutation molecule. Results were compared with the cytological and histopathological diagnoses in 24 patients submitted to surgery.
58% PTCs presented a genetic alteration either RET/PTC rearrangement, BRAF V599E mutation or both: three cases of PTCs (25%) presented a RET/PTC rearrangement; three cases of PTCs (25%) presented a BRAF V599E mutation and in one case (8%) both alterations were identified.
The present results suggest that searching for multiple molecular markers in thyroid aspirates may enhance the accuracy of FNAC and refine preoperative diagnosis of PTC.
The genes RET and RAS, and more recently BRAF, have been shown to be frequently mutated in human papillary thyroid carcinomas (PTC). The aim of this study was to genotype for these mutations a cohort of thyroid tumours collected at our institutions.
Thyroid tumours removed from 51 subjects were analysed, including 43 PTC and 8 non-PTC tumours [3 follicular adenomas (FA), 4 follicular carcinomas (FTC) and 1 anaplastic carcinoma (AC)].
RET/PTC1 and RET/PTC3 expression was evaluated by reverse transcriptase-polymerase chain reaction, whereas screening of BRAF (exon 15) and RAS (HRAS, KRAS2 and NRAS) mutations were performed, respectively, by single strand conformation polymorphism and denaturing high-pressure liquid chromatography.
RET/PTC expressions was positive in 5/43 (11.6%) PTC and in none of the non-PTC tumour. Similarly, BRAF mutations were positive only in PTC, but with a higher prevalence (24/43 positives, 55.8%). All but one BRAF mutation resulted in the prototypic substitution of valine 600 with a glutamic acid. In one case, a somatic in-frame insertion of three bases at codon 599 resulted in the insertion of an additional valine. RET/PTC expression and BRAF mutations were mutually exclusive. Screening of the RAS gene allowed identification of oncogenic mutations in 1/3 (33.3%) FA and 3/4 (75%) FTC. None of the PTCs was positive for RAS.
These data indicate that BRAF mutations are the most frequent genetic event in PTC and that RAS mutations, besides being a genetic hallmark of follicular tumours, are rare or completely absent in PTC from our area. Together, BRAF mutations and rarer RET rearrangements accounted for a genetic event in two-thirds of PTCs. This study showed a novel and presumably oncogenic mutation of BRAF, which is BRAF(V599Ins).
Papillary carcinoma is the most common type of thyroid malignancy. It has been recently shown that these tumors commonly have one of three genetic alterations: BRAF point mutations, RET/PTC rearrangements, or RAS point mutations. In this study, we analyze the relationship between these alterations and the microscopic features of papillary carcinomas, their clinical features, and prognostic characteristics. Ninety-seven papillary carcinomas were studied; in all cases, frozen tissue was available for nucleic acid extraction. Of 96 unselected cases, 42% were positive for BRAF, 18% for RET/PTC, and 15% for RAS mutations. Morphologic features were evaluated in detail in 61 cases and 6 characteristic nuclear features and 3 additional microscopic features were assessed quantitatively. At least 4 nuclear features were found in each tumor, with nuclear pseudoinclusions being the least frequent finding in all mutation groups. BRAF mutations were associated with older patient age, typical papillary appearance or the tall cell variant, a higher rate of extrathyroidal extension, and more advanced tumor stage at presentation. RET/PTC rearrangements presented at younger age and had predominantly typical papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases. Tumors with RAS mutations were exclusively the follicular variant of papillary carcinoma and correlated with significantly less prominent nuclear features and low rate of lymph node metastases. These findings demonstrate that BRAF, RET/PTC, and RAS mutations are associated with distinct microscopic, clinical, and biologic features of thyroid papillary carcinomas.
The oncogene BRAF(V600E) is the most frequent genetic event in papillary thyroid carcinoma (PTC) but its prognostic impact still remains to be elucidated. We evaluated a representative series of 67 individuals with PTC who underwent total thyroidectomy. BRAF-positive tumours correlated with early recurrences (32% vs 7.6%; P=0.02) during a median postoperative follow-up period of 3 years. Interestingly, within the recurrences, a significant majority had negative radioiodine ((131)I) total body scans, predicting a poorer outcome as treatment with (131)I is not effective. This last observation led us to investigate the role of BRAF(V600E) and the MEK-ERK pathway in thyroid dedifferentiation, particularly in Na(+)/I(-) symporter (NIS) impairment, as this thyroid-specific plasma membrane glycoprotein mediates active transport of I(-) into the thyroid follicular cells. A subset of 60 PTC samples was evaluated for NIS immunoreactivity and, accordingly, we confirmed a significant low NIS expression and impaired targeting to membranes in BRAF-positive samples (3.5% vs 30%; P=0.005). Furthermore, experiments with differentiated PCCl3 thyroid cells demonstrated that transient expression of BRAF(V600E) sharply impaired both NIS expression and targeting to membrane and, surprisingly, this impairment was not totally dependent on the MEK-ERK pathway. We have concluded that BRAF(V600E) is a new prognostic factor in PTC that correlates with a high risk of recurrences and less differentiated tumours due to the loss of NIS-mediated (131)I uptake.
Controversy remains as to the optimal management of patients with multiple thyroid nodules.
The objective of this study was to determine the prevalence, distribution, and sonographic features of thyroid cancer in patients with solitary and multiple thyroid nodules.
We describe a retrospective observational cohort study that was carried out from 1995 to 2003.
The study was conducted in a tertiary care hospital.
Patients with one or more thyroid nodules larger than 10 mm in diameter who had ultrasound-guided fine needle aspiration (FNA) were included in the study.
The main outcome measures were prevalence and distribution of thyroid cancer and the predictive value of demographic and sonographic features.
A total of 1985 patients underwent FNA of 3483 nodules. The prevalence of thyroid cancer was similar between patients with a solitary nodule (175 of 1181 patients, 14.8%) and patients with multiple nodules (120 of 804, 14.9%) (P = 0.95, chi(2)). A solitary nodule had a higher likelihood of malignancy than a nonsolitary nodule (P < 0.01). In patients with multiple nodules larger than 10 mm, cancer was multifocal in 46%, and 72% of cancers occurred in the largest nodule. Multiple logistic regression analysis of statistically significant features demonstrates that the combination of patient gender (P < 0.02), whether a nodule is solitary vs. one of multiple (P < 0.002), nodule composition (P < 0.01), and presence of calcifications (P < 0.001) can be used to assign risk of cancer to each individual nodule. Risk ranges from a 48% likelihood of malignancy in a solitary solid nodule with punctate calcifications in a man to less than 3% in a noncalcified predominantly cystic nodule in a woman.
In a patient with one or more thyroid nodules larger than 10 mm in diameter, the likelihood of thyroid cancer per patient is independent of the number of nodules, whereas the likelihood per nodule decreases as the number of nodules increases. For exclusion of cancer in a thyroid with multiple nodules larger than 10 mm, up to four nodules should be considered for FNA. Sonographic characteristics can be used to prioritize nodules for FNA based on their individual risk of cancer.
The activating BRAF(V600E) mutation is the most common genetic alteration reported in papillary thyroid carcinoma (PTC). While some reports suggest the BRAF(V600E) mutation is associated with factors predicting a poor prognosis and recurrence, this remains a controversial issue.
To determine whether the presence of the BRAF(V600E) mutation is a prognostic indicator for clinical recurrence in low-risk patients with conventional PTC.
The study involved 203 conventional PTC patients who underwent total or near-total thyroidectomy followed by immediate 131I ablation of the remnants. Patients with antithyroglobulin antibodies and those with extracervical metastases at presentation were excluded. DNA was extracted from paraffin-embedded tumour specimens, and the presence of the BRAF(V600E) mutation was evaluated using PCR amplification and direct sequencing.
The BRAF(V600E) mutation was found to be present in 149 (73.4%) of 203 patients. The BRAF(V600E) mutation was correlated with male gender (P = 0.006) and with tumour size (P = 0.005). While there appeared to be an association between the BRAF(V600E) mutation and extrathyroid extension, this did not reach statistical significance (P = 0.062). During follow-up of the 203 patients (median 7.3 years; range 0.7-10.0 years), 36 (18%) patients experienced recurrence. While univariate analysis showed the BRAF(V600E) mutation was associated with tumour recurrence (21% with mutation vs 7% without mutation; P = 0.037), this association was not shown following multivariate analyses adjusting for the clinicopathological prognostic factors of age, gender, tumour size, extrathyroid extension, multifocality and lymph node metastasis.
Although the BRAF(V600E) mutation was found to be associated with a higher clinical recurrence of disease in low-risk conventional PTC patients, it was not an independent predictor.
BRAF mutations have been detected in 30% to 80% of papillary thyroid carcinomas (PTC). Several detection methods for BRAF mutation have been reported, but a direct comparison between different assay methods has not been previously reported. In this study, we examined the diagnostic utility of BRAF (T1799A) mutation in 71 cases of thyroid fine needle aspiration specimens using 4 different methods, including direct sequencing, Colorimetric Mutector Assay, real-time LightCycler polymerase chain reaction (LC PCR) with fluorescence resonance energy transfer probes, and an allele-specific LC PCR with CYBR green 1. BRAF mutation was detected in 31 of 58 cases of PTC, but not in 13 cases of non-PTC lesions. The 4 assay methods used in this study were sensitive, reliable, and comparable with each other (100% of specificity and 53.5% of sensitivity). PTC harboring BRAF mutation had higher extrathyroidal invasion and/or lymph node metastasis than PTC with wild-type BRAF. BRAF mutation analysis should be useful for the clinical diagnosis of PTC in cases of indeterminate fine needle aspiration specimen, because of the high degree of specificity. Our results indicate that there is similar sensitivity for the four detection methods. However, the allele-specific LC PCR with CYBR green 1 method is most rapid, easier to perform, and least expensive technique, and it can be readily performed in most molecular diagnostic laboratories.
Between 10 and 30% of the fine needle aspiration biopsies (FNABs) of thyroid nodules are diagnosed as 'indeterminate'. A molecular diagnostic method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is the classic papillary type and the BRAF(V600E) mutation is highly prevalent.
To evaluate the role of pre-operative detection of BRAF(V600E) mutation in the FNAB specimens of thyroid nodules in a BRAF(V600E) mutation-prevalent geographical area.
In 137 specimens of FNAB (107 papillary thyroid carcinomas (PTC); 3 follicular thyroid carcinomas (FTC); 2 undifferentiated thyroid carcinomas; 25 benign lesions), both direct DNA sequencing and PCR-RFLP were used for detecting the BRAF(V600E) mutation. The sensitivity and specificity were calculated. We analysed the association between BRAF(V600E) mutation and the clinico-pathological parameters.
The BRAF(V600E) mutation was present in 93 (83%) of 112 thyroid cancers. Direct DNA sequencing showed a sensitivity of 83.0% and a specificity of 96.0%. The sensitivity and specificity of PCR-RFLP were 78.6% and 80.0%, respectively. Among 25 cases with indeterminate FNAB cytology, 8 patients had malignant lesions (5 PTC and 3 FTC). Three (60%) of 5 PTCs and 1 out of 17 benign lesions had BRAF(V600E) mutation (only one false positive case and the definitive pathology showed atypical nodular hyperplasia that could be a premalignant lesion). The diagnostic accuracy of this molecular method in only the 25 indeterminate nodules was 76% (19/25). No mutation was found in 3 FTCs. Among 107 PTCs, there was no significant association of the BRAF(V600E) mutation with the known risk factors.
Detection of the BRAF(V600E) mutation in FNAB specimens refines the FNAB-cytology diagnosis, especially in a BRAF(V600E) mutation-prevalent area. Direct DNA sequencing was a more reliable method than PCR-RFLP for detecting the BRAF(V600E) mutation with a high sensitivity and specificity.
Fine-needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20-40% of cases the FNAB result is uncertain.
We investigated whether a search for the oncogenes RET/PTC, TRK and BRAF(V600E) in thyroid aspirates could refine an uncertain diagnosis.
A total of 132 thyroid aspirates, including colloid nodules, inadequate samplings, indeterminate and suspicious for malignancy were analysed by reverse transcription polymerase chain reaction (RT-PCR) and mutant allele-specific amplification techniques for the presence of oncogenes.
No oncogenes were detected in 48 colloid nodules, 46 inadequate and 19 indeterminate FNABs, then confirmed to be benign at histology. No oncogenes were detected in one follicular thyroid cancer (FTC) with indeterminate cytology. Five out of six papillary thyroid cancers (83%) with FNAB suspicious for malignancy were correctly diagnosed by the presence of oncogenes. Among these, four (67%) contained the BRAF mutation and one (17%) contained RET/PTC-3. On final analysis, no false-positive results were reported in 131 samples and five out of seven carcinomas (71%) were correctly diagnosed. The finding of oncogenes in FNAB specimens suspicious for malignancy guided the extent of surgical resection, changing the surgery from diagnostic to therapeutic in five cases.
Detection of RET/PTC, TRK and BRAF(V600E) in FNAB specimens is proposed as a diagnostic adjunctive tool in the evaluation of thyroid nodules with suspicious cytological findings.
BRAF(V600E) mutation and p27(kip1) expression have been introduced as novel indicators that may predict prognosis in different tumors, as well as in papillary thyroid carcinomas.
Tissue samples from 214 consecutive patients who underwent total or near-total thyroidectomy with histological diagnosis of papillary thyroid carcinoma (PTC) <or=1 cm were analyzed for BRAF(V600E) mutation by a real-time, allele-specific amplification and for p27(kip1) expression by immunohistochemistry.
The BRAF(V600E) mutation was detected in 88 of the tumors examined, with significant differences between groups with and without lymph node (LN) metastases; the mean age of patients with BRAF(V600E) mutation was significantly higher than that of patients without mutations. A significant association was found between low p27(Kip1) protein expression and multifocality, bilaterality, and extrathyroidal extension, in addition to LN metastasis. In 42 cases with LN metastases, 23 harbored the BRAF(V600E) mutation in the metastatic tumor and presented a wider diameter of the largest metastatic area, a higher number of involved LNs, and a higher percentage of metastatic lesions with extracapsular extension of LN (ECE-LN). A significantly lower mean value of p27(Kip1) was observed in LNs harboring the BRAF(V600E) mutation and in ECE-LN; an inverse correlation was found between p27(Kip1) and the number of metastatic LNs, as well as the diameter of the largest metastatic area in LN.
The authors' data suggested that BRAF(V600E) mutation and p27(Kip1) down-regulation in cancer cells of PTC <or=1 cm may be factors that facilitate tumor-cell growth and progression once these are seeded in the LNs.
To summarize the definitions of and management recommendations for low-risk thyroid cancer made by the American and European Thyroid Associations and synthesize this information with the recent literature, including systematic evaluations of tumor staging systems guiding therapy.
The American Thyroid Association and European Thyroid Association guidelines were compared and pertinent literature since 2005 was reviewed.
Of papillary thyroid microcarcinomas (PTMC), up to 50% breach the thyroid capsule, 64% have lymph node metastases, up to 43% are multifocal, and as many as 2.8% have distant metastases. Locoregional and distant recurrences are, respectively, as high as 5.9% and 1.5%. As many as 1 in 4 patients with a papillary thyroid carcinoma 1.5 cm or smaller develop persistent disease. Cancer-related mortality rates are usually less than 1%, but are as high as 2% in some reports. Tumor staging systems are too inaccurate to guide therapy.
It is unlikely that many patients will forgo treatment after understanding their risk, especially when total thyroidectomy and radioiodine (131I) therapy can reduce the PTMC recurrence or persistence disease rate to zero. Preoperatively diagnosed PTMC should be treated with total or near-total thyroidectomy, regardless of tumor size. For very low-risk patients with unifocal PTMC smaller than 1 cm that is removed by chance during surgery to treat benign thyroid disease, lobectomy alone without 131I therapy may be sufficient therapy if there are no concerning histologic features and no tumor extension beyond the thyroid, metastases, history of head and neck irradiation, or positive family history--any of which requires total or near-total thyroidectomy and remnant ablation with 30 mCi.