A High-Resolution Melting Protocol for Rapid and Accurate Differential Diagnosis of Thyroid Nodules
2012; Elsevier BV; Volume: 14; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2012.03.003
ISSN1943-7811
AutoresIrene Mancini, Pamela Pinzani, Cinzia Pupilli, Luisa Petrone, Maria Laura De Feo, Lapo Bencini, Mario Pazzagli, Gianni Forti, Claudio Orlando,
Tópico(s)Thyroid Cancer Diagnosis and Treatment
ResumoA large majority of thyroid nodules are benign, and only 5% have malignant features on cytological examination. Unfortunately, fine-needle aspiration is inconclusive in approximately 30% of all thyroid biopsies, because the cytological features are indeterminate (suspicious for malignancy but not completely diagnostic or nondiagnostic). Wide panels of somatic mutations have been identified in thyroid cancers, and detection of genetic alterations in fine-needle aspirate has been demonstrated to improve diagnostic accuracy. Nevertheless, the relatively high number of genetic targets to be investigated, in comparison with the low percentage of malignant samples, makes the usual diagnostic protocol both time-consuming and expensive. We developed a reliable and sensitive protocol based on high-resolution melting analysis for the rapid screening of mutations of KRAS, HRAS, NRAS, and BRAF oncogenes in thyroid fine-needle aspirations. The entire procedure can be completed in approximately 48 hours, with a dramatic reduction in costs. The proposed protocol was applied to the analysis of 260 consecutive fine-needle aspiration biopsy (FNAB) samples. In 35 of 252 samples, 36 sequence variants were detected for BRAF (17 samples), NRAS (6 samples), HRAS (3 samples), KRAS codon 12 (9 samples), and KRAS codon 61 (1 sample). A large majority of thyroid nodules are benign, and only 5% have malignant features on cytological examination. Unfortunately, fine-needle aspiration is inconclusive in approximately 30% of all thyroid biopsies, because the cytological features are indeterminate (suspicious for malignancy but not completely diagnostic or nondiagnostic). Wide panels of somatic mutations have been identified in thyroid cancers, and detection of genetic alterations in fine-needle aspirate has been demonstrated to improve diagnostic accuracy. Nevertheless, the relatively high number of genetic targets to be investigated, in comparison with the low percentage of malignant samples, makes the usual diagnostic protocol both time-consuming and expensive. We developed a reliable and sensitive protocol based on high-resolution melting analysis for the rapid screening of mutations of KRAS, HRAS, NRAS, and BRAF oncogenes in thyroid fine-needle aspirations. The entire procedure can be completed in approximately 48 hours, with a dramatic reduction in costs. The proposed protocol was applied to the analysis of 260 consecutive fine-needle aspiration biopsy (FNAB) samples. In 35 of 252 samples, 36 sequence variants were detected for BRAF (17 samples), NRAS (6 samples), HRAS (3 samples), KRAS codon 12 (9 samples), and KRAS codon 61 (1 sample). Thyroid cancer is the most common malignancy of the endocrine system, accounting for approximately 1% of all malignancies in Western countries.1Sherman S.I. Thyroid carcinoma.Lancet. 2003; 361: 501-511Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar The incidence of thyroid cancer has increased 2.6-fold in the last 30 years. This change is attributed not only to an increment in papillary thyroid carcinoma,2Cramer J.D. Fu P. Harth K.C. Margevicius S. Wilhelm S.M. Analysis of the rising incidence of thyroid cancer using the Surveillance, Epidemiology and End Results national cancer data registry.Surgery. 2010; 148: 1147-1152Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar but also to more wisely conducted medical surveillance and improvements in diagnostic tools.3Davies L. Welch H.G. Increasing incidence of thyroid cancer in the United States, 1973–2002.JAMA. 2006; 295: 2164-2167Crossref PubMed Scopus (2656) Google Scholar The large majority of thyroid nodules, as discovered with the use of new diagnostic imaging techniques, are asymptomatic and benign. In this context, diagnostic studies are becoming essential to identify the small fraction of thyroid nodules that harbor malignant disease and to predict when surgery is indicated. Fine-needle aspiration biopsy (FNAB) has emerged over the past 30 years as an accurate and cost-effective procedure for the preoperative screening of thyroid nodules, representing the gold standard for differential diagnosis of benign and malignant nodules.4Tallini G. Gallo C. Fine-needle aspiration and intraoperative consultation in thyroid pathology: when and how?.Int J Surg Pathol. 2011; 19: 141-144Crossref PubMed Scopus (11) Google Scholar Under recent guidelines,5Gharib H. Papini E. Paschke R. Duick D.S. Valcavi R. Hegedüs L. Vitti P. AACE/AME/ETA Task Force on Thyroid NodulesAmerican Association of Clinical Endocrinologists, Associazione Medici Endocrinologi, and European Thyroid Association medical guidelines for clinical practice for the diagnosis and management of thyroid nodules.Endocr Pract. 2010; 16: 1-43Crossref PubMed Scopus (338) Google Scholar, 6Cooper D.S. Doherty G.M. Haugen B.R. Kloos R.T. Lee S.L. Mandel S.J. Mazzaferri E.L. McIver B. Pacini F. Schlumberger M. Sherman S.I. Steward D.L. Tuttle R.M. American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules and Differentiated Thyroid CancerRevised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer [Erratum appeared in Thyroid 2010, 20:674–675].Thyroid. 2009; 19: 1167-1214Crossref PubMed Scopus (5399) Google Scholar cytological smears are classified in five categories for the diagnostic report: Thy 1, nondiagnostic; Thy 2, benign or negative for malignant cells; Thy 3, all follicular lesions (including atypia/follicular lesion of undetermined significance and follicular neoplasm or suspicious for follicular neoplasm); Thy 4, suspicious; and Thy 5, diagnostic for malignancy. Although the overall accuracy of FNAB is considered excellent, approximately 30% of cytological aspirates do not allow definitive diagnosis of malignancy, because of intrinsic and unavoidable characteristics of samples.7Kim S.K. Kim D.L. Han H.S. Kim W.S. Kim S.J. Moon W.J. Oh S.Y. Hwang T.S. Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules.Diagn Mol Pathol. 2008; 17: 118-125Crossref PubMed Scopus (122) Google Scholar The major limitations of FNAB procedures are linked to inadequate and indeterminate specimens and, in that sense, are also linked respectively to the nondiagnostic or follicular lesions categories.8Gharib H. Papini E. Thyroid nodules: clinical importance, assessment, and treatment.Endocrinol Metab Clin North Am. 2007; 36 (vi): 707-735Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 9Layfield L.J. Cibas E.S. Gharib H. Mandel S.J. Thyroid aspiration cytology: current status.CA Cancer J Clin. 2009; 59: 99-110Crossref PubMed Scopus (134) Google Scholar Thus, a clinical need emerges for the characterization of aspirates with suspicious features but with unsatisfactory cellularity, to allow accurate distinction of benign from malignant forms of follicular lesions. Several somatic mutations have been identified in thyroid cancer, stimulating the search for genetic alterations in FNAB that could increase the diagnostic accuracy of traditional cytology. Numerous studies have demonstrated that identification of specific mutations in cytological specimens can assist in the diagnosis by FNAB and in the clinical decision to excise the nodule and to intensify the follow-up.10Cantara S. Capezzone M. Marchisotta S. Capuano S. Busonero G. Toti P. Di Santo A. Caruso G. Carli A.F. Brilli L. Montanaro A. Pacini F. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology.J Clin Endocrinol Metab. 2010; 95: 1365-1369Crossref PubMed Scopus (235) Google Scholar, 11Ohori N.P. Nikiforova M.N. Schoedel K.E. LeBeau S.O. Hodak S.P. Seethala R.R. Carty S.E. Ogilvie J.B. Yip L. Nikiforov Y.E. Contribution of molecular testing to thyroid fine-needle aspiration cytology of "follicular lesion of undetermined significance/atypia of undetermined significance".Cancer Cytopathol. 2010; 118: 17-23Crossref PubMed Scopus (216) Google Scholar, 12Moses W. Weng J. Sansano I. Peng M. Khanafshar E. Ljung B.M. Duh Q.Y. Clark O.H. Kebebew E. Molecular testing for somatic mutations improves the accuracy of thyroid fine-needle aspiration biopsy.World J Surg. 2010; 34: 2589-2594Crossref PubMed Scopus (144) Google Scholar, 13Zatelli M.C. Trasforini G. Leoni S. Frigato G. Buratto M. Tagliati F. Rossi R. Cavazzini L. Roti E. degli Uberti E.C. BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies.Eur J Endocrinol. 2009; 161: 467-473Crossref PubMed Scopus (100) Google Scholar, 14Shibru D. Chung K.W. Kebebew E. Recent developments in the clinical application of thyroid cancer biomarkers.Curr Opin Oncol. 2008; 20: 13-18Crossref PubMed Scopus (52) Google Scholar, 15Yip L. Nikiforova M.N. Carty S.E. Yim J.H. Stang M.T. Tublin M.J. Lebeau S.O. Hodak S.P. Ogilvie J.B. Nikiforov Y.E. Optimizing surgical treatment of papillary thyroid carcinoma associated with BRAF mutation.Surgery. 2009; 146: 1215-1223Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Nikiforov Y.E. Steward D.L. Robinson-Smith T.M. Haugen B.R. Klopper J.P. Zhu Z. Fagin J.A. Falciglia M. Weber K. Nikiforova M.N. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules.J Clin Endocrinol Metab. 2009; 94: 2092-2098Crossref PubMed Scopus (566) Google Scholar In most cases, genetic alterations are represented by activating mutations of oncogenes that are mutually exclusive and linked to distinct histological subtypes, with a demonstrated pathogenic role in thyroid cell transformation as effectors of the RAS/RAF/MAPK signaling cascade. The presence of BRAF mutations, a frequent alteration in papillary carcinoma (PTC), evolves into an unregulated activation of the intracellular MAPK pathway that can promote tumorigenesis and tumor progression. The main mutation of BRAF, identified exclusively in PTC, affects nucleotide 1799 in exon 15 and results in thymine-to-adenine transversion, which translates into valine-to-glutamate substitution at residue 600 (p.V600E).17Kimura E.T. Nikiforova M.N. Zhu Z. Knauf J.A. Nikiforov Y.E. Fagin J.A. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma.Cancer Res. 2003; 63: 1454-1457PubMed Google Scholar The adjacent p.K601E mutation has rarely been identified,18Trovisco V. Soares P. Preto A. de Castro I.V. Lima J. Castro P. Máximo V. Botelho T. Moreira S. Meireles A.M. Magalhães J. Abrosimov A. Cameselle-Teijeiro J. Sobrinho-Simões M. Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients' age but not with tumour aggressiveness.Virchows Arch. 2005; 446: 589-595Crossref PubMed Scopus (242) Google Scholar and no mutation in exon 11 has been described in thyroid cancers. BRAF mutations are associated with poor clinical prognosis due to extrathyroid invasion and higher risk of relapse and metastasis.19Frasca F. Nucera C. Pellegriti G. Gangemi P. Attard M. Stella M. Loda M. Vella V. Giordano C. Trimarchi F. Mazzon E. Belfiore A. Vigneri R. BRAF(V600E) mutation and the biology of papillary thyroid cancer.Endocr Relat Cancer. 2008; 15: 191-205Crossref PubMed Scopus (200) Google Scholar Mutations in the family of RAS oncogenes, which encode for G-proteins that also convey signals to the MAPK pathway, are more common in follicular carcinomas (FTC)20Vasko V. Ferrand M. Di Cristofaro J. Carayon P. Henry J.F. de Micco C. Specific pattern of RAS oncogene mutations in follicular thyroid tumors.J Clin Endocrinol Metab. 2003; 88: 2745-2752Crossref PubMed Scopus (239) Google Scholar and in follicular variant of papillary carcinoma (fvPTC).21Zhu Z. Gandhi M. Nikiforova M.N. Fischer A.H. Nikiforov Y.E. Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma An unusually high prevalence of ras mutations.Am J Clin Pathol. 2003; 120: 71-77Crossref PubMed Scopus (345) Google Scholar Point mutations in the HRAS, KRAS, and NRAS genes are associated with specific domains of the protein and are able either to increase its affinity for substrate (substitution in residues 12 and 13) or to inactivate the autocatalytic GTPase function (residue 61). RAS mutations seem to be related to benign as well as malignant growth of nodules; however, it is becoming evident that alterations in this family of oncogenes are competent to lead toward anomalous cellular transformation, through mechanisms of genomic instability22Fagin J.A. Minireview: branded from the start-distinct oncogenic initiating events may determine tumor fate in the thyroid.Mol Endocrinol. 2002; 16: 903-911Crossref PubMed Scopus (122) Google Scholar, 23Saavedra H.I. Knauf J.A. Shirokawa J.M. Wang J. Ouyang B. Elisei R. Stambrook P.J. Fagin J.A. The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway.Oncogene. 2000; 19: 3948-3954Crossref PubMed Scopus (153) Google Scholar and promotion of additional mutations.24Finney R.E. Bishop J.M. Predisposition to neoplastic transformation caused by gene replacement of H-ras1.Science. 1993; 260: 1524-1527Crossref PubMed Scopus (113) Google Scholar Because of the frequency and clinical relevance of BRAF mutations in thyroid papillary carcinomas (∼45% of all cases) and RAS mutations in follicular subtypes (40% to 50% of these tumors),25Nikiforova M.N. Nikiforov Y.E. Molecular genetics of thyroid cancer: implications for diagnosis, treatment and prognosis.Expert Rev Mol Diagn. 2008; 8: 83-95Crossref PubMed Scopus (233) Google Scholar the detection of these genetic alterations in FNABs has been widely adopted, to increase the specificity of testing. Development of rapid and accurate molecular methods could therefore be important for the screening of the large number of samples routinely collected by FNAB, to obtain a molecular diagnosis in a time frame compatible with clinical decision-making. High-resolution melting (HRM) analysis is a technique recently developed for mutation scanning of PCR products.26Margraf R.L. Mao R. Highsmith W.E. Holtegaard L.M. Wittwer C.T. RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.J Mol Diagn. 2007; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 27Krypuy M. Ahmed A.A. Etemadmoghadam D. Hyland S.J. DeFazio A. Fox S.B. Brenton J.D. Bowtell D.D. Dobrovic A. Australian Ovarian Cancer Study GroupHigh resolution melting for mutation scanning of TP53 exons 5–8.BMC Cancer. 2007; 7: 168Crossref PubMed Scopus (115) Google Scholar, 28Takano T. Ohe Y. Tsuta K. Fukui T. Sakamoto H. Yoshida T. Tateishi U. Nokihara H. Yamamoto N. Sekine I. Kunitoh H. Matsuno Y. Furuta K. Tamura T. Epidermal growth factor receptor mutation detection using high-resolution melting analysis predicts outcomes in patients with advanced non small cell lung cancer treated with gefitinib.Clin Cancer Res. 2007; 13: 5385-5390Crossref PubMed Scopus (97) Google Scholar, 29Simi L. Pratesi N. Vignoli M. Sestini R. Cianchi F. Valanzano R. Nobili S. Mini E. Pazzagli M. Orlando C. High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer.Am J Clin Pathol. 2008; 130: 247-253Crossref PubMed Scopus (162) Google Scholar The discrimination between wild-type and variant sequences is obtained by the comparison of the dissociation shape of amplicons when exposed to increasing temperature. A change in melting profile, generated by signal of fluorescent dyes intercalating only double-stranded DNA, is caused by a variation in the sequence, relative to the reference sample.30Reed G.H. Kent J.O. Wittwer C.T. High-resolution DNA melting analysis for simple and efficient molecular diagnostics.Pharmacogenomics. 2007; 8: 597-608Crossref PubMed Scopus (524) Google Scholar Our aim in the present study was to assess an inexpensive HRM analysis platform for the accurate analysis of a consistent number of cytological samples. The principal objectives were the optimization of an accurate test for a rapid screening of mutation-positive thyroid nodules and the evaluation of a molecular marker panel to refine the diagnostic accuracy among categories of cytological specimens. Nodule biopsies were obtained from 260 consecutive patients [53 men and 207 women; mean age, 55.1 years (range, 21–80 years)] undergoing FNAB for suspicious thyroid nodules. Eight samples out of the series, resulted negative at thyroglobulin assay, were excluded from subsequent molecular analysis. FNABs were performed under ultrasound guidance using a 21- to 23-gauge needle by performing five or six passes. FNAB was performed on all single nodules with a diameter >5 mm. In 13 patients with multiple nodules, FNAB was performed on a maximum of two dominant nodules for each patient. One biopsy was performed for each nodule. All samples to be submitted to cytopathology analysis were processed according to thin-layer cytology technique, as described previously.31Petrone L. Petrone L. Mannucci E. De Feo M.L. Parenti G. Biagini C. Panconesi R. Vezzosi V. Bianchi S. Boddi V. Di Medio L. Pupilli C. Forti G. A simple us score for the identification of candidates to fine needle aspiration of thyroid nodules.J Endocrinol Invest. 2011; ([Epub ahead of print])https://doi.org/10.3275/7978Crossref PubMed Scopus (7) Google Scholar The widest diameter of all nodules ranged from 6 to 75 mm (23.8 ± 11.0, mean ± SD). The sample obtained from biopsies was used for classical cytology and the needle washing was used for molecular assay. Fifty patients randomly selected underwent a second FNAB, which was used for molecular analysis (on both tissue and needle washing). All FNAB samples were collected in a single tube containing 500 μL of RNAlater stabilizing reagent (Qiagen, Milan, Italy). After one night at 4°C, samples were stored at −80°C until extraction of nucleic acids. The study was approved by the local ethics committee. Informed consent had been obtained previously from all patients. Cytological material was centrifuged at 15,890 × g for 15 minutes, and then the buffer was removed without disturbing the pellet. Immediately, 700 μL of RLT lysis buffer from an RNeasy micro kit (Qiagen) was added to the samples. Resuspended samples were divided into two 350-μL aliquots, one for DNA and one for RNA extraction. DNA was extracted using a Qiagen QIAamp DNA micro kit according to manufacturer's protocol for isolation of genomic DNA from tissue. Similarly, RNA was extracted using a Qiagen RNeasy micro kit according to the manufacturer's protocol for purification of total RNA from animal and human tissues. The entire RNA sample was reverse-transcribed using MuLV Reverse Transcriptase (Applied Biosystems-Life Technologies, Foster City, CA) and random hexamer primers in a final volume of 40 μL. Before testing for chromosomal rearrangements, 2.5 μL of cDNA was evaluated for GAPDH, using Applied Biosystems TaqMan control reagents, and for thyroglobulin gene expression (TaqMan gene expression assays; Hs00794359_m1, NM_003235.4). RNA from the human papillary thyroid carcinoma cell line TPC1 and from the human prostatic carcinoma cell line PC3 was used as positive and negative control, respectively, for thyroglobulin expression. For investigation of DNA point mutations, positive controls were obtained from cell lines harboring sequence variation in the target genes. DNA from the human T-cell lymphoblast-like cell line CCRF-CEM and the human colorectal adenocarcinoma cell line SW948 was selected as reference for the KRAS codon 12 (p.G12D, heterozygous) and 61 (p.Q61R, heterozygous) variants, respectively. CCRF-CEM was also used as a control for HRAS (p.A59A, heterozygous). DNA from the human bladder carcinoma cell line HT1197 was used as mutated reference for the NRAS codon 61 variant (p.Q61R, heterozygous). Finally, a reconstituted sample of human skin melanoma cell line SK-MEL-28 (BRAF p.V600E, homozygous) mixed with human breast cancer cell line MCF-7 DNA (wild-type BRAF) was used as heterozygous reference for exon 15 BRAF gene. HT1197, MCF-7, SK-MEL28 cell lines were supplied by Banca Biologica e Cell Factory (IRCCS Azienda Ospedaliera Universitaria San Martino – IST Istituto Nazionale per la Ricerca sul Cancro). Moreover, CCRF-CEM and SW948 cell lines were provided by ATCC-LGC Standards Partnership. DNA was extracted from all cell lines using a QIAamp DNA mini kit (Qiagen). The primer sets covering the hot-spot sites of genes were as listed in Table 1. During primer design (Primer3Plus software; http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi), the acceptable annealing temperature was set to be identical among different pairs and the product size was considered optimal within a range of 150 to 200 bp. Each amplicon was tested to exclude amplification of sequence homolog regions [UCSC Genome Browser applications In-Silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr?org=Human&db=hg19&hgsid=285213467) and BLAT Search (http://www.genome.ucsc.edu/cgi-bin/hgBlat?command=start)]. PCR reactions were performed in an ABI 2720 thermal cycler (Applied Biosystems-Life Technologies) using 10 ng DNA in a total volume of 20 μL containing a final concentration of 1× PCR buffer II (10 mmol/L Tris-HCl, pH 8.3; 50 mmol/L KCl) (Applied Biosystems-Life Technologies), 1.5 mmol/L MgCl2 solution, 0.2 mmol/L each dNTP, 0.5 μmol/L each primer, 0.5 μmol/L SYTO 9 green fluorescent nucleic acid stain (Invitrogen-Life Technologies, Carlsbad, CA), and 1 U AmpliTaq Gold DNA polymerase (Applied Biosystems-Life Technologies). Cycling conditions entailed an initial denaturation at 95°C for 10 minutes, followed by 45 cycles at 95°C for 20 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, with a final elongation step at 72°C for 20 minutes.Table 1Primer Sets and Conditions for Hot Spots of Interest in HRM Analysis of Thyroid NodulesGeneCodonSequenceProduct size (bp)Melting temperature⁎Annealing temperature was consistently 58°C. (°C)BRAF600–601Fwd: 5′-TGCTTGCTCTGATAGGAAAATG-3′17375–86Rev: 5′-CCACAAAATGGATCCAGACA-3′HRAS61Fwd: 5′-ATGGCAAACACACACAGGAA-3′14079–95Rev: 5′-GATTCCTACCGGAAGCAGGT-3′NRAS61Fwd: 5′-CCCCTTACCCTCCACACC-3′16277–88Rev: 5′-TGGCAAATACACAGAGGAAGC-3′KRAS12–13Fwd: 5′-GTCACATTTTCATTATTTTTATTATAAGG-3′15575–86Rev: 5′-TTTACCTCTATTGTTGGATCATATTC-3′KRAS61Fwd: 5′-ACTGTGTTTCTCCCTTCTCAGG-3′16177–88Rev: 5′-ATGGCAAATACACAAAGAAAGC-3′ Annealing temperature was consistently 58°C. Open table in a new tab The DNA from FNAB samples was screened by HRM analysis in a RotorGene 6000 system (Qiagen, Hilden, Germany). The denatured samples, obtained by an initial hold of 3 minutes at 95°C and 3 minutes at 40°C, were analyzed by the acquisition of fluorescence signal in a temperature range experimentally determined for each tested gene (Table 1). The melting ramp was set at 0.08 degrees/each step for each assay. All samples were run in duplicate. To confirm HRM analysis results, sequencing analysis was also performed in all samples. After HRM, samples were purified with a PCR purification kit (Qiagen) and submitted to cycle sequencing with 2 μL of BigDye Terminator ready reaction mix (Applied Biosystems-Life Technologies) and the same primers used in PCR at a final concentration of 0.16 μmol/L in a volume of 20 μL. After purification with a DyeEx 2.0 spin kit (Qiagen), samples were analyzed with an ABI Prism 310 genetic analyzer (Applied Biosystems-Life Technologies). The screening of chromosomal rearrangements (RET/PTC1, RET/PTC3, and PAX8/PPARG) was developed using primer sets reported by Nikiforov et al.16Nikiforov Y.E. Steward D.L. Robinson-Smith T.M. Haugen B.R. Klopper J.P. Zhu Z. Fagin J.A. Falciglia M. Weber K. Nikiforova M.N. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules.J Clin Endocrinol Metab. 2009; 94: 2092-2098Crossref PubMed Scopus (566) Google Scholar Amplification reactions were performed in simplex assay on a 7900HT fast real-time PCR system (Applied Biosystems-Life Technologies) using 2.5 μL of cDNA in a total volume of 12.5 μL containing a final concentration of 1× QuantiTect SYBR Green from a PCR kit (Qiagen) and 300 nmol/L of each primer. PCR was performed as follows: an initial hold at 95°C for 15 minutes, followed by 45 two-step cycles at 95°C for 15 seconds and 60°C for 60 seconds. The amplification products of chromosomal rearrangements assays were submitted to melting analysis to confirm the specificity of the fluorescence signal obtained during the amplification of cDNA. The thermal profile consisted of a denaturing/annealing stage (95°C for 15 seconds, 60°C for 15 seconds), followed by a dissociation stage from 60°C to 95°C (ramp rate, 2%). The quality of nucleic acids purified from 260 cytological biopsies indicated that the collection procedure and the extraction systems were suitable for development of mutation screening. In fact, it was possible to amplify by PCR all the DNA samples, as well as the RNA reverse-transcribed into cDNA extracted from the related biopsy. The performance of RNA extraction was verified by the amplification of the control gene, GAPDH; thyroglobulin expression was evaluated with a thyroid-specific marker to confirm the presence of thyroid cells in the FNAB specimens. Only eight samples out of the series (3%) were negative for thyroglobulin assay and therefore unreliable for molecular testing. Five of the eight samples had been classified as inadequate biopsies during the previous cytological diagnosis. The HRM analysis, set up by using DNAs from cell lines, was useful in the recognition of DNA mutations along all hot-spot regions investigated (Figure 1). Screening the FNAB samples provided a satisfactory resolution of melting for all of the DNA sites of interest and allowed the amplification of all genes simultaneously. To evaluate the theoretical sensitivity of our method, a detection limit was calculated, as described previously,29Simi L. Pratesi N. Vignoli M. Sestini R. Cianchi F. Valanzano R. Nobili S. Mini E. Pazzagli M. Orlando C. High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer.Am J Clin Pathol. 2008; 130: 247-253Crossref PubMed Scopus (162) Google Scholar by using serial dilution of positive controls (cell lines CCRF-CEM, SW948, HT1197, SK-MEL28) in wild-type DNA (from MCF-7 cell line) for each gene under study. We were able to detect the presence of mutated DNA up to 5% in a background of wild-type DNA (data not shown). Examples of HRM profiles for mutated samples and corresponding sequencing results, obtained after HRM analysis, are shown in Figure 2. The dideoxy-sequencing always confirmed HRM genotyping of mutated samples, including those cases in which the electropherogram demonstrated a faint peak corresponding to a mutated allele. Using a simple and rapid application of HRM analysis, we identified 38 mutations in a total of 37 nodules. Number and type of the genetic variants divided on the basis of the cytological category and histological findings are reported in Tables 2 and 3, respectively. Only one sample simultaneously carried mutations in two different genes (BRAF p.V600E and KRAS p.G12D). The BRAFV600E mutation was confirmed as the most frequent alteration in FNAB samples, particularly in the cytological categories of suspicious (Thy 4) and malignant nodules (Thy 5), with a frequency of 44.4% and 100%, respectively. In NRAS, only the most recurrent p.Q61R was identified, in a total of six samples (three Thy 2 and three Thy 3) (Table 2). Conversely, mutations caused by different nucleotide substitutions were detected by HRM analysis in the HRAS and KRAS genes (HRAS p.Q61R and p.Q61K; KRAS p.G12R and p.G12D) (Figure 2). Nodules positive for a mutation in RAS genes varied in distribution across cytological categories and were more frequently found in indeterminate specimens (Thy 3, with a frequency of 16.7%). Within the Thy 3 category, the BRAF p.V600E mutation was detected in two samples (Table 2), both of which were classified histologically as PTC.Table 2Genetic Variants Identified by HRM Analysis of Thyroid Nodules, by Cytological CategoryThy 1 [no. (%)]Thy 2 [no. (%)]Thy 3 [no. (%)]Thy 4 [no. (%)]Thy 5 [no. (%)]All samples [no. (%)]Total sample31 (12.3)154 (61.1)48 (19.0)9 (3.6)10 (4.0)252 (100)Samples with mutation4 (12.9)8 (5.2)11 (22.9)5 (55.6)10 (100.0)38 (15.1) BRAF (codon 600–601)01241017 (6.7) NRAS (codon 61)033006 (2.4) HRAS (codon 61)011103 (1.2) KRAS (codon 12–13)323019 (3.6) KRAS (codon 61)001001 (0.4) RET/PTC111003 (1.2)One sample carried two mutations simultaneously (BRAF p.V600E and KRAS p.G12D). Open table in a new tab Table 3Genetic Variants Identified by HRM Analysis of Thyroid Nodules, by Histological FindingsCategoryTotal samples (no.)Histology available (no.)Histology findings (no.)Mutations in related cytological sample (no.)Thy 215441 positive1 BRAF3 negative1 RAS; 2 wild typeThy 348389 positive2 BRAF; 2 RAS; 1 RET/PTC; 4 wild type29 negative3 RAS; 26 wild type Thy 4998 positive4 BRAF; 1 RAS1 negative1 wild typeThy 51055 positive5 BRAF0 negative Open table in a new tab One sample carried two mutations simultaneously (BRAF p.V600E and KRAS p.G12D). In two cases the analysis by HRM analysis and sequencing identified unusual in tandem mutations in KRAS, caused by the substitution of adjacent nucleotides (Figure 3) and resulting in the presence of both a synonymous
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