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International Journal of Surgery xxx (2014) 1e4

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Original research

Detection of RAS mutation by pyrosequencing in thyroid cytology samples Q4

Anna Guerra a, Mario Carrano b, Elisabetta Angrisani b, Alessandro Puzziello a, Giulia Izzo a, Vincenzo Di Crescenzo a, Alessandro Vatrella a, Mario Vitale a, * a b

Department of Medicine and Surgery, University of Salerno, Via Allende, 84081 Baronissi, Salerno, Italy S.C. Endocrinologia, AOU San Giovanni di Dio e Ruggi d'Aragona, Salerno, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 March 2014 Accepted 3 May 2014 Available online xxx

Introduction: Fine-needle aspiration cytology (FNAC) is the primary means to distinguish benign from malignant thyroid nodules. However, adjunctive diagnostic tests are needed as 20e40% of FNAC are inconclusive. RAS mutations have been described in differentiated thyroid cancer and they could be used as tumor markers. However, their prevalence varies widely among studies, probably as a result of the detection methods used. We investigated whether the pyrosequencing method can be applied to detect NRAS and KRAS mutations in thyroid aspirates. Patients and methods: A total of 37 thyroid aspirates, including benign hyperplastic nodules (HBN, N ¼ 16) and follicular thyroid carcinomas (FTC, N ¼ 21) were analyzed for the presence of NRAS61 and KRAS13 mutations. Results: A RAS mutation was found in 31% and 62% of BN and FTC respectively. Most samples displayed a percentage of mutated alleles lower than 50% (median ¼ 30.8% and 15.3% in FTC and HBN respectively), a result compatible with the presence of extra-nodular cells contaminating the FNA or with the subclonal nature of both types of thyroid nodules. Discussion: Pyrosequencing is a reliable assay to detect RAS mutations in fine-needle thyroid aspirates. Conclusions: The low specificity and sensitivity limit the power of this test to distinguish between FTC and benign nodules in inconclusive FNACs. © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

Keywords: Thyroid carcinoma RAS FNAC

1. Introduction Fine-needle aspiration cytology (FNAC) is the primary diagnostic means in a large number of different tissues lesions, including thyroid nodules [1e4]. A correct diagnosis is not always achieved by microscope observation of conventionally stained cytology smears, and alternative tools are needed [5e7]. Inconclusive cytology occurs in about 20% of thyroid FNAC, especially when Hashimoto's thyroiditis is concomitant [8,9]. Diagnostic thyroidectomy, a disabling surgical procedure sometime accompanied by complications is necessary in inconclusive FNAC [10e12]. In the last decades, the diagnostic, prognostic and therapeutic utility of a number of proteins and altered genes expressed in thyroid cancer have been investigated [13e16].

* Corresponding author. E-mail address: [email protected] (M. Vitale).

Among others, RAS mutations are promising as they are expressed in papillary thyroid cancer (PTC) and more importantly in follicular thyroid cancer (FTC) [17e20]. Besides FTC, RAS mutations have been detected also in thyroid adenomas, limiting its clinical utility as diagnostic tool. The prevalence of RAS mutations varies widely among studies, probably as a result of the detection methods used. This is especially true for tissue samples containing a mixture of thyroid and non-thyroid cells as occurs in the presence Hashimoto's i. A previous study demonstrated that pyrosequencing is a reliable assay to detect a single nucleotide polymorphism in a thyroid sample contaminated by lymphoid cells [21]. In this study, we determined whether pyrosequencing analysis can be applied in fine-needle thyroid aspirates to distinguish FTC from benign hyperplastic nodules (HBN). To this purpose we searched for the presence of NRAS61 and KRAS [13] mutations by pyrosequencing in a series of FTC and HBN fineneedle thyroid aspirates.

http://dx.doi.org/10.1016/j.ijsu.2014.05.045 1743-9191/© 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: A. Guerra, et al., Detection of RAS mutation by pyrosequencing in thyroid cytology samples, International Journal of Surgery (2014), http://dx.doi.org/10.1016/j.ijsu.2014.05.045

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2. Patients, materials and methods

Table 2 Patients and definitive histology diagnosis.

2.1. Patients and FNAC Patients were entered in the study after giving their consent and with approval from the institutional review boards. Cytology samples were obtained using a syringe with a 22-gauge needle passed three to four times. Aspirates were used for cytological examination and the needle was then washed in TRI Reagent buffer (Sigma) and stored at 20  C until DNA extraction. After definitive histological diagnosis, 37 samples were retrieved and used (16 benign hyperplastic nodules HBN; 21 follicular thyroid carcinomas, FTC). Lymphoreticular cells in the FNAC of selected samples were sporadic and less than 1% of thyrocytes.

2.2. DNA extraction and detection of RAS mutations DNA extraction was performed according to the TRI Reagent manufacturer's recommendations. The final pellet was resuspended in 10 ml diethyl-pyrocarbonate (DEPC) water. Pyrosequencing was performed as described in detail [22]. Briefly, 50e100 ng genomic DNA was amplified by PCR with, forward primer and reverse 5-biotinylated primer at 10 mM concentration, and 2.5 U Taq Polymerase Recombinant (VWR, Milan, Italy) (Table 1). PCR were performed in a TC-4000 Thermal Cycler (Bibby Scientific, Milan, Italy), with an initial denaturation of 5 min at 94  C and subsequent denaturation for 20 s at 94  C, annealing for 20 s at 61  C, and extension for 30 s at 72  C. Twenty microliters of biotinylated PCR product were immobilized on streptavidin-coated Sepharose high-performance beads (Diatech), processed to obtain a single stranded DNA using the PSQ 96 Sample Preparation Kit (Diatech), according to the manufacturer's instructions, and incubated under shaking at room temperature for 10 min in binding buffer. Hybridization to sequencing primers and sequencing-bysynthesis reaction of the complementary strand was automatically performed on a PSQ 96MA instrument (Biotage, Uppsala, Sweden). The cut-off was set at 10%, corresponding to the mean percentage of normal tissues plus 2 SD. Samples were considered positive when the RAS mutated alleles were 10% with a SD < 10%.

3. Results We analyzed for the presence of NRAS mutation at position 61 and KRAS at position 13, a total of 37 thyroid aspirates from 16 HBN and 21 FTC (Table 2). The search for RAS mutations was performed by pyrosequencing in all 37 samples in triplicate (Table 3). A total of 13 mutations was detected. Of 16 HBN, NRAS61 mutation was detected in 1 and KRAS [13] in 4. A RAS mutation was detected in 8 of the 21 FTC (38%), of which 5 NRAS61 and 3 KRAS [13] mutations. Mutated NRAS61alleles were present in the range 50 to 11.3% of total NRAS with a median of 33.6%. Mutated KRAS [13]alleles were

Table 1 PCR primers and sequencing primers. Gene

PCR primers

Sequencing primers

NRAS exon 2

Forward: 50 -biotin-GGTGAAACCTG TTTGTTGGACATA-30 Reverse: 50 -AATACATGAGGAC AGGCGAAGG-30 Forward: 50 -TATAAACTTGTGGTA GTTGGA-30 Reverse: 50 -biotin-GATCATATTC GTCCACAAAATGA-30

50 -AGAAGAGTACAGTGC CATG-30

KRAS exon 1

50 -TGTGGTAGTTGG AGC-30

N Gender (F/M) Stage

HBN

FTC

16 13/3

21 16/5 5 9 3 0

1 2 3 4

HBN, benign hyperplastic nodules; FTC, follicular thyroid carcinoma.

Table 3 RAS mutation in cytology specimens. N, (%). Assays were performed in triplicates. Mutations

HBN N ¼ 16

FTC N ¼ 21

NRAS61 KRAS13 Total RAS

1, (6.2) 4, (25.0) 5, (31.2)

5, (23.8) 3, (14.3) 8, (38.0)

HBN, benign hyperplastic nodules; FTC, follicular thyroid carcinoma.

present in the range 29 to 14.6% of total KRAS alleles with a median of 15.3%.

4. Discussion Ras proto-oncogenes are key components in the regulation of cell growth and differentiation of a number of different cell types [23]. Activating mutations of the Ras proto-oncogene have been identified in up to 35% human tumors. They are present in different benign and malignant tumors and represent a possible hallmark of tumor transformation, potentially useful for diagnostic and prognostic purposes. RAS mutations have been demonstrated in human thyroid tumors and their oncogenic potential has been demonstrated in both in vivo and in vitro experiments [17,19,24]. However, the prevalence and specificity of RAS mutations are quite variable in the literature, making difficult its utility as diagnostic tool. A major limitation for the study of RAS mutations in thyroid tumors is the different detection methods employed. The most popular are the direct sequencing of genomic DNA by dye-terminator and by singlestrand conformation polymorphism (SSCP). However, both have limitations. Direct sequencing is the gold standard to detect a single nucleotide mutation. However, this method is not sensitive and it is unable to detect the mutation in thyroid aspirates when largely contaminated by non-tumoral cells [25,26]. Conventional sequencing usually detects genetic mutations at a rate of >20%, a threshold that can limit its application to thyroid biopsies. Contamination with non-tumor cells occurs regularly in fineneedle biopsies when the needle track through the gland is long or when a Hashimoto's thyroiditis is concomitant [21,22]. In these circumstances, a more sensitive method such as pyrosequencing is more appropriate. Besides sample contamination by non-tumoral cells, the detection of genetic alterations can be impaired by the heterogeneity of the tumor. Experimental evidences are in favor of a genetic intratumor heterogeneity of benign thyroid nodules and papillary thyroid carcinomas [27e32]. BRAF mutation at position 1799 (BRAFV600E) has been reported to be a subclonal event in some recent studies, sometime concomitant with other oncogenes. Concomitant BRAFV600E and RET/PTC rearrangement has been detected in 19.4% of PTC, while concomitant BRAFV600E and RAS is infrequently [28,33,34]. The subclonal nature of BRAF and RAS mutation is not restricted to PTC. Single cell analysis demonstrated a subclonality of both oncogenes in a relevant fraction of

Please cite this article in press as: A. Guerra, et al., Detection of RAS mutation by pyrosequencing in thyroid cytology samples, International Journal of Surgery (2014), http://dx.doi.org/10.1016/j.ijsu.2014.05.045

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melanomas [35e37]. Although our data demonstrate that most of samples analyzed are composed of a mixture of cells with and without RAS mutations, the presence of non-thyroid or nonnodular cells in the samples prevents to state that although RAS mutation, as BRAFV600E is a subclonal event. In the light of these considerations and our results, its high sensitivity makes pyrosequencing a reliable method to detect RAS mutations if FNA. However, its clinical utility as a diagnostic tool is strongly limited by the unrestricted occurrence of RAS mutations to FTC. Benign hyperplastic nodules harboring RAS mutations have been proposed to be transition lesions requiring an aggressive treatment. In the absence of clear evidence that such nodules can turn into malignant nodules, they should be considered benign and subjected to a more stringent follow up. In conclusion, pyrosequencing is a reliable method to detect RAS mutations in FNA. It is not helpful to refine an inconclusive cytology, however it can identify benign nodules that need a more stringent follow up.

Q1

Ethical approval This is a retrospective study based only on the analyses of recorded data and then no Ethical Approval was necessary.

Author contribution Anna Guerra: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data; also participated substantially in the drafting and editing of the manuscript. Mario Carrano: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data. Elisabetta Angrisani: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data; also participated substantially in the drafting and editing of the manuscript. Alessandro Puzziello: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data; also participated substantially in the drafting and editing of the manuscript. Giulia Izzo: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data. Vincenzo Di Crescenzo: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data; also participated substantially in the drafting and editing of the manuscript. Alessandro Vatrella: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data. Pio Zeppa: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data. Mario Vitale: Participated substantially in conception, design, and execution of the study and in the analysis and interpretation of data; also participated substantially in the drafting and editing of the manuscript.

Funding All Authors have no source of funding.

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Detection of RAS mutation by pyrosequencing in thyroid cytology samples.

Fine-needle aspiration cytology (FNAC) is the primary means to distinguish benign from malignant thyroid nodules. However, adjunctive diagnostic tests...
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