Multiplex PCR approach to simultaneously identify several mutations in fine needle cytology thyroid samples

// Emilia Vuttariello 1 , Marco Borra 2 , Elvira Mauriello 2 , Celeste Calise 3 , Barbara D’Andrea 2, 3 , Anna Capiluongo 1 , Franco Fulciniti 4 , Anna Cipolletta 5 , Mario Monaco 1 , Luciano Pezzullo 6 and Gennaro Chiappetta 1 1 Functional Genomics Unit, Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, Naples, Italy 2 Molecular Biology and Bioinformatics Unit, Stazione Zoologica “A.Dorhn”, Naples, Italy 3 CMO, Naples, Italy 4 Clinical Cytopathology Service, Institute of Pathology, Locarno, Switzerland 5 SC Anatomia Patologica e Citopatologia, Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, Naples, Italy 6 Thyroid Surgery Unit, Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, Naples, Italy Correspondence to: Emilia Vuttariello, email: e.vuttariello@istitutotumori.na.it Gennaro Chiappetta, email: chiappettagennaro@gmail.com Keywords: multiplex PCR, thyroid, fine needle cytology, genetic testing, Sanger sequencing Received: November 24, 2016      Accepted: April 19, 2017      Published: May 07, 2017 ABSTRACT The most frequent initial manifestation of thyroid cancer is the appearance of a nodule. More than 20% of the general population has a palpable thyroid nodule and the percentage rises to 70% based on ultrasound identification. In 95% of cases the nodule is simply a hyperplastic or benign lesion. The most reliable diagnostic test for thyroid nodules is fine needle aspiration (FNA), but cytological discrimination between malignant and benign follicular neoplasms remains difficult. Cytological analysis is now, almost routinely, being combined with molecular genetics to enable the pathologist to make a more objective diagnosis. In this study, we performed the molecular analysis using a new simplified procedure that involves a panel of BRAF , RAS , RET and RET/PTC gene mutations in easily obtainable FNA samples, in the attempt to improve the efficacy of the FNA diagnosis of thyroid nodules and thus patient management. In this new procedure, PCR and sequencing analysis are used to detect point mutations, and, in parallel, RT-PCR is used to detect the chimeric RET/PTC1 and RET/PTC3 transcripts in RNA extracted from FNA.