RESEARCH POSTER PRESENTATION DESIGN © 2015 www.PosterPresentations.com FNAC- Routine investigation for pre-surgical diagnosis of thyroid nodules and triaging Molecular testing is proposed for Indeterminate categories- Cat 3/4/5 of TBSRTC Most common gene mutations: BRAF V600E (in PTC); RAS (FA, FC, PDC), TERT promoter (ATC) and TP53 Introduction Aims and Objectives DNA extraction – Qiagen Dneasy blood & Tissue kit Quantified- Sprectophotometer (absorbance at 260 nm) Purity of DNA- 260/280= 1.8 – 2.0 (acceptable) Real time PCR- Agilent technologies- Aria Mx Real time PCR system and EntroGen thyroid mutation analysis kit (THDNA-RT64) Reaction well- Primer set and Probes- labelled with fluorochromes- – FAM- 6-Carboxyfluoresence – VIC- 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy- fluorescein Analysis- AGILENT Aria Mx 1.0 software Materials and Methods References Contact: [email protected] ; [email protected] Mahajan, S., et al., Risk of Malignancy and Risk of Neoplasia in the Bethesda Indeterminate Categories: Study on 4,532 Thyroid Fine-Needle Aspirations from a Single Institution in India. Acta Cytol, 2017. 61(2): p. 103-110. Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid : official journal of the American Thyroid Association. 2017 Nov;27(11):1341-6. WHO Classification of Tumours of Endocrine Organs, 4 ed. R.V. Lloyd, R.Y. Osamura, G. Kloppel, Rosai J, editors. Lyon, France: International Agency for Research on Cancer (IARC) 69372 Lyon Cedex 08, France; 2017. Nikiforov YE, Ohori NP, Hodak SP, Carty SE, LeBeau SO, Ferris RL, et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. The Journal of clinical endocrinology and metabolism. 2011 Nov;96(11):3390-7. Cantara, S., et al., Molecular Signature of Indeterminate Thyroid Lesions: Current Methods to Improve Fine Needle Aspiration Cytology (FNAC) Diagnosis. Int J Mol Sci, 2017. 18(4) Censi, S., et al., Frequency and Significance of Ras, Tert Promoter, and Braf Mutations in Cytologically Indeterminate Thyroid Nodules: A Monocentric Case Series at a Tertiary-Level Endocrinology Unit. Front Endocrinol (Lausanne), 2017. 8: p. 273. Aim: To evaluate the diagnostic utility of BRAF and RAS gene mutations in thyroid FNA samples. Objectives: 1. Feasibility of molecular testing – BRAF V600E and – RAS (H-RAS, K-RAS and N-RAS) mutations in thyroid FNAC 2. Correlation of mutation type and frequency to the cytopathological diagnosis of different thyroid FNA TBSRTC categories. 3. Correlation to histopathology wherever available. Finally, to evaluate the utility of molecular testing for the indeterminate category 3, 4 and 5 of TBSRTC. Departments of Cytology 1 , Histopathology 2 and Otolaryngology and Head & Neck Surgery 3 Postgraduate Institute of Medical Education and Research, Chandigarh, INDIA Ojas Gupta 1 , Upasana Gautam 1 , Muralidaran C 1 , Arvind Rajwanshi 1 , Manish Rohilla 1 , Uma Nahar Saikia 2 , Bishan Dass Radotra 2 , Roshan Verma 3 , Radhika Srinivasan 1 Molecular testing for BRAF and RAS mutations from Fine needle aspirates of thyroid nodules: Can it improve the pre-surgical diagnosis? Study Design Real Time PCR [N=82] Mutation POSITIVE TEST: MUTATION POSITIVE (FAM probe) VIC probes: INTERNAL CONTROL : POSITIVE Results 30yr/ F Cytodiagnosis: Follicular lesion (Cat.3) NRAS Follicular adenoma – NRAS mutation positive Follicular Carcinoma – HRAS mutation positive HRAS 43 yr/M Cytodiagnosis: Follicular neoplasm (Cat. 4) Mutational Profile of Thyroid FNA cohort N=82 BRAF 26% NRAS 23% HRAS 5% KRAS 1% None detected 45% BRAF and RAS mutations in different TBSRTC [N=82] TBSRTC category No. of cases No mutation detected Mutation Detected BRAF NRAS HRAS KRAS 2 2 2 0 0 0 0 0 3 18 9 9 (56.25%) 1 6 1 1 4 9 3 6 (66.7%) 0 3 3 0 5* 16 9 7 (43.75%) 3 4 0 0 6 27 10 17 (66.7%) 12 5 0 0 Lymph nodal metastases 10 4 6 (55.6%) 5 1 0 0 Total 82 37 (45.1%) 45 (54.9%) 21 (46.7%) 19 (42.2%) 4 (8.9%) 1 (2.2%) Correlation of Gene mutation with histological outcome in Indeterminate category- Cat 3/4/5 [N=24] Histological outcome Mutation Positive Mutation Negative Total Benign* 2 8 10 Malignant 8 6 14 Total 10 14 24 *Includes Follicular and Hurthle cell adenomas • No correlation of gene mutation detection with malignant histological outcome (chi-square,3.311; p=0.068817) • Sensitivity 57.1%, Specificity 80% , Positive Predictive Value (PPV) 80%, Negative Predictive Value (NPV) 57.1%. CONCLUSION Molecular testing is easily performed on thyroid FNA samples either on a direct sample or on cell scrapes. The commercially available multiplex RT-PCR assay kit is more sensitive than Sanger sequencing and also economises on the amount of DNA. BRAF mutations are seen predominantly in PTC and in nodal metastasis of PTC Among the indeterminate category, molecular testing showed a PPV of 80% making it a good Rule- in test Gene mutation detected in Indeterminate category Cat 3/4/5 [N=43] Most common mutation in Indeterminate category Cat 3/4/5 was NRAS mutation Papillary Thyroid Carcinoma BRAFV600E mutation positive BRAF V600E VIC probes: INTERNAL CONTROL : POSITIVE MUTATION NEGATIVE (FAM probe) Mutation NEGATIVE Referred for Thyroid and LN FNAC FNAC performed Smears MGG (air dried) At least 6 clusters of cells (20 cells/cluster) Cell scrape (CS)- using sterile blade H & E (alcohol fixed) Direct sample (in RNA later solution) DNA Extraction N=82 Mostly prospective cases 2015-2018 Mostly retrospective cases Cases: TBSRTC Cat 3,4,5,6, Metastatic PTC in lymph node N=86 BRAF 9% HRAS 9% KRAS 3% NRAS 30% None detected 49% 1 FA 2 FC 1 FVPTC 1 FC 1 PTC 1 FVPTC 1 FA 1 PTC 4 FC 4 FA 1 FH 1 HCA 1 CG 2 FVPTC 2 PTC 2 cases: DNA QNS