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: ojas.bhu@gmail.com; drsradhika@gmail.com

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 Cytology1, Histopathology2 and Otolaryngology and Head & Neck Surgery3

Postgraduate Institute of Medical Education and Research, Chandigarh, INDIA

Ojas Gupta1, Upasana Gautam1, Muralidaran C1, Arvind Rajwanshi1, Manish Rohilla1, Uma Nahar Saikia2, Bishan Dass Radotra2, Roshan Verma3, Radhika Srinivasan1

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

BRAF26%

NRAS23%

HRAS5%

KRAS1%

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

metastases10 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 CarcinomaBRAFV600E 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

BRAF9%

HRAS9% KRAS

3%

NRAS30%

None detected49%

1 FA2 FC

1 FVPTC1 FC1 PTC

1 FVPTC1 FA1 PTC4 FC

4 FA1 FH1 HCA1 CG2 FVPTC2 PTC

2 cases: DNA QNS