Sgariglia, Roberta (2018) DEVELOPMENT, VALIDATION, COMPARISON AND CLINICAL IMPLEMENTATION OF DIFFERENT MULTIGENE ASSAYS FOR THE PRE-SURGICAL RISK STRATIFICATION OF INDETERMINATE THYROID FINE NEEDLE ASPIRATIONS. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: DEVELOPMENT, VALIDATION, COMPARISON AND CLINICAL IMPLEMENTATION OF DIFFERENT MULTIGENE ASSAYS FOR THE PRE-SURGICAL RISK STRATIFICATION OF INDETERMINATE THYROID FINE NEEDLE ASPIRATIONS.
Creators:
CreatorsEmail
Sgariglia, Robertaroberta.sgariglia@unina.it
Date: 11 December 2018
Number of Pages: 40
Institution: Università degli Studi di Napoli Federico II
Department: Sanità Pubblica
Dottorato: Sanità pubblica e medicina preventiva
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
Montagnani, Stefaniamontagna@unina.it
Tutor:
nomeemail
Troncone, GiancarloUNSPECIFIED
Date: 11 December 2018
Number of Pages: 40
Uncontrolled Keywords: Thyroid Cancer, Next Generation Sequencing, Fine Needle Aspirations, Custom Panel
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/08 - Anatomia patologica
Date Deposited: 19 Dec 2018 10:54
Last Modified: 23 Jun 2020 09:26
URI: http://www.fedoa.unina.it/id/eprint/12644

Abstract

Background: The aim of this PhD dissertation is to show the development, validation, comparison and preliminary clinical implementation of different multigene-based assays for the pre-surgical risk stratification of indeterminate thyroid fine-needle aspirates (FNA). In particular, this work was focused on the validation and comparison of two commercially multigene assays available for the study of the molecular alterations occurring in thyroid neoplasms, and on the development of a custom next-generation sequencing genomic panel. Methods: The two commercially available assays, one based on a real-time PCR (RT-PCR) technology and one based on a next-generation sequencing (NGS) platform were tested on a series of indeterminate thyroid FNA. Moreover, a custom NGS gene panel was designed and tested on a different series of retrospective and prospective FNA samples. Results: The commercial NGS panel showed parametric output data that were not sufficient to reliably use this panel on our clinical routine samples. Thus, the RT-PCR assay, which asses BRAF, N-H-KRAS, RET/PTC and PAX8/PPARG genomic alterations, were chosen for the subsequent clinical validation on a prospective series of n=1172 thyroid FNAs. In order to calculate the pre- and post- test risk of malignancy (ROM), only FNA with available histological follow-up (207/1006 adequate FNA, 20.6%) were included in the final study. The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was adopted for the microscopic diagnosis. FNA classified as atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) showed a 25.9% pre-test ROM whereas post-test ROM was 42.6% in mutation-positives (MT-pos) and 14.5% in mutation-negatives (MT-neg) cases, respectively. Considering the MT-positive cases, the cases harbouring BRAF-like mutations (BRAFV600E, RET/PTC1, RET/PTC3) showed a statistically significant higher ROM (80%) than those with RAS-like mutations (N-H-KRAS, PAX8/PPARγ) (32.4%, p=0.010). Follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) FNA showed a 44.1% pre-test ROM. Conversely, FN/SFN post-test ROM was 80% in MT-pos and 29.1% in MT-neg cases. Although BRAF- and RAS-like mutations were associated to different ROM even in FN/SFN cases (100% vs 71.4%), this difference did not reach a statistical significance (p=1,0). Suspicious for malignancy (SFM) FNA showed a 93% pre-test ROM; this latter figure almost overlapped to post-test ROM of both MT-pos (100%) and MT-neg (84.6%) FNAs. In particular, BRAFV600E-mutated FNAs were consistently associated with a papillary carcinoma on histology, irrespective of TBSRTC categories. Moreover, in order to switch from a RT-PCR based technology, that allows the study of alteration in only 7 genes, to a more comprehensive NGS-based multigene assay, we designed and analytically tested the performance of a custom panel. This latter, include beyond the 7-genes, additional genes which may give an additional predictive value when performed on indeterminate thyroid FNAs. Conclusions: Our preliminary data show that the 7-gene RT-PCR based test may contribute to the risk stratification in AUS/FLUS and FN/SFN categories, thanks to the significant difference in post-test ROM between MT-pos and MT-neg FNAs, confirming the high positive predictive value of BRAFV600E and BRAF-like mutations over the RAS-like genomic alterations. Moreover, the preliminary results of the custom NGS panel were satisfactory enough to expect its effective future adoption on our routine clinical samples.

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