DEVELOPMENT, VALIDATION, COMPARISON AND CLINICAL IMPLEMENTATION OF DIFFERENT MULTIGENE ASSAYS FOR THE PRE-SURGICAL RISK STRATIFICATION OF INDETERMINATE THYROID FINE NEEDLE ASPIRATIONS.

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

RNA-Based Assay for Next-Generation Sequencing of Clinically Relevant Gene Fusions in Non-Small Cell Lung Cancer

Gene fusions represent novel predictive biomarkers for advanced non-small cell lung cancer (NSCLC). In this study, we validated a narrow NGS gene panel able to cover therapeutically-relevant gene fusions and splicing events in advanced-stage NSCLC patients. To this aim, we first assessed minimal complementary DNA (cDNA) input and the limit of detection (LoD) in different cell lines. Then, to evaluate the feasibility of applying our panel to routine clinical samples, we retrospectively selected archived lung adenocarcinoma histological and cytological (cell blocks) samples. Overall, our SiRe RNA fusion panel was able to detect all fusions and a splicing event harbored in a RNA pool diluted up to 2 ng/µL. It also successfully analyzed 46 (95.8%) out of 48 samples. Among these, 43 (93.5%) out of 46 samples reproduced the same results as those obtained with conventional techniques. Intriguingly, the three discordant results were confirmed by a CE-IVD automated real-time polymerase chain reaction (RT-PCR) analysis (Easy PGX platform, Diatech Pharmacogenetics, Jesi, Italy). Based on these findings, we conclude that our new SiRe RNA fusion panel is a valid and robust tool for the detection of clinically relevant gene fusions and splicing events in advanced NSCLC

Implications of U.S. monetary policy normalization on international capital flows : evidence from South Korea

Treball fi de màster de: Master's Degree in Specialized Economic AnalysisDirectors: Fernando Broner; Antonio Ciccone; Jaume VenturaIn this thesis, we provide evidence on the transmission of U.S. monetary policy shocks to the Korean economy. First, we show that there are spillovers of U.S. monetary policy shocks to Korean domestic credit and real economic conditions. We find a drop in credit supply, an increase in lending rate, risk premia and a drop in asset prices in response to U.S. monetary policy shocks. Thereafter, we calculate the response of different capital inflows and outflows to identify which capital flows are likely to transmit the monetary policy shock. In line with the theory, we find that portfolio debt securities and especially short term banking flows drop significantly in response to U.S. monetary policy shocks.En aquesta tesi, proporcionem evidència de la transmissió dels xocs de la política monetària dels EUA a l'economia coreana. En primer lloc, ens mostren que hi ha efectes secundaris dels xocs de la política monetària dels EUA al crèdit nacional coreà i condicions econòmiques reals. Trobem una baixada en l'oferta de crèdit, un augment en la taxa d'interès, primes de risc i una disminució en les preus dels actius en resposta als xocs de la política monetària dels Estats Units. Per tant, calculem la resposta de diferents entrades i sortides de capital per identificar quins fluxos de capital són propensos a transmetre el xoc de la política monetària. En consonància amb la teoria, trobem que els títols de deute en cartera i especialment els fluxos bancaris a curt termini cauen significativament en resposta als xocs de la política monetària dels Estats Units

Molecular Testing of Thyroid Fine-Needle Aspiration: Local Issues and Solutions. An Interventional Cytopathologist Perspective

Molecular testing has acquired a relevant role for diagnostic and prognostic stratification of indeterminate thyroid nodules. Besides the available commercial solutions marketed in the United States, various local testing strategies have been developed in the last decade. In this setting, the modern interventional cytopathologist, the physician who performs the both aspirate and the morphologic interpretation plays a key role in the correct handling of fine-needle aspiration (FNA) samples not only for microscopy but also for molecular techniques. This review summarizes experiences with local approaches to the molecular testing of thyroid FNA, highlighting the role of the modern interventional cytopathologist

EGFR mutation detection by microfluidic technology: a validation study.

Advanced non-small cell lung cancer samples are tested for epidermal growth factor receptor (EGFR) gene mutations. Their detection by direct sequencing is time-consuming. Conversely, the length analysis of fluorescently labelled PCR products is easier. To avoid labelled primers and the automated capillary electrophoresis apparatus, we validated a fast and sensitive chip-based microfluidic technology. The limit of detection of fragment length assay on microfluidic device was 5%, more sensitive than direct sequencing (12.5%). The novel methodology showed high accuracy in the analysis of samples whose mutational status was known. The accuracy in quantifying mutated alleles (mA) was evaluated by PCR products subcloning; the mA% provided by direct sequencing of subcloned PCR products showed a close correlation with the mA% provided by the microfluidic technology for both exon 19 (R-2=0.9) and 21 (R-2=0.9). Microfluidic-based on-chip electrophoresis makes EGFR testing more rapid, sensitive and cost-effective

Less frequently mutated genes in colorectal cancer: Evidences from next-generation sequencing of 653 routine cases

Aims: The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers. Methods: Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes. Results: A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%). Conclusions: In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments

Multiplex digital colour-coded barcode technology on RNA extracted from routine cytological samples of patients with non-small cell lung cancer: pilot study

In the advanced stages of non-small cell lung cancer (NSCLC), molecular testing is often performed on archival cytological smears. The nCounter system (NanoString Technologies) is a new promising multiplex digital colour-coded barcode technology. However, its feasibility to evaluate the RNA expression of clinical relevant biomarkers on routine cytological smears is still uncertain. To this end, RNA was extracted from 12 NSCLC routine stained cytological smears, and nCounter analysis performed by using a 48-gene panel. Overall, 11/12 (92%) of the smears were adequate for the secondary analysis, fulfilling the quality check parameter analysis of nSolver software. This pilot study shows that RNA nCounter analysis is feasible on routine cytological smears preparing the field for the implementation of this technology in the routine setting