Diagnòstic molecular de biomarcadors predictius en pacients amb càncer de pulmó de cèl·lula no petita

[cat] El càncer de pulmó de cèl·lula no petita (CPCNP) es pot definir a nivell molecular per les alteracions genètiques que es produeixen en múltiples oncogens, inclosos els reordenaments dels gens ALK i ROS1. Per optimitzar el maneig clínic, el perfil molecular ha d'estar disponible per a tots els pacients per tal de fer accessibles les teràpies dirigides mitjançant tractaments inhibidors específics. Des de 2011, el test estàndard per detectar els reordenaments d’ALK i ROS1 ha estat la hibridació in situ fluorescent (FISH) utilitzant sondes de trencament i més endavant es va estandarditzar la detecció de sobreexpressió per immunohistoquímica (IHQ). Les dues tècniques tenen limitacions i han d'usar-se dins d'un context diagnòstic apropiat després de validacions per part del laboratori. En aquesta tesi doctoral es va confirmar la baixa prevalença dels reordenaments del gen ROS1 en la població espanyola i es va identificar una alta prevalença de les alteracions del nombre de còpies de ROS1 en els casos no reordenats sense que afectés a l'expressió de la proteïna ROS1 ni presentés implicacions clíniques associades en els pacients. Més recentment, l'ús de panells de seqüenciació massiva (NGS) per avaluar múltiples anomalies genòmiques clínicament rellevants ha demostrat ser viable per a la detecció d'aquests reordenaments genètics en les mostres d'adenocarcinoma de pulmó. La comparació amb les tècniques prèviament estandarditzades de FISH i IHQ s'ha investigat en el present treball de tesi doctoral per a la seva aplicació clínica. S'han trobat discrepàncies entre les tècniques, especialment en els casos ALK i ROS1 reordenats amb patró de FISH positiu per senyals 3 ' extra. Com l'efectivitat de les teràpies dirigides contra les cinases d’ALK i ROS1 depèn de la correcta selecció dels pacients segons les seves alteracions moleculars, proposem que es detalli en l'informe de resultats els patrons de FISH en el cas de positivitat. A més, segons els nostres resultats, la tècnica de NGS representa un enfocament pràctic i fiable a nivell clínic per a la detecció d’aquests reordenaments en el seu ús en mostres de teixit procedent de pacients amb CPCNP. Aquesta nova metodologia també permet avaluar en paral·lel els possibles mecanismes de resistència genètics relacionats amb les teràpies dirigides i facilitar l’accés dels pacients al tractament més adequat.[eng] Non-small cell lung cancer (NSCLC) can be defined at the molecular level by recurrent 'driver' mutations that occur in multiple oncogenes, including ALK and ROS1. To improve outcome, molecular profiling should be available to all patients in order to make targeted therapies accessible. Since 2011, the standard test to detect ALK- and ROS1-rearranged tumors has been fluorescence in situ hybridization (FISH) using break-apart probes and later on, immunohistochemistry (IHC). Both techniques have strengths and limitations and should be used within an appropriate diagnostic context and after proper laboratory validations. In this thesis, we confirmed the low prevalence of ROS1 rearrangements in the Spanish population and identified a high prevalence of ROS1 copy number alterations that neither affect ROS1 protein expression nor present associated clinical implications. More recently, the use of next-generation sequencing (NGS) panels to assess multiple clinically relevant genomic abnormalities has shown feasibility to detect gene rearrangements in lung adenocarcinoma. Comparison with widely accepted FISH and IHC methodologies has been tested herein for clinical application. Discrepancies between techniques have been found, especially in ALK and ROS1 rearranged cases with isolated 3’ signals FISH pattern. As the effectiveness of therapies targeting ALK and ROS1 is highly dependent upon appropriate selection of patients, we propose a detailed reporting of positive FISH patterns. NGS represents a practical and reliable ALK and ROS1 testing approach for use with routine NSCLC tissue specimens, enabling patients to receive optimal therapies. This novel methodology can also assess genomic-related mechanism of resistance to targeted therapies and guide the appropriate treatment selection

Measuring institutional thickness in tourism: An empirical application based on social network analysis

This article uses social network analysis to measure institutional thickness in a regional tourism destination in Colombia. Through the analysis of 107 institutions, the empirical findings show that the configuration of formal interaction spaces determine the governance system of the destination turning certain institutions into hubs or authorities. The contribution of this research is two-fold. Firstly, it provides a new approach to the study of institutional thickness by applying a social network analysis methodology making possible to identify the components theoretically defined such as the role of institutional presence, levels of interaction, structures of domination, and common agendas in tourism. Secondly, it highlights the importance of understanding the role of the regional institutional environment and the governance framework of tourism destinations, to better plan and manage their dynamics and effects

MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC).

OBJECTIVE: We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation. METHODS: Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation. RESULTS: Median MET H-score was 140 (range 0-400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25-50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively). CONCLUSIONS: MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation.This work was supported by RD12/0036/0051 /FEDER, PI13/00140/FEDER, a grant from Fundacio Marato de TV3. Ref.666/C/2013 and 2014 SGR 740. the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC).” JA is recipient of intensification programme ISCIII

ROS1 copy number alterations are frequent in non-small cell lung cancer

OBJECTIVES: We aimed to determine the prevalence and partners of ROS1 rearrangements, to explore the correlation between FISH and IHC assays, and to investigate clinical implications of ROS1 copy number alterations (CNAs). METHODS: A total of 314 NSCLC patients were screened using ROS1 FISH break-apart probes. Of these, 47 surgical tumors were included in TMAs to analyze ROS1 heterogeneity assessed either by FISH and IHC, and chromosome 6 aneusomy. To characterize ROS1 partners, probes for CD74, EZR, SLC34A2 and SDC3 genes were developed. ROS1 positive FISH cases were screened also by IHC. RESULTS: Five patients were ROS1 positive (1.8%). We identified two known fusion partners in three patients: CD74 and SLC34A2. Four out of five ROS1 rearranged patients were female, never smokers and with adenocarcinoma histology. Rearranged cases were also positive by IHC as well. According to ROS1 CNAs, we found a prevalence of 37.8% gains/amplifications and 25.1% deletions. CONCLUSIONS: This study point out the high prevalence of ROS1 CNAs in a large series of NSCLC. ROS1 gains, amplifications and deletions, most of them due to chromosome 6 polysomy or monosomy, were heterogeneous within a tumor and had no impact on overall survival.This work was supported in part by a grant from (1) Fundació La Marató de TV3 (666/C/2013), from (2) Red Temática de Investigación Cooperativa en Cáncer (RD12/0036/0044), from (3) Instituto de Salud Carlos III FEDER (PT13/0010/0005), (4) from Xarxa de Bancs de Tumors (XBTC), (5) from Agència de Gestió d’Ajuts Universitaris i de Recerca (2014SGR740) and (6) from Fondo de Investigaciones Sanitarias (FIS-ISCIII) (PI13/00140)

Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer

Introduction: Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. Methods: There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. Results: The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Discussion: Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications

MET expression and copy number heterogeneity in nonsquamous non-small cell lung cancer (nsNSCLC).

OBJECTIVE: We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation. METHODS: Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation. RESULTS: Median MET H-score was 140 (range 0-400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25-50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival (p = 0.042 and 0.047, respectively). CONCLUSIONS: MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation.This work was supported by RD12/0036/0051 /FEDER, PI13/00140/FEDER, a grant from Fundacio Marato de TV3. Ref.666/C/2013 and 2014 SGR 740. the “Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC).” JA is recipient of intensification programme ISCIII

HER-family ligands promote acquired resistance to trastuzumab in gastric cancer

Despite the clinical benefit of trastuzumab, eventually all HER2-amplified gastric cancer tumors develop drug resistance. We aimed to identify molecular mechanisms of acquired resistance to trastuzumab in gastric cancer by using well-established cell line-based preclinical models, as well as samples from patients with HER2-positive gastric cancer treated with trastuzumab. We studied trastuzumab resistance in NCI-N87 and OE19, two gastric cancer cell lines that overexpress HER2 receptor and are trastuzumab sensitive. Differences at protein, DNA, and RNA levels between the parental and resistant cells were characterized and functional studies were performed. Paired pre- and post-trastuzumab blood and tissue samples from patients with gastric cancer treated with trastuzumab were analyzed. We found that resistant cells were associated with increased activation of MAPK/ERK and PI3K/mTOR pathways driven by SRC activation. Upstream, resistant cells showed increased coexpression of multiple HER-family ligands that allowed for compensatory activation of alternative HER receptors upon HER2 blockade. Simultaneous inhibition of EGFR, HER2, and HER3 by the novel antibody mixture, Pan-HER, effectively reverted trastuzumab resistance in vitro and in vivo Similarly, an increase in HER-family ligands was observed in serum and tumor from patients with gastric cancer after trastuzumab therapy. We propose that trastuzumab resistance in gastric cancer is mediated by HER-family ligand upregulation that allows a compensatory activation of HER receptors and maintains downstream signaling activation despite trastuzumab therapy. Resistance is reverted by simultaneous inhibition of EGFR, HER2, and HER3, thereby revealing a potential therapeutic strategy to overcome trastuzumab resistance in patients with gastric cancer