Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform

Extracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 μL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs

Hsp90 inhibitors enhance the antitumoral effect of osimertinib in parental and osimertinib-resistant non-small cell lung cancer cell lines

Background: Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. Methods: MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. Results: Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. Conclusions: Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition

BRAF mutations classes I, II, and III in NSCLC patients included in the SLLIP trial : The need for a new pre-clinical treatment rationale

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decision

Common Co-activation of AXL and CDCP1 in EGFR-mutation-positive Non-smallcell Lung Cancer Associated With Poor Prognosis.

Epidermal growth factor receptor (EGFR)-mutation-positive non-smallcell lung cancer (NSCLC) is incurable, despite high rates of response to EGFR tyrosine kinase inhibitors (TKIs). We investigated receptor tyrosine kinases (RTKs), Src family kinases and focal adhesion kinase (FAK) as genetic modifiers of innate resistance in EGFR-mutation-positive NSCLC. We performed gene expression analysis in two cohorts (Cohort 1 and Cohort 2) of EGFR-mutation-positive NSCLC patients treated with EGFR TKI. We evaluated the efficacy of gefitinib or osimertinib with the Src/FAK/Janus kinase 2 (JAK2) inhibitor, TPX0005 in vitro and in vivo. In Cohort 1, CUB domain-containing protein-1 (CDCP1) was an independent negative prognostic factor for progression-free survival (hazard ratio of 1.79, p=0.0407) and overall survival (hazard ratio of 2.23, p=0.0192). A two-gene model based on AXL and CDCP1 expression was strongly associated with the clinical outcome to EGFR TKIs, in both cohorts of patients. Our preclinical experiments revealed that several RTKs and non-RTKs, were up-regulated at baseline or after treatment with gefitinib or osimertinib. TPX-0005 plus EGFR TKI suppressed expression and activation of RTKs and downstream signaling intermediates. Co-expression of CDCP1 and AXL is often observed in EGFR-mutation-positive tumors, limiting the efficacy of EGFR TKIs. Co-treatment with EGFR TKI and TPX-0005 warrants testing

Validation of liquid biopsy-based analysis on the NanoString nCounter platform

L'avaluació dels marcadors moleculars en teixit tumoral per predir el pronòstic del càncer i la resposta al tractament, també coneguda com a tractament personalitzat, ha transformat la pràctica clínica per a molts tipus de càncer. Es va descobrir que aquesta teràpia dirigida per genotipar, millora la supervivència del pacient i per tant s'han introduït diverses plataformes tècniques en els laboratoris clínics. No obstant això, no tots els tumors es poden biopsiar i, sovint, les quantitats de teixit són insuficients per a la caracterització del tumor. L'ARN, l'ADN i les proteïnes lliures circulants de biòpsies líquides, es poden extreure dels fluids corporals i poden reemplaçar o complementar les biòpsies de teixit. Les biòpsies líquides tenen diversos avantatges: la possibilitat de realització d’ estudis de serie o mínimament invasiva i permet analitzar l'heterogeneïtat tumoral. Malauradament, encara hi ha una gran bretxa entre la recerca bàsica i la implementació clínica de biòpsies líquides, principalment a causa de la manca de metodologies estandarditzades. A més, les plataformes tècniques que s'utilitzen actualment, no sempre són adequades per analitzar la baixa quantitat i qualitat de material del tumor derivat d'una biòpsia líquida. En conseqüència, la validació i implementació dels assajos de biomarcadors en biòpsies líquides en els laboratoris clínics, requereixen una plataforma tècnica estandarditzada que sigui sensible, ràpida, fàcil d'utilitzar, relativamente econòmica, flexible i amb poca quantitat de mostra. La plataforma nCounter es pot utilitzar per analitzar tota classe de molècules, inclosos ARN, ADN i proteïnes. La hibridació de codis de barres codificats per colors pels objectius d'interès permet una lectura directa dels nivells d'expressió de gens i proteïnes o la detecció de mutacions. El desenvolupament d'assaigs de biomarcadors en teixits, usant el nCounter, va conduir a l'aprovació per la FDA de l'assaig Prosigna™ per a ús clínic en la tipificació del càncer de mama. Els esforços anteriors també han destacat el potencial d'aquesta plataforma per analitzar molècules derivades i amplificades de biòpsies líquides, encara que es necessiten estudis de validació en l'entorn clínic. En aquesta tesi validem l'ús de la plataforma NanoString nCounter per analitzar material de biòpsies líquides i desenvolupar assajos de biomarcadors clínicament rellevants.La evaluación de los marcadores moleculares en tejido tumoral para el pronóstico del cáncer y la predicción de respuesta al tratamiento (lo que habitualmente se conoce como tratamiento personalizado) ha transformado la práctica clínica a la hora de tratar muchos tipos de cáncer. Son numerosos los trabajos que desde hace tiempo respaldan el efecto que esta terapia dirigida por genotipo tiene sobre los pacientes oncológicos mejorando la supervivencia del paciente; consecuentemente, un amplio rango de plataformas técnicas han sido implementadas en los laboratorios clínicos en los últimos años. Sin embargo, no todos los tumores se pueden biopsiar y, a menudo, las cantidades de tejido son insuficientes para la caracterización del tumor. Las biopsias líquidas, como el ARN, el ADN o las proteínas circulantes tanto libres como encapsuladas en una membrana, pueden extraerse de los fluidos corporales reemplazando o complementando de este modo las tradicionales biopsias de tejido. Las biopsias líquidas tienen varias ventajas: ofrecen la posibilidad de realizar estudios seriados, son mínimamente invasivas y permiten analizar la heterogeneidad tumoral. Desafortunadamente, todavía existe una gran brecha entre la investigación básica y la implementación clínica de las biopsias líquidas, principalmente debido a la falta de metodologías estandarizadas. Además, las plataformas técnicas que se utilizan actualmente no siempre son adecuadas para analizar la baja cantidad y calidad de material del tumor procedente de una biopsia líquida. En consecuencia, la validación e implementación de los ensayos de biomarcadores en biopsias líquidas en los laboratorios clínicos requieren una plataforma técnica estandarizada que sea sensible, rápida, fácil de usar, viable económicamente, flexible y que requiera un aporte inicial de ácidos nucleicos bajo, debido a la baja concentración que normalmente se obtiene en las biopsias líquidas. La plataforma nCounter se puede utilizar para analizar todo tipo de moléculas, incluyendo ARN, ADN y proteínas. La hibridación de diferentes códigos formados por moléculas de colores siguiendo patrones específicos con secuencias de interés permite una lectura directa de los niveles de expresión de genes y proteínas o la detección de mutaciones. El desarrollo de ensayos de biomarcadores en tejidos usando nCounter condujo a la aprobación por la administración de fármacos y alimentos de los Estados Unidos (FDA) del ensayo Prosigna ™ para su uso clínico en la tipificación del cáncer de mama. Numerosos estudios han destacado el potencial de esta plataforma para analizar moléculas derivadas y amplificadas de biopsias líquidas, aunque estudios de validación en el entorno clínico aun son necesarios. El objeto de esta tesis es la validación del uso de la plataforma NanoString nCounter para analizar material de biopsias líquidas y desarrollar ensayos de biomarcadores clínicamente relevantes.The assessment of predictive- and prognostic molecular markers in tumor tissue, also known as personalised treatment, has transformed clinical practice for many cancer types. This genotype-directed therapy was found to improve patient survival, and several technical platforms have been introduced in clinical laboratories since then. However, not all tumors can be biopsied and tissue quantities are often insufficient for tumor characterisation. Liquid biopsies, such as membrane-encapsulated- or circulating free RNA, DNA and proteins, can be derived from body fluids and can replace or complement tissue biopsies. They have several advantages, such as repeated sampling, a minimally invasive character and heterogeneous profiling. Unfortunately, there is still a big gap between basic research and clinical implementation of liquid biopsies, mainly due to the lack of standardised methodologies. In addition, currently used technical platforms are not always suitable to analyze the low quantity and quality of tumor-derived material that can be found in a liquid biopsy. In consequence, large-scale validation and clinical implementation of liquid biopsy-based biomarker assays requires a sensitive, quick, easy-to-use, relatively cheap, flexible and standardized technical platform with low input requirements. The nCounter platform can be used to analyze all types of molecules, including RNA, DNA and proteins. Binding of color coded barcodes to targets of interest allows for either a direct read-out of gene- or protein expression levels or the detection of mutations. Tissue-based biomarker assay development on nCounter led to the FDA approval of the Prosigna™ assay for clinical use in breast cancer subtyping. Previous efforts have also highlighted the potential of this platform to analyze amplified liquid biopsy-derived molecules, although validation studies in the clinical setting are needed. In this thesis we validated the use of the NanoString nCounter platform to analyze material from liquid biopsies and develop clinically relevant biomarker assays.Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicin

Validation of liquid biopsy-based analysis on the NanoString nCounter platform

L'avaluació dels marcadors moleculars en teixit tumoral per predir el pronòstic del càncer i la resposta al tractament, també coneguda com a tractament personalitzat, ha transformat la pràctica clínica per a molts tipus de càncer. Es va descobrir que aquesta teràpia dirigida per genotipar, millora la supervivència del pacient i per tant s'han introduït diverses plataformes tècniques en els laboratoris clínics. No obstant això, no tots els tumors es poden biopsiar i, sovint, les quantitats de teixit són insuficients per a la caracterització del tumor. L'ARN, l'ADN i les proteïnes lliures circulants de biòpsies líquides, es poden extreure dels fluids corporals i poden reemplaçar o complementar les biòpsies de teixit. Les biòpsies líquides tenen diversos avantatges: la possibilitat de realització d' estudis de serie o mínimament invasiva i permet analitzar l'heterogeneïtat tumoral. Malauradament, encara hi ha una gran bretxa entre la recerca bàsica i la implementació clínica de biòpsies líquides, principalment a causa de la manca de metodologies estandarditzades. A més, les plataformes tècniques que s'utilitzen actualment, no sempre són adequades per analitzar la baixa quantitat i qualitat de material del tumor derivat d'una biòpsia líquida. En conseqüència, la validació i implementació dels assajos de biomarcadors en biòpsies líquides en els laboratoris clínics, requereixen una plataforma tècnica estandarditzada que sigui sensible, ràpida, fàcil d'utilitzar, relativamente econòmica, flexible i amb poca quantitat de mostra. La plataforma nCounter es pot utilitzar per analitzar tota classe de molècules, inclosos ARN, ADN i proteïnes. La hibridació de codis de barres codificats per colors pels objectius d'interès permet una lectura directa dels nivells d'expressió de gens i proteïnes o la detecció de mutacions. El desenvolupament d'assaigs de biomarcadors en teixits, usant el nCounter, va conduir a l'aprovació per la FDA de l'assaig Prosigna™ per a ús clínic en la tipificació del càncer de mama. Els esforços anteriors també han destacat el potencial d'aquesta plataforma per analitzar molècules derivades i amplificades de biòpsies líquides, encara que es necessiten estudis de validació en l'entorn clínic. En aquesta tesi validem l'ús de la plataforma NanoString nCounter per analitzar material de biòpsies líquides i desenvolupar assajos de biomarcadors clínicament rellevants.La evaluación de los marcadores moleculares en tejido tumoral para el pronóstico del cáncer y la predicción de respuesta al tratamiento (lo que habitualmente se conoce como tratamiento personalizado) ha transformado la práctica clínica a la hora de tratar muchos tipos de cáncer. Son numerosos los trabajos que desde hace tiempo respaldan el efecto que esta terapia dirigida por genotipo tiene sobre los pacientes oncológicos mejorando la supervivencia del paciente; consecuentemente, un amplio rango de plataformas técnicas han sido implementadas en los laboratorios clínicos en los últimos años. Sin embargo, no todos los tumores se pueden biopsiar y, a menudo, las cantidades de tejido son insuficientes para la caracterización del tumor. Las biopsias líquidas, como el ARN, el ADN o las proteínas circulantes tanto libres como encapsuladas en una membrana, pueden extraerse de los fluidos corporales reemplazando o complementando de este modo las tradicionales biopsias de tejido. Las biopsias líquidas tienen varias ventajas: ofrecen la posibilidad de realizar estudios seriados, son mínimamente invasivas y permiten analizar la heterogeneidad tumoral. Desafortunadamente, todavía existe una gran brecha entre la investigación básica y la implementación clínica de las biopsias líquidas, principalmente debido a la falta de metodologías estandarizadas. Además, las plataformas técnicas que se utilizan actualmente no siempre son adecuadas para analizar la baja cantidad y calidad de material del tumor procedente de una biopsia líquida. En consecuencia, la validación e implementación de los ensayos de biomarcadores en biopsias líquidas en los laboratorios clínicos requieren una plataforma técnica estandarizada que sea sensible, rápida, fácil de usar, viable económicamente, flexible y que requiera un aporte inicial de ácidos nucleicos bajo, debido a la baja concentración que normalmente se obtiene en las biopsias líquidas. La plataforma nCounter se puede utilizar para analizar todo tipo de moléculas, incluyendo ARN, ADN y proteínas. La hibridación de diferentes códigos formados por moléculas de colores siguiendo patrones específicos con secuencias de interés permite una lectura directa de los niveles de expresión de genes y proteínas o la detección de mutaciones. El desarrollo de ensayos de biomarcadores en tejidos usando nCounter condujo a la aprobación por la administración de fármacos y alimentos de los Estados Unidos (FDA) del ensayo Prosigna ™ para su uso clínico en la tipificación del cáncer de mama. Numerosos estudios han destacado el potencial de esta plataforma para analizar moléculas derivadas y amplificadas de biopsias líquidas, aunque estudios de validación en el entorno clínico aun son necesarios. El objeto de esta tesis es la validación del uso de la plataforma NanoString nCounter para analizar material de biopsias líquidas y desarrollar ensayos de biomarcadores clínicamente relevantes.The assessment of predictive- and prognostic molecular markers in tumor tissue, also known as personalised treatment, has transformed clinical practice for many cancer types. This genotype-directed therapy was found to improve patient survival, and several technical platforms have been introduced in clinical laboratories since then. However, not all tumors can be biopsied and tissue quantities are often insufficient for tumor characterisation. Liquid biopsies, such as membrane-encapsulated- or circulating free RNA, DNA and proteins, can be derived from body fluids and can replace or complement tissue biopsies. They have several advantages, such as repeated sampling, a minimally invasive character and heterogeneous profiling. Unfortunately, there is still a big gap between basic research and clinical implementation of liquid biopsies, mainly due to the lack of standardised methodologies. In addition, currently used technical platforms are not always suitable to analyze the low quantity and quality of tumor-derived material that can be found in a liquid biopsy. In consequence, large-scale validation and clinical implementation of liquid biopsy-based biomarker assays requires a sensitive, quick, easy-to-use, relatively cheap, flexible and standardized technical platform with low input requirements. The nCounter platform can be used to analyze all types of molecules, including RNA, DNA and proteins. Binding of color coded barcodes to targets of interest allows for either a direct read-out of gene- or protein expression levels or the detection of mutations. Tissue-based biomarker assay development on nCounter led to the FDA approval of the Prosigna™ assay for clinical use in breast cancer subtyping. Previous efforts have also highlighted the potential of this platform to analyze amplified liquid biopsy-derived molecules, although validation studies in the clinical setting are needed. In this thesis we validated the use of the NanoString nCounter platform to analyze material from liquid biopsies and develop clinically relevant biomarker assays.Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicin

Column Re-use_Apogee_Calibrations

Apogee A60 - MESF and size (Rosetta) calibration files and output</p

STAT3 as a potential immunotherapy biomarker in oncogene-addicted non-small cell lung cancer

Immune checkpoint blockade has modified the treatment landscape for many types of tumors, including lung cancer. Still our knowledge on the biology of the interaction between tumor cells and the microenvironment is limited, preventing the optimal use of these new compounds and the maximum benefit that the patients can derive from them. We have actively worked on the role of STAT3, a transcriptional factor that causes innate resistance to targeted therapies in oncogene-addicted tumors. In this short review we take the opportunity to express our opinion and review existing knowledge on the immune role of STAT3 and the possible implications that this may have for the discovery of new biomarkers to predict response to immunotherapy, as well as new partners to combine with and increase the efficacy of immune checkpoint inhibitors

PIM-1 inhibition with AZD1208 to prevent osimertinib-induced resistance in EGFR-mutation positive non-small cell lung cancer

Aim: The progression free survival of non-small cell lung cancer (NSCLC) patients has been doubled over the last years, but still single epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) lead to incomplete responses. Compensatory signaling pathways are activated upon single EGFR TKIs. We have shown that compounds, which inhibit these pathways, are synergistic with EGFR TKIs. Proviral integration site for Moloney murine leukemia virus (PIM) has been connected to cancer therapy resistance, being involved in receptor tyrosine kinase, signal transducer and activator of transcription 3 (STAT3) and interleukin-6 signaling. We hypothesized that combined PIM and EGFR inhibition may improve the upfront therapy of EGFR-mutation positive NSCLC.Methods: We reviewed the literature on PIM kinases, and performed cell viability assays, gene expression analyses, and immunoblotting experiments to reveal the mechanisms of action of the PIM inhibitor (AZD1208) alone and combined with osimertinib in five EGFR-mutation positive NSCLC cell lines.Results: Osimertinib alone induced the activation of signal transducer and activator of transcription 3 (STAT3) as well as other signaling nodes. Combined osimertinib and AZD1208 yielded moderate synergistic effects in all NSCLC cell lines investigated. Among the EGFR-mutation positive cell lines examined, the H1975 and PC9 cell lines had the highest PIM1 and STAT3 mRNA expression. The combination decreased the osimertinib-induced STAT3 phosphorylation.Conclusion: This study provides a short review on PIM kinases, and shows our results of combined PIM and EGFR inhibition in EGFR-mutation positive NSCLC cell lines. The combination was moderately synergistic but decreased STAT3 phosphorylation, an important signaling node in therapy resistance