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Background ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. Methods EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. Results Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. Conclusions ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

Multiple questions about SARS-CoV-2 humoral and cellular immunity remain unanswered. One key question is whether preexisting memory T or B cells, specific for related coronaviruses in SARS-CoV-2-unexposed individuals, can recognize and suppress COVID-19, but this issue remains unclear. Here, we demonstrate that antibody responses to SARS-CoV-2 antigens are restricted to serum samples from COVID-19 convalescent individuals. In contrast, cross-reactive T cell proliferation and IFN-γ production responses were detected in PBMCs of around 30% of donor samples collected prepandemic, although we found that these prepandemic T cell responses only elicited weak cTFH activation upon stimulation with either HCoV-OC43 or SARS-CoV-2 NP protein. Overall, these observations confirm that T cell cross-reactive with SARS-CoV-2 antigens are present in unexposed people, but suggest that the T cell response to HCoV-OC43 could be deficient in some important aspects, like TFH expansion, that might compromise the generation of cross-reactive TFH cells and antibodies. Understanding these differences in cellular responses may be of critical importance to advance in our knowledge of immunity against SARS-CoV-2., This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028)and grants from Madrid Regional Government “IMMUNOTHERCAN”[S2017/BMD-3733-2(M.V.G.)];the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER,EU):RTI2018-093569-B-I00(M.V.G.),SAF2017-82940-R(J.M.R.F.),SAF2017-83265-R(H.T.R.);SAF2017-82886-R(F.S.M.)];RETICSProgramofISCIII[RD16/0012/0006;RIER(J. M. R. F.)]. A. F. G. J. is a recipient of a fellowship (FPU18/01698)from the Spanish Ministry of Science and Education.D.F.S.isarecip-ientofafellowship (PRE2018-083200)from the Spanish Ministry of Science and Innovation. Both are graduate students in the Molecular Biosciences doctoral program of the Autonomous University of Madrid

BackgroundExtracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors.ResultsHere we describe a method that, using just a few microliters of patient’s plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs.ConclusionsThis optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.Graphical Abstract

Many new aspects of COVID-19 disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein and the main cysteine-like protease. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected patients from vaccine responders., This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028) and grants from Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (JMRF); PI19/00549 (AA)]. The study was also funded by “La Caixa Banking Foundation” (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM.

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19., This work was supported by: Spanish National Research Council (CSIC-202020E079, CSIC-COVID19-028); Madrid Regional Government “IMMUNOTHERCAN” [S2017/BMD-3733-2 (MVG)]; Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU, RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (EMGC); PI19/00549 (AA)]; “La Caixa Bank Foundation” (HR17-00016), Fondo Supera COVID (CRUE-Banco de Santander), both to FSM.

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2 specific antibodies are based on serum detection of antibodies to either the viral spike glycoprotein (the major target for neutralising antibodies) or the viral nucleocapsid protein that are known to be highly immunogenic in other coronaviruses. Conceivably, exposure of antigens released from infected cells could stimulate antibody responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other non-structural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titre IgG, IgM and IgA antibody responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher antibody titres in these assays associated with more severe disease and no cross-reactive antibodies against prior betacoronavirus were found. Remarkably, IgG antibodies specific for Mpro and other SARS-CoV-2 antigens can also be detected in saliva. In conclusion, Mpro is a potent antigen in infected patients that can be used in serological tests and its detection in saliva could be the basis for a rapid, non-invasive test for COVID-19 seropositivity.

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2–specific Abs are based on serum detection of Abs to either the viral spike glycoprotein (the major target for neutralizing Abs) or the viral nucleocapsid protein that is known to be highly immunogenic in other coronaviruses. Conceivably, exposure of Ags released from infected cells could stimulate Ab responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other nonstructural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titer IgG, IgM, and IgA Ab responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher Ab titers in these assays associated with more-severe disease, and no cross-reactive Abs against prior betacoronavirus were found. Remarkably, IgG Abs specific for Mpro and other SARS-CoV-2 Ags can also be detected in saliva. In conclusion, Mpro is a potent Ag in infected patients that can be used in serological tests, and its detection in saliva could be the basis for a rapid, noninvasive test for COVID-19 seropositivity., This work was supported by the Spanish National Research Council (Project 202020E079) and grants from the Madrid Regional Government (IMMUNOTHERCAN S2017/BMD-3733-2 [to M.V.-G.]), the Spanish Ministry of Science and Innovation (MCIU/AEI/FEDER, EU: RTI2018-093569-B-I00 [to M.V.-G.], SAF2017-82940-R [to J.M.R.F.], SAF2017-83265-R [to H.T.R.], and SAF2017-82886-R [to F.S.-M.]), and RETICS Program of Instituto de Salud Carlos III (RD16/0012/0006; RIER [to J.M.R.F.], RD16/0011/0012 and PI18/0371 [to I.G.-A.], and PI19/00549 [to A.A.]). The study was also funded by La Caixa Banking Foundation (HR17-00016 to F.S.-M.) and Banco Santander Supera COVID to F.S.-M. This work has been cofunded by grant Covid 2019 from the Madrid Regional Government to Health Institute “La Princesa.”

Background: Identification of ALK rearrangements in NSCLC patients has important implications as it significantly expands their therapeutic opportunities. However, the availability of tumors biopsies is sometimes limited in the clinical setting. The analysis of liquid biopsies can potentially overcome such limitation. Nevertheless, the identification of ALK rearrangements using liquid biopsies remains challenging as fusion transcripts are rapidly degraded by RNAses present in the blood. Nonetheless, encapsulated RNA inside extracellular vesicles (EVs) is protected from RNAses. The main objective of this study is to explore the potential value of exosomes for non-invasive ALK testing. Methods: H3122 and H2228 cell lines, derived from NSCLC patients with EML4-ALK variant 1 (E13;A20) and variant 3 (E6a/b;A20) respectively, were used to isolate exosomes by the sequential centrifugation of cell culture supernatants. Briefly, supernatants were centrifuged twice for 10 min at 200xg, twice for 500xg for 10 min, once for 30 min at 10,000xg and a final ultracentrifugation at 100,000×g for 2 h at 4 °C. Purified exosomes were verified in terms of morphology and size by transmission electron microscopy (TEM). In addition, specific EVs markers, namely CD63, CD81 and CD9, were measured by flow cytometry and Western-Blot. EML4-ALK variants were analyzed at the protein level by Western blot using 5A4 antibody, and RNA level by digital PCR (dPCR), using a QuantStudio® 3D Digital PCR System and specific TaqMan® assays for EML4-ALK variant 1 and 3. Samples were also submitted to NGS analysis using the Oncomine™ Pan-Cancer Cell-Free Assay kit. Results: TEM showed spherical vesicles around 100 nm in both cell lines. Flow cytometry and Western blot confirmed expression of the exosomal specific markers CD63, CD81 and CD9 tetraspanins. Western blot also showed EML4-ALK variant 1 (120 kDa) and variant 3 (87 kDa) in H3122 and H2228 purified exosomes, respectively. Isolated exosome RNA profile was composed of small RNA (< 200 nt), although 18S and 28S rRNA peaks were also present. dPCR and NGS confirmed EML4-ALK rearrangements in exosome derived from both cell lines. Conclusions: EML4-ALK fusions can be detected at the RNA levels and at the protein levels analyzing EVs derived from H3122 and H2228 cell lines. These results set the stage for the development of EV-based non-invasive ALK testing in NSCLC patients. Citation Format: Estela Sánchez-Herrero, Carmen Campos-Silva, Yaiza Cáceres Martell, Sandra Sanz-Moreno, Roberto Serna-Blasco, Alejandro Rodríguez Festa, Miguel Ángel Molina-Vila, Mariano Provencio, Mar Valés-Gómez, Atocha Romero. ALK fusions can be clearly detected in extracellular vesicles from NSCLC cell lines: A become of hope for non-invasive ALK testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 561.