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Immune-checkpoint inhibitors (ICIs) are an effective therapeutic strategy, improving the survival of patients with lung cancer compared with conventional treatments. However, novel predictive biomarkers are needed to stratify which patients derive clinical benefit because the currently used and highly heterogenic histological PD-L1 has shown low accuracy. Liquid biopsy is the analysis of biomarkers in body fluids and represents a minimally invasive tool that can be used to monitor tumor evolution and treatment effects, potentially reducing biases associated with tumor heterogeneity associated with tissue biopsies. In this context, cytokines, such as transforming growth factor-β (TGF-β), can be found free in circulation in the blood and packaged into extracellular vesicles (EVs), which have a specific delivery tropism and can affect in tumor/immune system interaction. TGF-β is an immunosuppressive cytokine that plays a crucial role in tumor immune escape, treatment resistance, and metastasis. Thus, we aimed to evaluate the predictive value of circulating and EV TGF-β in patients with non-small-cell lung cancer receiving ICIs.Plasma samples were collected in 33 patients with advanced non-small-cell lung cancer before and during treatment with ICIs. EV were isolated from plasma by serial ultracentrifugation methods and circulating and EV TGF-β expression levels were evaluated by enzyme-linked immunosorbent assay.Baseline high expression of TGF-β in EVs was associated with nonresponse to ICIs as well as shorter progression-free survival and overall survival, outperforming circulating TGF-β levels and tissue PD-L1 as a predictive biomarker.If validated, EV TGF-β could be used to improve patient stratification, increasing the effectiveness of treatment with ICIs and potentially informing combinatory treatments with TGF-β blockade.Treatment with immune-checkpoint inhibitors (ICIs) has improved the survival of some patients with lung cancer. However, the majority of patients do not benefit from this treatment, making it essential to develop more reliable biomarkers to identify patients most likely to benefit. In this pilot study, the expression of transforming growth factor-β (TGF-β) in blood circulation and in extracellular vesicles was analyzed. The levels of extracellular vesicle TGF-β before treatment were able to determine which patients would benefit from treatment with ICIs and have a longer survival with higher accuracy than circulating TGF-β and tissue PD-L1, which is the currently used biomarker in clinical practice.

Background: Immune-checkpoint inhibitors (ICIs) changed the therapeutic landscape of patients with lung cancer. However, only a subset of them derived clinical benefit and evidenced the need to identify reliable predictive biomarkers. Liquid biopsy is the non-invasive and repeatable analysis of biological material in body fluids and a promising tool for cancer biomarkers discovery. In particular, there is growing evidence that extracellular vesicles (EVs) play an important role in tumor progression and in tumor-immune interactions. Thus, we evaluated whether extracellular vesicle PD-L1 expression could be used as a biomarker for prediction of durable treatment response and survival in patients with non-small cell lung cancer (NSCLC) undergoing treatment with ICIs. Methods: Dynamic changes in EV PD-L1 were analyzed in plasma samples collected before and at 9 ± 1 weeks during treatment in a retrospective and a prospective independent cohorts of 33 and 39 patients, respectively. Results: As a result, an increase in EV PD-L1 was observed in non-responders in comparison to responders and was an independent biomarker for shorter progression-free survival and overall survival. To the contrary, tissue PD-L1 expression, the commonly used biomarker, was not predictive neither for durable response nor survival. Conclusion: These findings indicate that EV PD-L1 dynamics could be used to stratify patients with advanced NSCLC who would experience durable benefit from ICIs., Center for Thoracic Oncology Icahn School of Medicine at Mount Sinai, Borsa Dottorati FSE XXXII ciclo Unime, United States Department of Health & Human Services, National Institutes of Health (NIH) - USA, NIH National Cancer Institute (NCI) P30CA016672, University of Pittsburgh Hillman Cancer Center, Hillman Cancer Center's NCI Cancer Center Support Grant (CCSG) P30CA047904, A.S.S.O. (Associazione Siciliana Sostegno Oncologico) Onlus, National Cancer Institute-Cancer Center Support Grant (CCSG) P30CA134274, Merck & Company

Osimertinib is a third generation EGFR-TKI inhibitor approved in the first-line setting for patients with advanced non-small cell lung cancer (NSCLC). Additionally, it represents the treatment of choice in patients who present with T790M mutations and evidence of relapse of the disease. Effectiveness and safety of this drug have been studied in multiple clinical trials and observational studies, however, information regarding outcomes among Hispanic patients treated with Osimertinib is scarce. The objective of this study was to examine real-world effectiveness and safety of first-line Osimertinib in a cohort of Hispanic patients with NSCLC, emphasizing post-progression outcomes.This is a multicenter, multinational, retrospective cohort study of Hispanic patients treated with Osimertinib as first-line for EGFR-mutated NSCLC. Patients with a confirmed diagnosis of metastatic EGFR-mutated NSCLC who received Osimertinib (80mg/day until evidence of disease progression or presence of intolerable adverse effects) were identified and included. NGS was performed in tumor samples or liquid biopsies among patients who had disease progression. The primary outcome was progression-free survival, and the secondary outcome was post-progression survival.A total of 94 patients from Mexico, Argentina, Costa Rica, Colombia, Panama, Chile and the USA were included, with a median age of 59 years. Identified mutations included EGFR Exon 19 deletions and EGFR pL858R point mutations. Median progression-free survival (PFS) was 14.4 months (95%CI 12.4-18.2 months). Lung/pleura and lymph nodes were the most common sites of progression. Median post-progression survival was 7.73 months (95%CI 4.07 months-Not reached). Factors which negatively affected PFS included presence of liver metastases at diagnosis and a tumor mutational burden5 mut/Mb.Treatment with first line osimertinib represents an effective and safe option for Hispanic patients with metastatic NSCLC. Liver metastases and a higher tumor mutation burden were associated with a lower PFS. Despite effectiveness, different mechanisms of resistance were identified among the patients in this cohort, including mutations which can be targeted by other therapeutic options.

Background: Platelets are active players in tumorigenesis, although the exact interactive mechanisms and their direct impact on tumor cells remain largely unknown. Methods: Bidirectional transference of lipids, proteins and RNA between platelets and tumor cells and its impact on tumor cell behavior and tumor process are analyzed in this work. Phenotypic, genetic and functional modifications induced by platelets were analyzed both in tumor cell lines and in circulating tumor cells (CTCs). Results: Data from these assays showed that platelets transferred structural components to tumor cells with higher efficiency than tumor cells to platelets (p = 0.001). This biological interplay occurred by direct contact, internalization or via extracellular vesicles. As a result, tumor cells acquired platelet markers (CD61 and CD42), showed decreased EpCAM, expressed epithelial-to-mesenchymal transition markers, and increased proliferation rates. Moreover, we were able to detect CD61 in CTCs from early and advanced prostate cancer. Conclusions: Our results demonstrated, for the first time, that platelets educate tumor cells by highly efficient transference of lipids, proteins and RNA through different mechanisms. These results suggest that tumor cells and CTCs might acquire highly dynamic and aggressive phenotypes due to platelets interaction including EMT, stem-like phenotype and high proliferative rates.

Background: Mutations in STK11 (STK11(Mut)) and, frequently co-occurring, KEAP1 mutations (KEAP1(Mut)) are associated with poor survival in metastatic Non-small Cell Lung Cancer (mNSCLC) patients treated with immunotherapy. However, there are limited data regarding the prognostic or predictive significance of these genomic alterations among Hispanics. Methods: This retrospective study analyzed a cohort of Hispanic patients (N = 103) diagnosed with mNSCLC from the US and seven Latin American countries (LATAM) treated with immune checkpoint inhibitors (ICI) alone or in combination as first-line (Cohort A). All cases were treated in routine care between January 2016 and December 2021. The main objectives were to determine the association of mutations in STK11 or KEAP1 in these patients' tumors with overall (OS) and progression-free survival (PFS), presence of KRAS mutations, tumor mutational burden (TMB), and other relevant clinical variables. To compare outcomes with a STK11(Wt)/KEAP1(Wt) population, historical data from a cohort of Hispanic patients (N = 101) treated with first-line ICI was used, matching both groups by country of origin, gender, and Programed Death-ligand 1 (PD-L1) expression level (Cohort B). Results: Most tumors had mutations only in STK11 or KEAP1 (45.6%) without KRAS co-mutation or any other genomic alteration. Besides, 35%, 8.7%, 6.8%, and 3.9% were KRAS(Mut) + STK11(Mut), KRAS(Mut) + STK11(Mut) + KEAP1(Mut), STK11(Mut) + KEAP1(Mut), and KRAS(Mut) + KEAP1(Mut), respectively. Based on KRAS status, STK11 alterations were associated with significantly lower PD-L1 expression among those with KRAS(Wt) (p = 0.023), whereas KEAP1 mutations were predominantly associated with lower PD-L1 expression among KRAS(Mut) cases (p = 0.047). Tumors with KRAS(Mut) + KEAP1(Mut) had significantly higher median TMB when compared to other tumors (p = 0.040). For Cohort A, median PFS was 4.9 months (95%CI 4.3-5.4), slightly longer in those with KEAP1(mut) 6.1 months versus STK11(Mut) 4.7 months (p = 0.38). In the same cohort, PD-L1 expression and TMB did not influence PFS. OS was significantly longer among patients with tumors with PD-L1 >= 50% (30.9 months), and different from those with PD-L1 1-49% (22.0 months), and PD-L1 < 1% (12.0 months) (p = 0.0001). When we compared the cohorts A and B, OS was significantly shorter for patients carrying STK1 [STK11(Mut) 14.2 months versus STK11(Wt) 27.0 months (p = 0.0001)] or KEAP1 [KEAP1(Mut) 12.0 months versus KEAP1(Wt) 24.4 months (p = 0.005)] mutations. PD-L1 expression significantly affected OS independently of the presence of mutations in STK11, KEAP1, or KRAS. TMB-H favored better OS. Conclusions: This is the first large Hispanic cohort to study the impact of STK11 and KEAP1 mutations in NSCLC patient treated with ICI. Our data suggest that mutations in the above-mentioned genes are associated with PD-L1 expression levels and poor OS.

Purpose of review Cancer vaccines are one of the most extensively studied immunotherapy type in solid tumors. Despite favorable presuppositions, so far, the use of cancer vaccines has been associated with disappointing results. However, a new generation of vaccines has been developed, promising to revolutionize the immunotherapy field. Recent findings In this review, we aim to highlight the advances in cancer vaccines and the remaining hurdles to overcome. Summary Cancer vaccination has experienced tremendous progress in the last decade, with myriad promising developments. Future efforts should focus on optimization of target identification, streamlining of most appropriate vaccination strategies, and adjuvant development, as well as predictive biomarker identification. Cautious optimism is warranted in the face of early successes seen in recent clinical trials for oncolytic vaccines. If an approach were to prove successful, it could revolutionize cancer therapy the way ICIs did in the previous decade. Purpose of review Cancer vaccines are one of the most extensively studied immunotherapy type in solid tumors. Despite favorable presuppositions, so far, the use of cancer vaccines has been associated with disappointing results. However, a new generation of vaccines has been developed, promising to revolutionize the immunotherapy field. Recent findings In this review, we aim to highlight the advances in cancer vaccines and the remaining hurdles to overcome. Summary Cancer vaccination has experienced tremendous progress in the last decade, with myriad promising developments. Future efforts should focus on optimization of target identification, streamlining of most appropriate vaccination strategies, and adjuvant development, as well as predictive biomarker identification. Cautious optimism is warranted in the face of early successes seen in recent clinical trials for oncolytic vaccines. If an approach were to prove successful, it could revolutionize cancer therapy the way ICIs did in the previous decade.

In light of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants potentially undermining humoral immunity, it is important to understand the fine specificity of the antiviral antibodies. We screened 20 COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. Importantly, mutations which typically occur in the B.1.351 “South African” variant, significantly reduced the binding of anti-RBD antibodies. Nine of 20 patients were critically ill and were considered high-risk (HR). These patients showed significantly higher levels of transforming growth factor beta (TGF-β) and myeloid-derived suppressor cells (MDSC), and lower levels of CD4+ T cells expressing LAG-3 compared to standard-risk (SR) patients. HR patients evidenced significantly higher anti-S1/RBD IgG antibody levels and an increased neutralizing activity. Importantly, a large proportion of S protein-specific antibodies were glycosylation-dependent and we identified a number of immunodominant linear epitopes within the S1 and N proteins. Findings derived from this study will not only help us to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response but will also enable us to design more meaningful immunomonitoring methods for anti-COVID-19 vaccines., Atanackovic and coauthors screen COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. They also characterised immune responses in these patients and their findings will help to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response as well as assist the design of immunomonitoring methods for anti-COVID-19 vaccines.

Introduction: The rise of the personalized era in lung cancer prompted the evaluation of novel diagnostic tools to overcome some of the limits of traditional tumor genotyping. Liquid biopsy refers to a multitude of minimally invasive techniques that can allow a real-time biomolecular characterization of the tumor through the analysis of human body fluids. Areas covered: Herein we provide a comprehensive overview of the role of liquid biopsy in lung cancer, mainly focusing on the most studied members of the liquid biopsy family, cell-free DNA (cfDNA) and circulating tumor cells (CTCs). Expert opinion: Among the different components of the large liquid biopsy family, cfDNA is the most studied and widely adopted source for tumor genotyping in lung cancer, already entered clinical practice for detection of both sensitizing and resistance EGFR mutations. However, the impressive technological advances made in the last few years are expanding its potential applications, allowing a more comprehensive plasma genotyping through next-generation sequencing and moving from advanced/metastatic disease to novel frontiers, such as early detection and minimal residual disease evaluation.

BackgroundImmune-checkpoint inhibitors (ICIs) revolutionized the treatment of advanced non-small cell lung cancer (NSCLC).1–3 To date, tissue PD-L1 immunohistochemistry is one of the leading biomarkers for prediction of ICIs response but has several limitations.4 5Extracellular vesicles (EVs) are cell-derived structures involved in cell communication and represent a potential minimally invasive alternative to predicting ICI response.6–9 Based on this and our preliminary results presented at SITC 2020,10 we hypothesize that EV PD-L1 predicts response to ICIs in NSCLC.MethodsThis study evaluates an exploratory cohort of advanced/metastatic NSCLC patients receiving ICIs (cohort A) and a validation cohort receiving Pembrolizumab+docetaxel or docetaxel alone (PROLUNG Phase 2 randomized trial) (cohort B).11 Plasma samples were collected pre-treatment (T1) and at 3 treatment cycles (T2) (figure 1A). Response was assessed by computed-tomography scan at 3 (cohort A) and 6–8 treatment cycles (cohort B) according to mono- or chemotherapy combination therapy. Patients were classified as responders (partial, stable, or complete response) or non-responders (progressive disease) by RECISTv1.1.12 EVs were isolated by serial ultracentrifugation and characterized following ISEV recommendations.13,14 Tissue PD-L1 expression was measured by standardized immunohistochemistry (SP263, 22C3, or 28–8 clones)5 and EV PD-L1 expression by immunoblot and its ratio was calculated as EV PD-L1 T2/T1. Cut-offs from the exploratory cohort were applied to the validation cohort, being EV PD-L1 ratio ResultsPaired samples from 30 ICIs, 23 pembrolizumab+docetaxel, and 15 docetaxel treated patients were analyzed. In cohort A, non-responders showed higher EV PD-L1 ratio than responders (p=0.012) (figure 1B) with an area-under-the-curve (AUC) of 77.3%, 83.3% sensitivity, and 61.1% specificity, while the tissue PD-L1 was not predictive (AUC=50%). As a validation, pembrolizumab+docetaxel treated non-responders showed higher EV PD-L1 ratio (p=0.036) than responders with an AUC=69.3%, sensitivity=75%, and specificity=63.6%, outperforming the tissue PD-L1 (figure 1C). No statistically significant differences were observed in the docetaxel group (p=0.885). Moreover, ICIs patients with higher EV PD-L1 ratio showed shorter progression-free survival (PFS) (HR=0.30, p=0.066) and overall survival (OS) (HR=0.17, p=0.016) (figure 1D) which was also observed in the pembrolizumab+docetaxel cohort with shorter PFS (HR=0.12, p=0.004) and OS (HR=0.23, p=0.010) (figure 1E). EV PD-L1 ratio did not predict survival in docetaxel-treated patients.Abstract 23 Figure 1(A) Study design and methodology. (B) EV PD-L1 ratio predicts response to ICIs in 30 NSCLC patients from the discovery cohort A and outperforms tissue PD-L1. (C) EV PD-L1 ratio is predictive for response to pembrolizumab+docetaxel in 23 NSCLC patients but not in 15 patients receiving docetaxel alone from cohort B. (D) Higher EV PD-L1 ratio predicts shorter PFS and OS in 30 patients from the discovery cohort A treated with ICIs. (E) Higher EV PD-L1 ratio is associated with shorter PFS and OS in 23 patients treated with pembrolizumab+docetaxel but not in patients treated with docetaxel alone. Abbreviations: CT: Computed tomography, EV: Extracellular vesicle; HR: Hazard Ratio; ICIs: Immune-checkpoint Inhibitors; IHC: Immunohistochemistry; NR: Non-Responders; OS: Overall Survival; p: p-value; PFS: Progression-free survival; R: Responders [Created with BioRender].ConclusionsWe demonstrated that treatment-associated changes in EV PD-L1 levels are predictive of response and survival in advanced NSCLC patients treated with ICIs. This model, if confirmed in a large prospective cohort, could have important clinical implications, guiding treatment decisions and improving the outcome of patients receiving ICIs.AcknowledgementsWe would like to extend our gratitude to the all the patients that participated in the study.ReferencesBorghaei H, Paz-Ares L, Horn L, Spigel DR, Steins M, Ready NE, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non–Small-Cell Lung Cancer. N Engl J Med 2015;373:1627–39.Herbst RS, Baas P, Kim DW, Felip E, Pérez-Gracia JL, Han JY, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): A randomised controlled trial. Lancet 2016;387:1540–50.Ruiz-Patiño A, Arrieta O, Cardona AF, Martín C, Raez LE, Zatarain-Barrón ZL, et al. Immunotherapy at any line of treatment improves survival in patients with advanced metastatic non-small cell lung cancer (NSCLC) compared with chemotherapy (Quijote-CLICaP). Thorac Cancer 2020;11:353–61.Doroshow DB, Bhalla S, Beasley MB, Sholl LM, Kerr KM, Gnjatic S, et al. PD-L1 as a biomarker of response to immune-checkpoint inhibitors. Nat Rev Clin Oncol 2021;18:345–362.Hirsch FR, McElhinny A, Stanforth D, Ranger-Moore J, Jansson M, Kulangara K, et al. PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the blueprint PD-L1 IHC assay comparison project. J Thorac Oncol 2017;12:208–222.Poggio M, Hu T, Pai CC, Chu B, Belair CD, Chang A, et al. Suppression of exosomal PD-L1 induces systemic anti-tumor immunity and memory. Cell 2019;177:414–427.e13.Cordonnier M, Nardin C, Chanteloup G, Derangere V, Algros MP, Arnould L, et al. Tracking the evolution of circulating exosomal-PD-L1 to monitor melanoma patients. J Extracell Vesicles 2020;9:1710899.Del Re M, Cucchiara F, Rofi E, Fontanelli L, Petrini I, Gri N, et al. A multiparametric approach to improve the prediction of response to immunotherapy in patients with metastatic NSCLC. Cancer Immunol Immunother 2020;70:1667–1678.Chen G, Huang AC, Zhang W, Zhang G, Wu M, Xu W, et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature. 2018;560:382–6.10 de Miguel Perez D, Russo A, Gunasekaran M, Cardona A, Lapidus R, Cooper B, et al. 31 Dynamic change of PD-L1 expression on extracellular vesicles predicts response to immune-checkpoint inhibitors in non-small cell lung cancer patients. 2020J Immunother Cancer;8(Suppl 3):A30–A30.Arrieta O, Barrón F, Ramírez-Tirado LA, Zatarain-Barrón ZL, Cardona AF, Díaz-García D, et al. Efficacy and safety of pembrolizumab plus docetaxel vs docetaxel alone in patients with previously treated advanced non–small cell lung cancer: the PROLUNG phase 2 randomized clinical trial. 2020JAMA Oncol;6:856–864.Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). 2009Eur J Cancer;45:228–47.Reclusa P, Verstraelen P, Taverna S, Gunasekaran M, Pucci M, Pintelon I, et al. Improving extracellular vesicles visualization: From static to motion. 2020Sci Rep;10:6494.Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. 2018J Extracell Vesicles;7:1535750Ethics ApprovalPatients consented to Institutional Review Board–approved protocol, A.O. Pappardo, Messina, Italy for cohort A and Thoracic Oncology Unit, Instituto Nacional de Cancerología (INCan), México City, México in case of the cohort B. Biological material was transferred to the University of Maryland School of Medicine, Baltimore for EV analysis under signed MTA between institutions MTA/2020–13111 & MTA/2020–13113.

Background Immune-checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced/metastatic non-small cell lung cancer patients (NSCLC), however, only a small subset of patients derives clinical benefit.1–3 To date, PD-L1 immunohistochemical evaluation is the gold-standard assay and the only approved biomarker, but associated with several limitations due to technical and biological factors such as spatial and temporal tumor heterogeneity.4 5 In this context, liquid biopsies emerge as novel powerful tools that could allow the non-invasive real-time characterization of the tumor PD-L1 status. In particular, extracellular vesicles (EVs), defined as cell-derived double-membrane structures involved in cell communication, hold strong potential as tissue surrogates. Recent studies have suggested that EV PD-L1 could stratify melanoma patients receiving ICIs, but none has showed the predictive value of this biomarker in NSCLC patients.6 7 We hypothesize that EV PD-L1 cargo can serve to stratify the response to ICIs in NSCLC patients. Methods This study enrolled advanced/metastatic NSCLC patients receiving ICI treatment. Plasma samples were obtained at baseline (T1) and at 8 weeks (T2) during the first response evaluation. Patients were classified as responders when showing partial, stable or complete response or as non-responders when manifesting progressive disease following RECIST v1.1.8 Plasma EVs were isolated by standard serial ultracentrifugation methods and characterized according to ISEV recommendations.9 10 Tissue PD-L1 expression was measured by immunohistochemistry while EV PD-L1 expression was measured by immunoblot. A predictive model was created by logistic-regression and a bootstrap corrected ROC curve to validate the results. Results Paired plasma samples from 21 patients were analyzed. PD-L1 tissue expression was not correlated with treatment response (p=0.394) nor matched the baseline EV PD-L1 levels (p=0.337) (figure 1.A). However, the dynamics of EV PD-L1 (T1-T2) correlated with the treatment response, observing an increase of PD-L1 expression in non-responders and a decrease or stable levels in responders (p=0.043) (figure 1.B). The predictive model reported an AUC=0.85, 90% CI=0.72–0.97, with 74.2% sensitivity and 73.5% specificity (figure 1.C). Moreover, the increase of EV PD-L1 was associated with shorter overall survival (HR=4.34, p=0.037) and shorter progression-free survival (HR=5.06, p=0.025) (figure 1 D & E). Conclusions Our preliminary-study showed, for the first time, the predictive and prognostic value of EV PD-L1 dynamic changes in immunotherapy-treated NSCLC patients. Although larger studies are needed to validate these results, this promising biomarker could have important clinical implications, guiding treatment decisions in near real-time and improving the outcome of patients that could benefit from ICIs. Acknowledgements We would like to extend our gratitude to the all the patients that participated in the study. Ethics Approval All patients consented to an Institutional Review Board–approved protocol (A.O. Papardo, Messina, Italy). Biological material was transfer to the University of Maryland, USA under signed MTA between both institutions (MTA/2020-13111). References Rittmeyer A, Barlesi F, Waterkamp D, et al. Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial. Lancet 2017;389:255–265. Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer. N Engl J Med 2015;373:1627–1639. Chen DS, Mellman I: Oncology Meets Immunology: The Cancer-Immunity Cycle. Immunity 2013, 39:1–10. Zou WP, Wolchok JD, Chen LP. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci Transl Med. 2016; 8:328rv4. Patel SP, Kurzrock R. PD-L1 Expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther 2015;14:847–56. Cordonnier M, Nardin C, Chanteloup G, et al. Tracking the evolution of circulating exosomal-PD-L1 to monitor melanoma patients. J Extracell Vesicles 2020;9:1710899. Del Re M, Marconcini R, Pasquini G, et al. PD-L1 mRNA expression in plasma-derived exosomes is associated with response to anti-PD-1 antibodies in melanoma and NSCLC. Br J Cancer 2018;118:820–824. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45:228–47. Reclusa P, Verstraelen P, Taverna S, et al. Improving extracellular vesicles visualization: From static to motion. Sci Rep 2020;10(1):6494. Thery C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for extracellular vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 2018;7:1535750.

Lung cancer has a high morbidity and mortality rate, and affected patients have a poor prognosis and low survival. The therapeutic approaches for lung cancer treatment, including surgery, radiotherapy, and chemotherapy, are not completely effective, due to late diagnosis. Although the identification of genetic drivers has contributed to the improvement of lung cancer clinical management, the discovery of new diagnostic and prognostic tools remains a critical issue. Liquid biopsy (LB) represents a minimally invasive approach and practical alternative source to investigate tumor-derived alterations and to facilitate the selection of targeted therapies. LB allows for the testing of different analytes such as circulating tumor cells, extracellular vesicles (EVs), tumor-educated platelets, and cell-free nucleic acids including DNAs, RNAs, and noncoding RNAs (ncRNAs). Several regulatory factors control the key cellular oncogenic pathways involved in cancers. ncRNAs have a wide range of regulatory effects in lung cancers. This review focuses on emerging regulatory ncRNAs, freely circulating in body fluids or shuttled by EVs, such as circular-RNAs, small nucleolar-RNAs, small nuclear-RNAs, and piwi-RNAs, as new biomarkers for early detection, prognosis, and monitoring of therapeutic strategy of lung cancer treatment.

e21144 Background: Immune-checkpoint inhibitors (ICIs) have revolutionized the therapeutic landscape of lung cancer patients. However, its low treatment efficacy is still an issue and the current standard of care tissue PD-L1 presents high variability. Liquid biopsy is a promising tool in the discovery of biomarkers in body fluids. In particular, extracellular vesicles (EVs) can present PD-L1 in their membranes, playing a role in the inhibition of the anti-tumor immune response. Likewise, TGF-β is crucial in the immune response found in the circulation and into EVs. On the other hand, radiomics analysis of conventional imaging provides information about tumor heterogeneity and immune response. Hence, we aimed to evaluate the predictive role of circulating biomarkers in lung cancer patients undergoing ICIs and the additional value of radiomics data. Methods: This is a retrospective analysis of 30 advanced/metastatic non-small lung cancer patients treated with ICIs. Plasma samples were collected at baseline and at 8 weeks during treatment, matching the first response evaluation. Patients with complete, partial response, or stable disease were classified as responders and those with progressive disease as non-responders following RECIST v1.1. EVs were isolated from plasma by ultracentrifugation and PD-L1 expression was revealed by immunoblot. Circulating and EV levels of TGF-β were analyzed by ELISA. Additionally, 400 radiomics features from target and non-target lesions were analyzed to evaluate response in 24 patients according to RECIST v1.1 and irRECIST. Robustness of predictive models was validated by ridge penalty and leave-one-out cross-validation. Results: The analysis of the dynamics of EV PD-L1 during treatment identified increased levels in non-responders in comparison to responders ( p= 0.012), while tissue PD-L1 levels were not associated to the response ( p= 0.585). The predictive model for EV PD-L1 reported a high accuracy with an area under the curve (AUC) = 77%, 91.7% sensitivity, and 61.1% specificity. The combination with radiomics, improved the accuracy and reported an AUC = 83%, 82% sensitivity, and 77% specificity. Additionally, the analysis of the association of the circulating biomarkers with the outcome revealed that increasing dynamics of EV PD-L1 and high baseline EV TGF-β were associated with shorter progression-free survival and overall survival, outperforming the circulating levels of TGF-β. Conclusions: This pilot study demonstrated that EV PD-L1 could serve as better predictive factor than tissue PD-L1 for the stratification of lung cancer patients undergoing ICIs and that it could be complemented with radiomics. Moreover, EV levels of PD-L1 and TGF-β have potential for the stratification and prognosis of patients treated with ICIs and their novel combinations with TGF-β blockade.

Introduction: Most non-small cell lung carcinoma (NSCLC) patients are diagnosed at advanced stages, when the prognosis is dismal. Thoracic irradiation in combination with chemotherapy have demonstrated to increase the survival of these patients, however, progression is still developed early. The failure of the treatment may be caused by inaccurate patient stratification due to the heterogeneous and evolving nature of tumors that tissue biopsy fails to reflect. Therefore, real-time predictive and prognostic biomarkers are needed to improve the survival of NSCLC patients. In this context, miRNAs are potential markers as they regulate the expression of cancer genes and can be selectively encapsulated into extracellular vesicles (EVs) by cancer cells and found in blood circulation. Similarly, circulating tumor cells (CTCs) are a key event in the metastatic process that shredded into the circulation resemble the features of the tissue of origin. Thus, CTCs can act as tissue surrogates and potential predictive and prognostic factors in these irradiated patients. Recent evidence has associated autophagy activation to an increased resistance to radiotherapy and chemotherapy. However, controversial reports showed a dual role of autophagy during tumor cell survival, thus requiring further research to be done. In this work, we aimed to identify the predictive and prognostic role of specific EV miRNAs and autophagic CTCs in advanced NSCLC patients under concomitant radio-chemotherapy. Methods: This study prospectively enrolled 38 locally advanced NSCLC patients under concomitant radio-chemotherapy (cisplatin-vinorelbine/carboplatin-taxol). Peripheral blood samples were drawn before, during, and after the treatment. CTCs were immunomagnetically isolated and autophagic activity was characterized by immunofluorescence. EVs were isolated by ultracentrifugation methods and specific miRNAs were analyzed by RT-PCR. Internal validation of the predictive model was performed by bootstrap approach. Results: The increase of EV-miR-375 and miR-200c was associated with the reduction of autophagic CTCs along the treatment (p=0.013 & p=0.025). Moreover, baseline levels of EV-miR-375, miR-200c, and miR-30c predicted the response to the treatment with an area under the curve (AUC) of 86%. The presence of autophagic CTCs and low EV-miR-30c were independent prognostic biomarkers for shorter relapse-free survival (p=0.002 & p=0.002) and overall survival (p=0.012 & p Citation Format: Diego de Miguel Perez, Rosario Guerrero Tejada, Alessandro Russo, Francisco Gabriel Ortega, Antonio Martínez-Única, Muthukumar Gunasekaran, Jose Antonio Lorente, Jose Exposito, Maria Jose Serrano Fernandez, Christian Rolfo. Extracellular vesicle miRNAs and autophagic CTCs: Predictive and prognostic biomarkers in radiotherapy treated NSCLC patients [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-063.

Introduction:The spread of SARS-CoV-2 virus continues to pose a major public health threat. Patients with cancer are thought to be at increased risk from SARS-COV-2 infection due to the immunodeficiency that results from the underlying neoplasm and treatment. The immune response to this infection has been the subject of great interest, with an extreme variation in clinical severity between infected individuals. Variation in the immune cell response (B, T, and NK lymphocytes, monocytes, and myeloid derived suppressor cells (MDSCs), among others) and their function have been hypothesized to be responsible for this range of presentation. Methods:Two patients with a history of hematologic malignancies were matched with three non-cancer patients with similar baseline clinical characteristics and severity of COVID related illness. The critical group (CG) was defined as those requiring mechanical ventilation (MV) due to COVID related respiratory failure and the non-critical group (NCG) were hospitalized but did not require MV. All samples studied were obtained from peripheral blood and processed within 4-hours of collection. Peripheral blood mononuclear cell (PBMC) were isolated using ficoll density gradient separation. Flowcytometric analysis using CytekTM Aurora was done on fresh PBMC samples. Thirty antibody-based flow markers were used to identify 54 distinct immune cell populations. IRB approval was obtained. Results: Critical Group (CG):The CG included case 1, a 47 year-old (y.o.) female (F) with a history (hx) of acute myeloid leukemia and had an matched related donor allogeneic hematopoietic stem cell transplant (alloHSCT) 10-years prior remaining in remission, with hematologic recovery, and off immunosuppressants treated with remdesivir and coritcosteroids for COIVD directed therapy; and case 2, a 55 y.o. F with a hx of HIV treated with corticosteroids for COIVD directed therapy (see Figure 1a). Non-Critical Group (NCG):The NCG included case 3, a 73 y.o male (M) with hx of relapsed/refractory Philadelphia chromosome negative Acute Lymphoblastic Leukemia with loss of CD19 and CD22 expression following treatment with blinatumumab and inotuzumab, and most recently treated with decitabine/venetoclax; case 4, a 66 y.o. M with hx of cardiomyopathy; and case 6, a 54 y.o. M with hx of obesity. None of the NCG cases were treated with COVID directed therapy. See Table 1 for further clinical information. Immunophenotypic expression:Flow cytometry gating strategy done as outlined in Fig 1a. Case 1 had a high proportion of B-cells, CD8+ T-cells, and cells with exhaustion markers (CD8+CD94+ T-cells, CD4+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1-CD94+ NK T-cells, Lag3+Cd11b- non-TB leukocytes) and MDSC immunophenotypes compared with matched case 2. Case 3 also had a high proportion of exhaustion markers (Lag3+CD39 low B-cells, CD8+PD1+ T-cells, CD8+CD94+PD1+ T-cells, CD4+PD1+CD94+ T-cells, PD1+CD94+ NK T-cells, PD1-CD94+ NK T-cells, Lag3+CD11b+, Lag3+CD11b- non-TB leukocytes) and high expression of immunosuppressive Treg and all MDSC; although high expression of granulocytic MDSC. Case 2 had a significant number of exhaustion and immunosuppressive cells as well. Cases 4 and 5 had a higher predominance of all T-cell subtypes and also had variable expression of exhaustion and immunosuppressive immunophenotypes (See Fib 1b). Conclusion:In our study of one critical and one non-critical patient with a history of hematologic malignancy matched with three non-cancer patients we demonstrate the high predominance of exhaustion markers (Lag3,PD1,CD94) and immunosuppressive cell types (Treg, granulocytic and monocytic MDSC). These findings are consistent with the fact that both CG and NCG, as hospitalized patients, represent the most severely ill COVID patient cohort. Of notable interest to the cancer population, cases 1 and 3 had a significant number of exhaustion and immunosuppressive immunophenotypes, suggestive of baseline exhaustion following alloHSCT even years after engraftment in case 1 and attenuated functional immunity in a patient undergoing active treatment in case 3. Interestingly, case 3 had lower expression of all MDSC, a known treatment effect of decitabine. Paired cytokine measurement and its effect on immunophenotype is underway. Additionally, we plan to present an atlas of the peripheral immune cell response on fifteen additional non-cancer COVID patients. Disclosures No relevant conflicts of interest to declare.