Circulating epithelial cell as viral infection and tissue origin marker in patients with severe COVID-19

Liquid biopsy (LB) is a minimally invasive procedure that detects biomarkers in body fluids for real-time monitoring of patients. This study developed a new LB approach to analyze Circulating Epithelial Cells (CECs) in Intensive Care Unit (ICU) patients with severe COVID-19 and High-Exposure Negative Population to COVID-19 (HENPC) as the control group. The CECs were characterized by multispectral imaging flow cytometry, and an anti-SARS-CoV-2 Spike S1 protein (ProtS) antibody was used to detect infection. The results showed that CECs were present in most ICU patients (p = 0.0412), and their median number was significantly higher (p = 0.0004) than in controls. CEC clusters were only identified in patients, and high positive ProtS expression was observed in CECs from ICU patients compared to negative controls. In conclusion, LB could be a minimally invasive tool for detecting tissue damage caused by infectious agents and could provide real-time biological information about disease status and evolution. However, further validation in a larger population of patients is needed

23 Validation of PD-L1 dynamic expression on extracellular vesicles as a predictor of response to immune-checkpoint inhibitors and survival in non-small cell lung cancer patients

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 <0.85 = Low.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

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

Importance: Because of socioeconomic factors, many patients with advanced non-small cell lung cancer (NSCLC) do not receive immunotherapy in the first-line setting. It is unknown if the combination of immunotherapy with chemotherapy can provide clinical benefits in immunotherapy-naive patients with disease progression after treatment with platinum-based chemotherapy. Objective: To evaluate the safety and efficacy of the combination of pembrolizumab plus docetaxel in patients with previously treated advanced NSCLC following platinum-based chemotherapy regardless of EGFR variants or programmed cell death ligand 1 status. Design, Setting, and Participants: The Pembrolizumab Plus Docetaxel for Advanced Non-Small Cell Lung Cancer (PROLUNG) trial randomized 78 patients with histologically confirmed advanced NSCLC in a 1:1 ratio to receive either pembrolizumab plus docetaxel or docetaxel alone from December 2016 through May 2019. Interventions: The experimental arm received docetaxel on day 1 (75 mg/m2) plus pembrolizumab on day 8 (200 mg) every 3 weeks for up to 6 cycles followed by pembrolizumab maintenance until progression or unacceptable toxic effects. The control arm received docetaxel monotherapy. Main Outcomes and Measures: The primary end point was overall response rate (ORR). Secondary end points included progression-free survival (PFS), overall survival, and safety. Results: Among 78 recruited patients, 32 (41%) were men, 34 (44%) were never smokers, and 25 (32%) had an EGFR/ALK alteration. Forty patients were allocated to receive pembrolizumab plus docetaxel, and 38 were allocated to receive docetaxel. A statistically significant difference in ORR, assessed by an independent reviewer, was found in patients receiving pembrolizumab plus docetaxel vs patients receiving docetaxel (42.5% vs 15.8%; odds ratio, 3.94; 95% CI, 1.34-11.54; P =.01). Patients without EGFR variations had a considerable difference in ORR of 35.7% vs 12.0% (P =.06), whereas patients with EGFR variations had an ORR of 58.3% vs 23.1% (P =.14). Overall, PFS was longer in patients who received pembrolizumab plus docetaxel (9.5 months; 95% CI, 4.2-not reached) than in patients who received docetaxel (3.9 months; 95% CI, 3.2-5.7) (hazard ratio, 0.24; 95% CI, 0.13-0.46; P <.001). For patients without variations, PFS was 9.5 months (95% CI, 3.9-not reached) vs 4.1 months (95% CI, 3.5-5.3) (P <.001), whereas in patients with EGFR variations, PFS was 6.8 months (95% CI, 6.2-not reached) vs 3.5 months (95% CI, 2.3-6.2) (P =.04). In terms of safety, 23% (9 of 40) vs 5% (2 of 38) of patients experienced grade 1 to 2 pneumonitis in the pembrolizumab plus docetaxel and docetaxel arms, respectively (P =.03), while 28% (11 of 40) vs 3% (1 of 38) experienced any-grade hypothyroidism (P =.002). No new safety signals were identified. Conclusions and Relevance: In this phase 2 study, the combination of pembrolizumab plus docetaxel was well tolerated and substantially improved ORR and PFS in patients with advanced NSCLC who had previous progression after platinum-based chemotherapy, including NSCLC with EGFR variations. Trial Registration: ClinicalTrials.gov Identifier: NCT02574598

La vesícula extracelular TGF-β basal es un biomarcador predictivo de la respuesta a los inhibidores del punto de control inmunitario y de la supervivencia en el cáncer de pulmón no microcítico

Antecedentes: Los inhibidores de los puntos de control inmunitarios (ICI) son una estrategia terapéutica eficaz que mejora la supervivencia de los pacientes con cáncer de pulmón en comparación con los tratamientos convencionales. terapéutica eficaz que mejora la supervivencia de los pacientes con cáncer de pulmón en comparación con los tratamientos convencionales. Sin embargo, se necesitan biomarcadores predictivos novedosos para estratificar qué pacientes obtienen un beneficio clínico, ya que el histológico PD-L1, actualmente utilizado y altamente heterogéneo, ha mostrado una baja precisión. La biopsia líquida es el análisis de biomarcadores en fluidos corporales y representa una herramienta mínimamente invasiva que puede utilizarse para monitorizar la evolución del tumor y los efectos del tratamiento, reduciendo potencialmente los sesgos asociados a la heterogeneidad tumoral asociada a las biopsias de tejidos. En este contexto citoquinas, como el factor de crecimiento transformante-β (TGF-β), pueden encontrarse libres en circulación en la sangre y empaquetadas en vesículas extracelulares (VE), que tienen un tropismo de administración específico y pueden afectar a la interacción entre el tumor y el sistema inmunitario. El TGF-β es una citocina inmunosupresora que desempeña un papel crucial en el escape inmunitario de los tumores, la resistencia al tratamiento y la metástasis. Así pues, nuestro objetivo era evaluar el valor predictivo predictivo del TGF-β circulante y EV en pacientes con cáncer de pulmón no microcítico que reciben ICI.Background: 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

Daily risk of adverse outcomes in patients undergoing complex lesions revascularization: a subgroup analysis from the RAIN-CARDIOGROUP VII study (veRy thin stents for patients with left mAIn or bifurcatioN in real life)

Introduction: Percutaneous coronary intervention (PCI) for complex lesions, including unprotected left main (ULM) and bifurcations, is gaining a relevant role in treating coronary artery disease with good outcomes, also thanks to new generation stents. The daily risk of adverse cardiovascular events and their temporal distribution after these procedures is not known.Methods: All consecutive patients presenting with a critical lesion of ULM or bifurcation treated with very thin struts stents, enrolled in the RAIN-Cardiogroup VII study, were analyzed. The daily risk of major acute cardiovascular events (MACE), target lesion revascularization (TLR) and stent thrombosis (ST) and their temporal distribution in the first year of follow-up was the primary endpoint. Differences among subgroups (ULM, patient presentation, kind of stent polymer) were the secondary endpoint.Results: 2745 patients were included, mean age 68 ± 11 years, 33.3% diabetics, 54.5% had an acute coronary syndrome (ACS); 88.5% of treated lesions were bifurcations, 27.2% ULM. Average daily risk was 0.022% for MACE, 0.005% for TLR and 0.004% for ST, in the first year. Bimodal distribution of adverse events, especially TLR, with an early peak in the first 50 days and a late one after 150 days, was observed. Patients with ULM presented a significantly higher daily risk of events, and ACS patients presented higher MACE risk. No difference emerged according to the type of stent polymer.Conclusions: The daily risk of adverse events in the first year after complex PCI in our study is acceptably low. PCI on ULM carries a higher risk of complications

Effects of exosomes released by NSCLC cells on osteoclasts differentiation.

Effects of exosomes released by NSCLC cells on osteoclasts differentiation Pucci M., Taverna S., Corrado C., Giallombardo M., Rolfo C. and Alessandro R. Non-small cell lung cancer (NSCLC) has a poorly 5-year survival rate, as a consequence of the delay in the detection of the disease. The majority of patients are diagnosed in an advanced disease stage. Bone metastasis is the most frequent complication in NSCLC resulting in osteolytic lesions. The perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in bone metastasis, inducing osteoclastogenesis. Most of the patients with lung cancer are treated with EGFR inhibitors (TKIs). Numerous studies show in patients with NSCLC the presence of somatic mutations at the level of the exons coding for tyrosine kinase domain of the EGFR. The cell line CRL 2868 presents the “activating” mutation of exon 19, that confers sensitivity to treatment with TKIs. Recent studies demonstrated that exosomes, nano-size vesicles that shuttle proteins, lipids and nucleic acids, have a key role in tumor progression and premetastatic niche formation. Exosomes are involved in modulation of metastasis formation; we investigated the role of NSCLC cell-derived exosomes in OCs differentiation. We show that CRL 2868 cells release exosomes which are actively internalized by RAW 264.7 cell line, a cellular model of preosteoclasts. CRL 2868 cell-derived exosomes positively modulate pre-osteoclast migration and play a significant pro-differentiative role of RAW 264.7 cells, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Our data indicate that CRL2868-exosomes modulate OCs function and differentiation. Further studies will be carried out to investigate the role of TKI in OCs differentiation

Targeted Therapies in Early Stage NSCLC: Hype or Hope?

Non-small-cell lung cancer (NSCLC) represents roughly 85% of lung cancers, with an incidence that increases yearly across the world. The introduction in clinical practice of several new and more effective molecules has led to a consistent improvement in survival and quality of life in locally advanced and metastatic NSCLC. In particular, oncogenic drivers have indeed transformed the therapeutic algorithm for NSCLC. Nearly 25% of patients are diagnosed in an early stage when NSCLC is still amenable to radical surgery. In spite of this, five-year survival rates for fully resected early stage remains rather disappointing. Adjuvant chemotherapy has shown a modest survival benefit depending on the stage, but more than half of patients relapse. Given this need for improvement, over the last years different targeted therapies have been evaluated in early-stage NSCLC with no survival benefit in unselected patients. However, the identification of reliable predictive biomarkers to these agents in the metastatic setting, the design of molecularly-oriented studies, and the availability of novel potent and less toxic agents opened the way for a novel era in early stage NSCLC treatment. In this review, we will discuss the current landscape of targeted therapeutic options in early NSCLC

Targeted Therapies in Early Stage NSCLC : Hype or Hope?

Non-small-cell lung cancer (NSCLC) represents roughly 85% of lung cancers, with an incidence that increases yearly across the world. The introduction in clinical practice of several new and more effective molecules has led to a consistent improvement in survival and quality of life in locally advanced and metastatic NSCLC. In particular, oncogenic drivers have indeed transformed the therapeutic algorithm for NSCLC. Nearly 25% of patients are diagnosed in an early stage when NSCLC is still amenable to radical surgery. In spite of this, five-year survival rates for fully resected early stage remains rather disappointing. Adjuvant chemotherapy has shown a modest survival benefit depending on the stage, but more than half of patients relapse. Given this need for improvement, over the last years different targeted therapies have been evaluated in early-stage NSCLC with no survival benefit in unselected patients. However, the identification of reliable predictive biomarkers to these agents in the metastatic setting, the design of molecularly-oriented studies, and the availability of novel potent and less toxic agents opened the way for a novel era in early stage NSCLC treatment. In this review, we will discuss the current landscape of targeted therapeutic options in early NSCLC

Curcumin induces selective packaging of miR-21 in exosomes released by Chronic Myelogenous Leukemia cells

Chronic myeloid leukemia is a myeloproliferative disorder that comes from a hematopoietic stem cell or a multipotent progenitor. Exosomes are biological nanovescicles (40-100 nm) able to modulate intercellular communication and tumour microenvironment; they also contain miRNAs that can influence gene expression in target cells. OncomiRNAs, such as miR-21, are often increased in cancer. Some observation indicate a possible cellular disposal of miRNAs by exosomes. Curcumin affects the expression of microRNAs in CML cells and according to our data may play a role in this process