BackgroundEnhancing anti-tumor immunity is a foundational therapeutic strategy against cancer.1 The Aliya Pulsed Electric Fields (PEF) proprietary system has been shown to promote local and systemic ...anti-tumor immune activation in pre-clinical murine models through the activation of immunogenic cell death (ICD) and the release of damage associated molecular patterns (DAMPs) and tumor-specific antigens.2 3 Unlike apoptosis, often immunosuppressive, ICD mechanisms generate a potent immune response by releasing DAMPs that are recognized by and attract immune cells.4 ICD mechanisms can suppress cancer cell proliferation and migration.5 6 In this study we evaluated PEF’s capability for modulating anti-tumor immunity in patients with NSCLC.MethodsThe treat-and-resect INCITE ES study enrolled patients with suspected or confirmed NSCLC stage IA2-IB and no cancer treatment history within two years. This two-arm study included 34 patients in the treatment group and 8 patients in the control group. Treatment group subjects received AliyaTM PEF (GTI-00018 investigational device; Galvanize Therapeutics) after the diagnostic biopsy, whereas the control group only had the biopsy. Blood, serum, and tissue samples were collected pre- and post-PEF at specific timepoints (figure 1).ResultsIngenuity Pathway Analysis (IPA) of serum cytokines predicts that host innate immunity pathways that drive immune cell trafficking, differentiation and activation are activated in PEF-treated samples, including the Acute Phase Response, IL-6, JAK-STAT and Th17/IL-17 signaling (figure 2A).7–9 scRNA-Seq from this initial cohort of patients additionally shows that PEF-treated tumors have a significant increase in the proportion of plasma B cells, T cells and neutrophils, with neutrophils expressing genes indicative of functional activation (figure 2B).7 In samples from PEF-treated patients, IPA further predicts activation of ICD mechanisms, such as pyroptosis and HMGB1 signaling (figure 3A,B).4 10 After this initial innate immune response, IPA predicts activation of Th2 signaling, responsible for dampening acute inflammation and orchestrating adaptive anti-tumor immunity (figure 4A).11 Flow cytometry analysis further suggests activation of adaptive immunity, as PEF samples have higher levels of circulating B cells and effector memory T cells, and lower Tregs (figure 4B). Within the tumor microenvironment, PEF tumors show increased proportion of cytotoxic CD8+ T cells, plasma B cells and tumor leukocytes overexpressing antigen-presentation genes (figure 4C).ConclusionsData from this initial cohort show the first clinical evidence that PEF may have the potential to induce inflammatory changes in the tumor microenvironment capable of engaging host innate and adaptive immune responses, which may elicit anti-tumor activity.AcknowledgementsThe authors wish to acknowledge Dr Cristina Teodosio and Julio Pozo for their assistance with the flow cytometry analysis.Trial RegistrationThe study is registered on clinicaltrials.gov (NCT04732520).ReferencesSanmamed MF, Chen L. A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization. Cell. 2018 Oct 4;175(2):313-326.M Silvestrini, S Tamakloe, C Pastori, T O’Brien, R Neal. Local Treatment with Pulsed Electric Fields Generates a Tumor Specific Response. Journal of Vascular and Interventional Radiology 2022 June;33(6 Supplement):S161-S162.C Pastori, M Wagh, E Nafie, F Murad, R Neal. Pulsed Electric Field (PEF) Ablation Invokes Different Immune Cytokine Profile and Tumor Response than Radiofrequency Thermal Ablation for Matched Ablation Volumes in the EMT6 Mouse Model. SIR 2023, Phoenix, AZ.Kroemer G, Galassi C, Zitvogel L, et al. Immunogenic cell stress and death. Nat Immunol 2022;23:487–500.Zhang Z, et al. Gasdermin E suppresses tumour growth by activating anti-tumour immunity. Nature 2020;579:415–420.Wang Q, et al. A bioorthogonal system reveals antitumour immune function of pyroptosis. Nature 2020;579:421–426.Mantovani A, Garlanda C. Humoral Innate Immunity and Acute-Phase Proteins. N Engl J Med. 2023 Feb 2;388(5):439-452.Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014 Sep 4;6(10):a016295.Gaffen SL, Jain R, Garg AV, Cua DJ. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol. 2014 Sep;14(9):585-600.Wei X, Xie F, Zhou X, et al. Role of pyroptosis in inflammation and cancer. Cell Mol Immunol 2022;19:971–992.Walker JA, McKenzie ANJ. TH2 cell development and function. Nat Rev Immunol. 2018 Feb;18(2):121-133.Xie Z, Bailey A, Kuleshov MV, Clarke DJB, Evangelista JE, Jenkins SL, Lachmann A, Wojciechowicz ML, Kropiwnicki E, Jagodnik KM, Jeon M, Ma'ayan A. Gene set knowledge discovery with Enrichr. Current Protocols, 2021;1:e90.Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol. 2010 Feb;11(2):136–40.Ethics ApprovalThis study was approved by C.E.I.M. reference 20/1615 (E.C.P.S.) (Spain); CUHK-NTEC CREC 2021.294-T (Hong Kong); CMO Arnhem-Nijmegen Region NL-number: NL76406.091.21 (Netherlands).ConsentThe patient was identified as an appropriate candidate and consented for study, standard of care procedures, and publication of the data for the Ethics Committee-approved INCITE ES trial (NCT04732520)Abstract 697 Figure 1Sample collection and time points used for examining local and systemic immune effects of PEF treatment. (A) Enrolled patients underwent a standard of care diagnostic biopsy to confirm their tumor was malignant. After malignancy was confirmed, patients in the treatment group received PEF. Tumor tissue was collected on the day of the diagnostic biopsy (Pre-PEF, labeled as ‘biopsy’ in the diagram) and on the day of tumor resection (22 ± 7 days average ± SD after PEF; labeled as ‘resected tissue’ in the diagram). Peripheral blood and serum samples were collected prior to PEF (day 0), and approximately on days 3, 10, 21, and on the day of tumor resection, as indicated by the blue (serum samples) and purple (blood samples) arrows. A CT-scan was performed 0 and prior to resection (gray arrows). Subjects in the control group received a biopsy on day 0 but no PEF, and all other samples were collected as in the treatment group. (B) Single-cell RNA-Seq (scRNA-Seq) from pre-PEF and post-PEF tumor samples (n=8 pre-PEF, n=9 post-PEF) was performed to exmaine PEF-induced changes in the relative cell frequency and gene expression of tumor immune cells.Flow cytometry and serum cytokine profiling were used to evaluate systemic changes in the immune cell populations and levels of 71 cytokines/chemokines at each timepoint relative to Day 0Abstract 697 Figure 2PEF energy transiently activates the innate immune response systemically and within the tumor microenvironment (TME). (A) Heatmap of serum cytokine Ingenuity Pathway Analysis showing the most significantly affected pathways predicted to be activated or inhibited in response to PEF energy delivery. Serum cytokines were analyzed using a 71-analyte Luminex multiplex assay (n=30 treated, n=6 control). Heatmap values correspond to Z-score activity predictions, based on the log2 ratio (PEF-treated samples/control samples) of the cytokine values at each timepoint with respect to the baseline values (Day 0). Orange and blue represent predicted activation and inhibition. respectively. A manually curated list of innate immune signaling pathways with absolute Z-score values >2 (significant) is shown, with early activation at days 3 or 10 that subsides by resection. (B) Signaling pathways enriched in genes upregulated in neutrophils from PEF-treated tumors compared to non-treated control tumors Top signaling pathways based on adjusted p-value significance from Enrichr MSigDB Hallmark 2020 are shown 12.Abstract 697 Figure 3PEF energy induces immunogenic cell death mechanisms. (A) Heatmap of serum cytokine Ingenuity Pathway Analysis showing the predicted pattern of activation of immunogenic cell death signaling pathways, pyroptosis, and HMGB1 signaling. Heatmap values correspond to Z-score activity predictions, based on the log2 ratio (PEF-treated samples/Control samples) of the cytokine values at each timepoint with respect to the baseline values (Day 0). Orange and blue represent predicted activation and inhibition, respectively. (B) Heatmap showing the relative expression of the TXNIP gene in the indicated populations obtained from scRNA-Seq. Values correspond to the normalized average expression of TXNIP in cells from biopsy samples (n=8, pre-PEF) and resected PEF-treated tumors (n=9, post-PEF). TXNIP encodes for thioredoxin-interacting protein, a major regulator of cellular redox signaling that binds the NLRP3 inflammasome and has essential functions in pyroptosis and as a tumor suppressor gene 13.Abstract 697 Figure 4Adaptive immune mechanisms induced by PEF. (A) Heatmap of serum cytokine Ingenuity Pathway Analysis showing the predicted activation pattern of the Th2 signaling pathway. Heatmap values correspond to Z-score activity predictions, based on the log2 ratio (PEF-treated samples/Control samples) of the cytokine values at each timepoint with respect to the baseline values (Day 0). Orange and blue represent predicted activation and inhibition, respectively. (B) Flow cytometry analysis of peripheral blood B cells, effector memory T cells, and Tregs in PEF-treated samples and control samples (n=3 treated, n=3 control) approximately 10 days after PEF energy delivery. (C) Cell populations showing significantly increased expression of the indicated antigen-presenting genes (HLA-DQA2, HLA-DQB1, HLA-DRB5, HLA-DRA) via scRNA-Seq in tumors treated with PEF (n=9, post-PEF) compared to biopsy samples (n=8, pre-PEF).
The great pulmonary affectation produced by the COVID-19 infection, requires a fast diagnostic tool that complements the diagnostic test by PCR and which is also useful in evaluating the progression ...of lung lesions. Since most of these are peripheral, in this consensus document we propose the use of thoracic ultrasound for early diagnosis and for the daily evaluation of the progression of lung lesions by a single explorer without the need to use the chest CT. In this consensus, it is proposed to carry out a systematic ultrasound examination of the thorax dividing it by quadrants and therefore identifying the ultrasound signs that are related to the type of parenchymal or pleural affectation that the patient has: A lines, B lines, parenchymal condensation, pleural line and pleural effusion. These findings will facilitate the decision making regarding the patient management, both when deciding the place of admission of the patient and the type of treatment to be prescribed.
Background Tertiary lymphoid structures (TLS) may develop in non-lymphoid tissues in response to a variety of different stimuli and can serve as foci for generating anti-tumor immunity.1 TLS ...formation is emerging as a strong prognostic and predictive biomarker2 associated with patient survival benefits in NSCLC.3,4Pulsed Electric Fields (PEF) have been reported to induce an immunogenic form of cell death and thus may enhance adaptive immunity in the setting of cancer. The treat-and-resect INCITE ES study enrolled adults with suspected or confirmed NSCLC stage IA2-IB (>1 to ≤4 cm) and without a history of treatment for cancer within the previous two years. Methods The INCITE ES study design includes both control and treatment groups with 8 enrolled control group subjects and 30 enrolled treatment group subjects. Treatment group subjects received PEF (AliyaTM System, GTI-00018 investigational device; Galvanize Therapeutics, San Carlos, CA) either percutaneously or endoscopically at time of biopsy prior to surgical resection. Blood, bronchoalveolar lavage (BAL) when applicable, and tissue samples were collected over the course of the study for appropriate pre- and post-PEF comparison. Serial histologic sections were obtained from an initial cohort of 12 patients (n=1 control, n=11 treatment group) on the day of surgery 17-21 days post-PEF delivery, stained for standard H&E as well as duplex stained for pan-cytokeratin (panCK) and CD20, and reviewed by an independent pathologist. Results TLS were identified and characterized according to their maturity and localization within or adjacent to the tumor (see criteria in Table 1). Intratumor TLS were observed admixed among tumor cells or within the invasive margin (figures 1 to 5), including within the cellular depletion zone induced by PEF (figures 6 and 7). Independent of tumor morphology, a significant quantity of 49.8 ± 55.8 TLS per tumor was observed post-PEF (n=11, average ± S.D.). TLS across treated tumors showed varying proportions of mature vs. immature TLS, using the criteria in Table 1. No TLS were identified in the available pre-PEF biopsy specimens (figures 2 and 8). TLS density was greater in PEF specimens compared to the non-treated control, where only three immature TLS were observed (figure 9). Conclusions This initial cohort suggests that PEF may induce the formation of TLS within the tumor, including proximal to the PEF delivery zone. The observed density and detection of mature TLS may suggest ongoing immune activity. As such, PEF has the potential to induce or enhance an immune response irrespective of tumor morphology. Trial Registration The study is registered on clinicaltrials.gov (NCT04732520). References Schumacher TN, Thommen DS. Tertiary lymphoid structures in cancer. Science. 2022;375(6576):eabf9419. Petitprez F, de Reynies A, Keung E. B cells are associated with survival and immunotherapy response in sarcoma. Nature. 2020;577(7791):556–560. Sautès-Fridman C, Petitprez F, Calderaro J. Tertiary lymphoid structures in the era of cancer immunotherapy. Nat Rev Cancer. 2019;19:307–325. Cottrell TR, Thompson ED, Forde PM. Pathologic features of response to neoadjuvant anti-PD-1 in resected non-small-cell lung carcinoma: a proposal for quantitative immune-related pathologic response criteria (irPRC). Ann Oncol. 2018;29(8):1853–1860. Ethics Approval This abstract discusses the INCITE ES clinical study. Participants gave informed consent before taking part in the study. The study obtained ethics approval from the Ethics Committee for Research with Drugs (CEIm) of the Salamanca Health Area (Salamanca, Spain, reference 20/1615 (E.C.P.S.), Committee on Research Involving Human Subjects (CMO) of Radboud University Medical Center (Nijmegen, the Netherlands, NL76406.091.21), and the Joint Chinese University of Hong Kong – New Territories East Cluster Research Ethics Committee (Hong Kong SAR, reference 2021.294-T). Abstract 702 Figure 1 Example of outlined resected tumor region and CDZ post-PEF. Tumors were resected post PEF delivery and underwent immunostaining. The duplex staining with PanCK (pink) identifies epithelial cells and CD20+ B cells (brown). Areas of dense pink coloration are tumor and the remaining tissue is non-cancerous lung parenchyma. The residual tumor area is outlined in maroon to the leading tumor edge adjacent to non-cancerous lung parenchyma. The PEF-induced cellular depletion zone (CDZ) is outlined in blue. On average, the CDZ measures 0.9 ± 0.3 cm (longest dimension) by 0.6 ± 0.2 cm (longest perpendicular dimension) after a single PEF delivery (n=9). Figure omitted. See PDF Abstract 702 Figure 2 Compilation of resected NSCLC tumors from INCITE ES study. Tumors underwent duplex PanCK and CD20+ staining. One non-treatment control specimen resected 35 days post biopsy is included (T10). Tumors after a single delivery of PEF energy were resected 17 to 21 days after PEF. Inset images are of pre-PEF biopsy specimens with duplex stain, when available. Colored asterisk (*) in each image denotes the estimated location of the cellular depletion zone. Acronyms ACA, mACA, and SCC indicate tumor histopathology adenocarcinoma, mucinous adenocarcinoma, and squamous cell carcinoma, respectively. Figure omitted. See PDF Abstract 702 Figure 3 TLS in tumor periphery 19 days after a single PEF delivery. Representative H&E and IHC images of a right upper lobe tumor. Characterization of the TLS indicates presence of CD20+ B cells (brown), CD3+ T-cells (red), FoxP3+ regulatory T-cells (Tregs, brown), CD4+ helper T-cells (red) and cytotoxic CD8+ T-cells (brown). Higher magnification images of the area denoted by the green box in A and B show two mTLS and two immature imTLS. Asterisks (*) in images shown in A and B denote the estimated location of the cellular depletion zone. Figure omitted. See PDF Abstract 702 Figure 4 Mature and immature TLS in a tumor post-PEF energy delivery. Representative H&E and IHC images of a left lower lobe tumor. Purple-outlined (left column) and green-outlined (middle column) boxes representing mature TLS (mTLS) and immature TLS (imTLS), respectively, are higher power magnification of the specimen sampled at approximately 7 o’clock and 12 o’clock, respectively, in the wide field view (upper right). Both mTLS and imTLS are identified by a collection of dense, small CD20+ B cells (brown, middle row), generally juxtaposed with T cell aggregate (CD4+ helper T-cells (red) and cytotoxic CD8+ T-cells (brown), bottom row). The mTLS contains a germinal center (GC) discernable via H&E. The center of the GC is shown in the right column (yellow-dashed outline represents area of higher magnification) to identify dendritic cells (black arrows) and CD8+ T-cells (bright green arrows). Mature CD20+ B cells within the GC exhibit the characteristic morphology of proliferating centroblasts (cyan arrows) and centrocytes (dark green arrow). A GC may also contain tingible body macrophages (dark blue arrow) licensed for phagocytosis by follicular dendritic cells within the germinal center. Figure omitted. See PDF Abstract 702 Figure 5 Representative mature TLS located intratumorally post-PEF. Images were captured from duplex PanCK and CD20, duplex CD4 and CD8, and H&E-stained tissue sections of tumor T4. The whole tissue from the resection sample is shown with red asterisk (*) denoting the estimated location of the cellular depletion zone and the yellow dashed box indicating the mTLS within the tumor bed that is shown at higher magnification as indicated in subsequent image panels. The mTLS contains a germinal center (GC) discernable via H&E. The high magnification image of the region denoted by the dashed yellow box in the H&E image demonstrates mature CD20+ B cells within the GC exhibiting the characteristic morphology of proliferating centroblasts and centrocytes. Accumulation of CD4+ and CD8+ T cells can be seen within and surrounding the mTLS. Figure omitted. See PDF Abstract 702 Figure 6 A mature TLS located at the CDZ-lung parenchyma interface. Images were captured from duplex PanCK and CD20, duplex CD4 and CD8, and H&E-stained tissue sections of tumor T9. The whole tissue from the resection sample is shown with the red asterisk (*) denoting the estimated location of the CDZ and the black dashed box indicating the mTLS that is shown at higher magnification as indicated in subsequent image panels. The mTLS contains a germinal center (GC) discernable via H&E. The high magnification image of the region denoted by the dashed yellow box in the H&E image demonstrates mature CD20+ B cells within the GC exhibiting the characteristic morphology of proliferating centroblasts and centrocytes. Accumulation of CD4+ and CD8+ T cells can be seen within and surrounding the mTLS. Figure omitted. See PDF Abstract 702 Figure 7 A mature TLS located within the cellular depletion zone (CDZ). Images were captured from duplex PanCK and CD20, duplex CD4 and CD8, and H&E-stained tissue sections of tumor T6. The whole tissue from the resection sample is shown with the black asterisk (*) denoting the estimated location of the CDZ and the black dashed box indicating the mTLS within the CDZ that is shown at higher magnification as indicated in subsequent image panels. The mTLS contains a germinal center (GC) discernable via H&E. The high magnification image of the region denoted by the dashed red box in the H&E image demonstrates mature CD20+ B cells within the GC exhibiting the characteristic morphology of proliferating centroblasts and centrocytes. Accumulation of CD4+ and CD8+ T cells can be seen within and surrounding the mTLS. Figure omitted. See PDF Abstract 702 Figure 8 Representative pre-PEF tumor biopsy specimens. Images were captured from duplex PanCK and CD20 and duplex CD4 and CD8 stained tissue sections of tumors T4, T5, T7, and T8. The black dashed box indicates the region of the tissue at shown at higher magnification in the lower panels for
La gran afectación pulmonar producida por la infección del COVID-19 hace necesaria una herramienta diagnóstica rápida que complemente el test diagnóstico mediante PCR y que además sea útil en la ...evaluación de la progresión de las lesiones pulmonares. Ya que la mayoría de estas son periféricas, en este documento de consenso proponemos el uso de la ecografía torácica para el diagnóstico precoz y la evaluación diaria de la progresión de lesiones pulmonares por un solo explorador sin necesidad de utilizar la TC de tórax.
En este consenso se propone la realización de una exploración sistemática ecográfica del tórax dividiéndolo por cuadrantes e identificando los signos ecográficos que se relacionen con el tipo de afectación parenquimatosa o pleural que tiene el paciente: líneas A, líneas B, condensación parenquimatosa, línea pleural y derrame pleural. Estos hallazgos nos facilitarán la toma de decisiones respecto al manejo del paciente, tanto en la decisión del lugar de ingreso del paciente como en el tipo de tratamiento que debemos pautar.
The great pulmonary affectation produced by the COVID-19 infection, requires a fast diagnostic tool that complements the diagnostic test by PCR and which is also useful in evaluating the progression of lung lesions. Since most of these are peripheral, in this consensus document we propose the use of thoracic ultrasound for early diagnosis and for the daily evaluation of the progression of lung lesions by a single explorer without the need to use the chest CT. In this consensus, it is proposed to carry out a systematic ultrasound examination of the thorax dividing it by quadrants and therefore identifying the ultrasound signs that are related to the type of parenchymal or pleural affectation that the patient has: A lines, B lines, parenchymal condensation, pleural line and pleural effusion. These findings will facilitate the decision making regarding the patient management, both when deciding the place of admission of the patient and the type of treatment to be prescribed
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