bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential use of IL-7 and IL-15 for ex vivo generation of LAK.
Authors: Filippova Svetlana Yu. , Kit Oleg I. , Sitkovskaya Anastasia O. , Zlatnik Elena Yu. , Novikova Inna A. , Vashchenko Larisa N. , Kechedzhieva Emma E. , Dashkova Irina R. , Ausheva Tatiana V. , Andreiko Elena , Saforyan Natalya S. , Vladimirova Liubov Yu
Published: May 2020

e15201

Background: A review of scientific literature has shown that IL-2 is most often used for the LAK generation, while the potential of other NK-stimulating interleukin cells remains poorly understood. In this study, we investigated the effect of IL-7 and IL-15 on ex vivo LAC generation. Methods: A fraction enriched in NK cells was isolated by magnetic cell sorting with the NK Cell Isolation Kit (#130-092-657, Miltenyi Biotec, Germany) from PBMC in 11 patients with stage II-III breast cancer without treatment. Cells were introduced into a 6-well 3x105 plate in RPMI medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA). Cytokines 40 ng/ml were added to the wells in 6 variants: 1) IL-15; 2) IL-2; 3) IL-7; 4) IL-15+IL-7; 5) IL-15+IL-7+IL-2; 6) control without cytokines. Cells were cultured at 5.0% CO2 and 37°C. Cells were counted with a hemocytometer daily for 5 days and on days 8, 9 and 10 of cultivation. Results: The number of NK cells in control samples gradually decreased: by 2 times on day 5 and by 3 times on day 10. On day 5, the number of NK cells was 1.5 times higher than in the control when cultured with IL-2, and 1.4 times higher when cultured with IL-7+IL-15. After 9 days, a statistically significant increase in the number of cells, compared to the control sample, was observed with the addition of IL-2 (1.6 times); IL-15 and IL-7+IL-15 (1.5 times). On day 10, significant differences from the control were found in most samples: the number of cells was higher in samples cultured with IL-2 and IL-7+IL-15 (1.9 times) and with IL-15 and IL-2+IL-7+IL-15 (1.7 times). IL-7 alone led to a gradual decrease in the number of cells, and on days 8, 9 and 10 it was lower than in the control samples. Conclusions: In general, the introduction of cytokines into the samples enriched with NK cells contributed to the preservation of this subpopulation on days 5-10 of cultivation. However, the use of IL-7 and IL-15, both alone and in combination, did not lead to a significant increase in LAK compared to the use of IL-2.
Proteome-wide biomarker quantification and target screening.
Authors: Quan Qimin , Geanacopoulos John , Ritchey Joshua , Clenow Mark , Wilkinson Joe , Boyce John
Published: May 2020

e15205

Background: Existing drug development programs are represented by only a few hundred protein targets. A large subset of the ~20,000 proteins encoded by the human genome remain undiscovered. Proteome-wide “druggability” screening may lead to new targets for therapeutics. Methods: The NanoMosaic platform is a digital immunoassay technology that achieves sub-pg/ml level sensitivity, whole-proteome level multiplexing capability, and 7 logs of dynamic range. The platform overcomes the sensitivity and dynamic range limitations of traditional protein arrays and mass spectrometry. Results: The NanoMosaic technology is powered by silicon nanoneedle biosensors that are densely integrated on a plate and manufactured with CMOS-compatible nanofabrication processes. Each nanoneedle is a label-free biosensor, functionalized with capture antibodies. Its scattering spectrum changes when an antigen binds to its surface. Each analyte specific sensing area consists a total of ~23k nanoneedles divided into a digital region (~20k nanoneedles) and an analog region (~3k nanoneedles). The digital nanoneedles provide the single molecule sensitivity. Therefore, at ultra-low concentration when antigens that are captured by the nanoneedles follow Poisson statistics, the number of antigens can be quantitated by counting the presence or absence of color changes of individual nanoneedles in a binary fashion. As the protein concentrations increase, the binding event counts increase accordingly and achieve saturation when all nanoneedles capture more than one protein. Above the digital saturation concentration, an adjacent section of analog nanoneedles perform quantitative analysis based on the level of color change, thus providing a wider dynamic range up to 1ug/ml. Ultrahigh level multiplex can be achieved by parallelizing the detection in a microarray format without loss of the sensitivity and dynamic range. A 20,000-plex proteome-wide study can be achieved with a total of 5 billion nanoneedles on a ~70mm by 70mm chip. Conclusions: In conclusion, proteome-wide biomarker quantification and target discovery can be performed on the NanoMosaic platform at higher sensitivity, wider dynamic range, lower cost and higher throughput than is currently possible by mass spectrometry or traditional immunoassays.
AVIDIO as a novel oncolytic immunotherapy platform for the treatment of colorectal cancer.
Authors: Almassian Bijan , Madina Bhaskara R , Chen Ju , Ye Xiaoyang , Krady Marie M , Ma Xianyong , Yarovinsky Timur O , Mason Stephen W , Nakaar Valerian , Wang Kepeng
Published: May 2020

e15210

Background: Colorectal cancer is the third deadliest of all cancers causing more than 50,000 deaths per year in the U.S. Oncolytic viruses have seen limited use for the treatment of cancers, and further improvement of these methods with immune-modulating activities may prove crucial for the effectiveness of these agents in the treatment of human malignancies. To this end, we developed an artificial virus for infectious diseases and immuno-oncology (AVIDIO) platform that employs virus-like vesicles (VLV) for both the delivery of immunomodulatory agents to tumors and oncolytic activity. Methods: The AVIDIO platform is comprised of in vitro evolved RNA-dependent RNA polymerase from an alphavirus, Semliki forest virus, and envelope glycoproteins from vesicular stomatitis virus, which together form VLVs. Both unarmed VLVs and VLVs armed with the p35 subunit of IL-12 (VLV-IL12p35), an immunomodulatory cytokine that can induce Th1-mediated immunity, were tested for oncolytic activity against various cancer cell lines, including MC38 colorectal cancer cells, in vitro. Using the MC38 syngeneic murine tumor model, we evaluated the antitumor activity of VLV-IL-12p35 in vivo. We used tumor growth measurements and analyses of tumor-infiltrating cells after consecutive treatments with VLV-IL-12p35 to monitor its antitumor and immunomodulatory activities, respectively. Results: VLV-IL-12p35 showed robust oncolytic activity against MC38 cells in vitro, killing over 80% of cells within 24 h. Treatment of intradermal MC38 tumors by intra-tumoral delivery of VLV-IL-12p35 resulted in more than 65% suppression of tumor growth within 2 weeks ( p< 0.05). VLV-IL-12p35-treated tumors also harbored significantly more CD8+ T cells, IFN-gamma-producing CD4+ T cells, and reduced numbers of Foxp3+ regulatory T cells. Conclusions: Our results show that VLV-IL-12p35 derived from the AVIDIO platform has oncolytic activity in vitro and antitumor and immunomodulatory activities in vivo. Therefore, AVIDIO is a promising platform for the delivery of immunomodulatory agents to tumors. Further optimization of the platform, including the addition of other immunomodulatory agents, is in progress to advance the AVIDIO platform to clinical applications for colorectal cancer.
R-DOTAP (Versamune): A novel enantiospecific cationic lipid nanoparticle that induces CD4 and CD8 cellular immune responses to whole protein and tumor-specific peptide antigens.
Authors: Wood Lauren Virginia , Gandhapudi Siva K , Sundarapandiyan Karuna , Bedu-Addo Frank K , Conn Gregory , Woodward Jerold G
Published: May 2020

e15211

Background: Immunotherapy approaches are limited in their ability to induce antigen-specific CD8+ T cells in vivo able to recognize and kill tumor cells. We developed a novel immunotherapy approach using enantiomerically pure, R-DOTAP cationic lipid nanoparticles and tumor-derived T cell antigens, and previously demonstrated that R-DOTAP formulations efficiently prime cytotoxic T cells through enhanced cross presentation and induction of type I interferons.[1] A phase I clinical trial of a R-DOTAP HPV16 peptide formulation confirmed induction of strong in vivo HPV-specific CD8+ cytolytic T-cells without associated systemic toxicities. In this study, we assessed R-DOTAP nanoparticle formulations containing whole protein (ovalbumin) or long multi-epitope peptides from the tumor antigen TARP (T-cell alternate reading frame protein): a 58-residue protein overexpressed in prostate and breast cancers, documented to be immunogenic in humans. Methods: R-DOTAP formulations were prepared containing ovalbumin (OVA) or TARP peptides. C57BL/6K mice were immunized with 10 μg/mouse of OVA plus R-DOTAP, CFA or sucrose on Days 0, 15 and 30. OVA-specific cellular and humoral responses following vaccination were assessed by measuring splenic CD4 and CD8 T cell IFN-γ production and circulating OVA-specific antibodies in serum. HLA-A2 transgenic mice (AAD mice) were vaccinated with long, multi-epitope TARP peptides delivered as an R-DOTAP admixture or with CFA or sucrose on Days 0 and 7. Antigen-specific T cell responses were measured by IFN-γ ELISpot assay. Results: OVA R-DOTAP formulations induced strong antigen-specific effector CD4 and CD8 immune and memory responses detected 7 and 30 days, respectively, following vaccination as well as OVA-specific antibody responses. In TARP peptide vaccinated mice, R-DOTAP formulations were able to present multiple CD8 T cell epitopes and stimulate responses that were superior to CFA. Conclusions: Our results suggest that R-DOTAP is a versatile immune activating therapy that can be formulated with long, multi-epitope tumor-derived peptides or whole proteins. R-DOTAP formulations induce quantitatively robust antigen-specific CD4 and CD8 T cells in vivo compared to well-established immune stimulants. Reference: 1.Gandhapudi SK, Ward M, Bush JP et al. Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response. J Immunol 2019;202:3524-3536
Characteristics of carrier cells associated with clinical response in children treated with adenoviral virotherapy administered in autologous marrow-derived mesenchymal cells.
Authors: Ramirez Manuel , Franco-Luzón Lidia , González-Murillo África , Melen Gustavo , Ruano David , Lassaletta Álvaro , Madero Luis
Published: May 2020

e15217

Background: We used oncolytic virotherapy carried by autologous marrow-derived mesenchymal cells to treat children with metastatic/refractory tumors (NCT01844661; 10.1016/j.ymthe.2020.01.019 and 10.1016/j.canlet.2015.11.036). Most patients did not show clinical response while a small group did. Methods: We have carried out a comprehensive study of the transcriptome of this new advanced therapy medicine (ATM) to find determinants that might be associated with clinical outcome. Results: The study of the ATM's transcriptome from a first cohort of patients identified signatures associated to biological processes in the infected carrier cells that distinguished responders from non-responders (viral infection and nucleic acid metabolism; immune regulatory routes; cell-cell adhesion; mitosis; cell metabolism; response to stress; and bone marrow fibrosis). Next, we used an approach based on systems biology and neural networks to identify candidates responsible for the differences found in gene expression signatures. We then validated the identified candidates using samples obtained from a second independent cohort of patients, by quantitative PCR and ELISA. Mesenchymal cells of the responding patients expressed significantly lower levels of the MAVS and NDRG1 genes compared with those of the non-responders after oncolytic adenoviral infection. Both genes are related to cellular antiviral responses and cellular metabolism. Accordingly, mesenchymal cells of responders produced significantly lower amounts of IL-6 and CCL-2 than non-responders’ cells. Conclusions: These results point to possible pretreatment biomarkers and the possibility of optimizing a universal version of the carrier cells for the oncolytic virus in this new ATM.
Predictive value of sonoelastography of secondary changed lymphous nodes in the assessment of effectiveness of immunotherapy.
Authors: Moiseenko Fedor Vladimirovich , Moiseyenko Vladimir , Chernobrivtceva Vera , Egorenkov Vitaliy , Zhegalina Arina , Artemeva Elizaveta
Published: May 2020

e15225

Background: With the use of existing predictors of the effectiveness of immunotherapy it is not possible to identify patients who will have a response to treatment or a rapid progression. The mechanism of the antitumor effect of the modern generation of immunotherapeutic drugs leads to significant changes in the cellular structure of the tumor and, as a consequence of this, in its physical characteristics. Sonoelastography allows to evaluate the stiffness of the tumor. Methods: The study included 17 patients with malignant neoplastic disease with the presence of secondarily affected peripheral lymph nodes (l.n.). 11/17 patients had melanoma, 2/17 - NSCLC, 3/17 - neck cancer. 8/9 - M / F, average age 61.8 years. 5/17 patients received combined immunotherapy (Ipilimumab + Nivolumab), 12/17 - anti-PD-1 (8 people Nivolumab, 4 people Pembrolizumab). Results: An ultrasound examination of the l. n. in a multimodal mode with compression elastography was performed on a Philips Epic 7 device before the first administration of the antitumor drug and every 3 weeks. Before the first objective assessment of the effect was done, in 5/17 patients was registered an increase in the size of the studied lymph nodes by 2 or more times, in 7/17 patients the size of the l. n. did not change, in 5/17 patients the affected l. n. decreased (26% - 64%).The results of control examination after 2 months of treatment: PR - 2/17, CR - 4/17, SD- 9/17, PD- 4/17.In the group with an objective response (PR, CR, SD - 13/17), the average value. St. Ratio before treatment was 5.7 ± 0.4; during treatment decreased to an average of 3.2 ± 0.5 (p < 0.05). In the group with progression during treatment, the average St. Ratio value before treatment was 4.1 ± 0.4, and during treatment increased on average to 5.7 ± 0.5 (p < 0.05).A qualitative analysis of tumor blood flow was carried out, the Resistance Index (RI) was studied. The RI in the first group of patients was 0.65 ± 0.4; after treatment, the RI increased to 0.74. In the second group of patients, the RI before and after treatment did not significantly change. Conclusions: During the immunotherapy treatment, a decrease in the stiffness of the tumor is observed, regardless of the change in size and an increase in the indices of peripheral resistance to blood flow in case of further regression or stabilization of the process. An early change in the sonographic characteristics of tumor foci can identify patients with a subsequent treatment effect, and can also distinguish true progression from pseudoprogression, which requires confirmation in larger studies.
Crosstalk between the MSI status and tumor microenvironment in colorectal cancer.
Authors: Luo Peng , Lin Anqi , Zhang Jian
Published: May 2020

e15236

Background: In recent years, cancer immunotherapy has been extensively studied, and colorectal cancer (CRC) patients have also derived clinical benefits from immunotherapy, especially CRC patients with mismatch repair deficiency (dMMR)/microsatellite instability-high (MSI-H), whose sensitivity to immune checkpoint inhibitors (ICIs) is significantly higher than that of patients with microsatellite-stable (MSS)/microsatellite instability-low (MSI-L) disease. This study suggests that patients with MSI-H CRC have a higher mutational burden and more immune cell infiltration than those with MSS/MSI-L disease. However, most studies have not systematically evaluated the immune characteristics and immune microenvironments of MSI-H and MSS/MSI-L CRC. Methods: A published CRC cohort with mutation and immunotherapy-related prognostic data was collected. We analyzed the relationship between the MSI status and prognosis of ICI treatment in an immunotherapy cohort. We then further used mutation data for the immunotherapy and The Cancer Genome Atlas (TCGA)-CRC (colon adenocarcinoma (COAD) + rectum adenocarcinoma (READ) cohorts. For mRNA expression, mutation data analysis of the immune microenvironment and immunogenicity under different MSI status was performed. Results: Compared with MSS/MSI-L CRC patients, patients with MSI-H CRC significantly benefited from ICI treatment. We found that MSI-H CRC had more immune cell infiltration, higher expression of immune-related genes and higher immunogenicity than MSS/MSI-L disease. The MANTIS score used to predict the MSI status was positively correlated with immune cells, immune-related genes, and immunogenicity. In addition, subtype analysis showed that COAD and READ might have different tumor immune microenvironments. Conclusions: MSI-H CRC may have an inflammatory tumor microenvironment and increased sensitivity to ICIs. Unlike those of MSI-H READ, the immune characteristics of MSI-H COAD may be consistent with those of MSI-H CRC. Furthermore, the possible mechanism underlying the prognostic differences among CRC patients receiving ICIs in relation to the immune microenvironment were elucidated to provide theoretical guidance for further improving the curative effect of ICIs treatment on MSI-H CRC patients in the future and solve the problems underlying why MSS/MSI-L CRC patients do not benefit from ICIs treatment.
Learning relevant H&E slide morphologies for prediction of colorectal cancer tumor mutation burden using weakly supervised deep learning.
Authors: Joshi Rohan P. , Kruger Andrew J. , Sha Lingdao , Kannan Madhavi , Khan Aly A. , Stumpe Martin
Published: May 2020

e15244

Background: Tumor mutation burden (TMB) is the number of non-synonymous mutations present in a cancer exome. In colorectal cancer (CRC), high TMB is associated with microsatellite instability (MSI), POLE mutations, and response to immunotherapy. TMB prediction from whole-slide images (WSIs) could aid workflows that determine MSI and POLE status. Deep learning has previously been used to predict MSI status from WSIs. This approach assumed the morphologies of all regions within the tumor are equally associated with MSI. Here, we predict TMB using a weakly supervised deep learning framework that relaxes this assumption and automatically learns relevant regions within the tumor that are most associated with TMB, potentially uncovering morphological associations. Methods: Weakly supervised learning methods facilitate classification of samples that contain many individual instances, only some of which are related to the sample label. Here, a given WSI has a single TMB-high or -low label and contains individual regions that may or may not be associated with TMB status. We implemented a ResNet18 attention-based, multiple-instance learning (MIL), convolutional neural network to simultaneously learn which tiles are important for prediction of the slide-level TMB and the tile features that are associated TMB-high and -low. We determined performance through 8-fold cross-validation within a Tempus dataset using a 75%-12.5%-12.5% split of ~940 WSIs for training, validation, and testing folds. Results: In the cross-validation, we observed a receiver operating characteristic area under the curve of 0.854 (95% CI 0.776-0.932), an average precision of 0.723 (95% CI 0.580-0.865), and an accuracy of 0.889 (95% CI 0.833-0.945) in the held-out test sets. Morphologies predicted as irrelevant for TMB include adipose tissue and WSI artifacts. Visualizations of model weights show morphologies determined to be most associated with TMB-high and -low, such as high tumor/lymphocyte content and vasculature/red blood cells, respectively. Conclusions: Attention-MIL shows high performance for the prediction of TMB in CRC from H&E images and potentially reveals the morphologies of CRC that are most associated with TMB. Future directions include further investigation of morphological associations, generalizing this model beyond Tempus acquired data, and re-training on the entire Tempus dataset.
PD-L1 status and efficacy of immune check-point inhibitors (ICIs) in advanced cancer patients: A pooled analysis of randomized trials.
Authors: Biello Federica , Pinato David James , Borra Gloria , Genestroni Silvia , Siffredi Guido , Repetti Ilaria , Martini Veronica , Varughese Feba mariam , Vachanaram Ajay Ram , Mora Edoardo , Nardin Simone , Bruzzi Paolo , Sica Antonio , Gennari Alessandra
Published: May 2020

e15263

Background: The introduction of immune check-point inhibitors (ICIs) in the treatment of a broad range of tumor types has led to a significant and clinically meaningful improvement in overall survival (OS) in advanced disease stages. However, the efficacy of these agents is not consistent across trials and in routine practice. The role of PD-L1 expression as a tumour-agnostic predictive correlate of response to ICIs remains unclear. We performed a pooled analysis of the efficacy of PD-1/PD-L1-targeted ICI regimens as compared to standard of care (SoC) therapy according to PD-L1 expression, based on landmark clinical studies. Methods: We searched literature databases to identify phase III randomized controlled trials that compared anti-PD-1/PD-L1 antibodies alone or in combination with chemotherapy or targeted agents against SoC therapy in the treatment of different tumor types. We reported efficacy data, in terms of OS, according to PD-L1 status. Log hazard ratios (HRs) were pooled across the studies overall and by PD-L1 status by inverse variance weighting. All statistical tests were two-sided. Results: Twenty-four studies including 17687 randomised patients with advanced lung, renal, urothelial, liver, breast, head and neck cancers and melanoma, were eligible for analysis. Efficacy according to PD-L1 immunohistochemical expression, with a 1% cutoff ( < 1% versus > 1%), was reported in 11 studies (7126 patients). Overall, ICI-containing regimes were significantly superior in terms of OS to SoC regimens (pooled HR = 0.64; 95% CI 0.60 to 0.68). When OS data were pooled according to PD-L1 expression, HR was 0.66; 95% CI 0.61 to 0.71 in PD-L1 < 1%, versus 0.62; 95% CI 0.56 to 0.68 in PD-L1 >1% (p = 0.7). Conclusions: The significant improvement in OS favoring the use of ICIs over SoC does not seem to be confined to patients with PD-L1-overexpressing tumours. The difference in efficacy according to PD-L1 status appears to be, at best, marginal.
Mechanisms of skin toxicity of paclitaxel: An in vitro preclinical assessment.
Authors: Perez-Fidalgo Jose Alejandro , Estornut Navarro Cristina , Sanz Garcia Celia , Perez-Leal Martin , Poveda Ferriols Jesus , Cervantes Andres , Milara Paya Javier , Cortijo Gimeno Julio
Published: May 2020

e15511

Background: Paclitaxel skin toxicity is a frequent side effect extensively evaluated in the clinical setting. However little is known about the preclinical mechanisms that lead to this toxicity. The endpoint of this study was to analyse the cutaneous mechanisms that drive paclitaxel toxicity in a preclinical model. Methods: Primary human keratinocytes were co-cultured with human dermal fibroblast in collagen gel under air-liquid interface conditions to generate a multilayered 3D epidermis. Paclitaxel was added to 3D epidermis at 0.3 µM, 3 µM and 30 µM and total RNA and protein was extracted after 24h of incubation. Markers of cell senescence (p21 and p53), anti-apoptotic mediators (Bcl-2), skin elasticity (tropoelastin ELN, collagen type I and fibronectin), hidratation (aquaporin 3 AQP3), oxidative stress (NOX4), antioxidant (SOD1, Nrf2), and angiogenic markers (VEGFR, eNOS) were evaluated by RT-PCR and western blot. NfKB phosphorylation was measured by western blot and inflammatory citokines IL1α, IL-6 and IL-8 were measured in cultured supernatants by ELISA. Human primary melanocytes were cultured and stimulated with paclitaxel to measure melanogenesis through the expression of of TYR, TYRP1 and DCT genes. The effect of paclitaxel on skin endothelial cell angiogenesis was measured by endothelial tube formation. Results: In human 3D keratinocytesPaclitaxel inhibited the expression of Bcl-2, and increased the expression of p53 and p21. The angiogenic markers VEGF and eNOS were decreased. The expression of oxidative stress marker NOX4 was increased and the anti-oxidant mediators Nrf2 and SOD1 were decreased. Markers of elasticity, collagen type I, fibronectin and FN1 were decreased as occurs with AQP3 hidratation marker. Paclitaxel increased the phosphorylation of NfkB and elevated the secretion of IL1α, IL-6 and IL-8 cytokines. In skin endothelial cells, paclitaxel reduced the endothelial tube formation. In melanocytes, paclitaxel increased skin pigmentation represented by the increase of RNA expression of TYR, TYRP1 and DCT genes. Conclusions: This preclinical 3D model showed that paclitaxel impacts on the expression of proteins related with angiogenics, elasticity and senescence in human kerantinocytes. Moreover higher doses of paclitaxel increased inflammation parameters and confirmed phototoxic and anti-angiogenenic effects. This preclinical model could be a valuable tool to assess skin toxicity of new antineoplastic agents.
MicroRNAs involved in immune response as prognostic markers in early and metastatic breast cancer.
Authors: Papadaki Chara , Thomopoulou Konstantina , Koronakis George , Monastirioti Alexia A , Papadaki Maria A. , Kalbakis Kostas , Agelaki Sofia , Mavroudis Dimitrios
Published: May 2020

e15528

Background: MicroRNAs (miRNAs) are involved in the regulation of immune response and have an important role in immune escape. We analyzed the expression levels of plasma miR-10b, miR-126, miR-19α, miR-20α and miR-155, which regulate tumor-immune interactions and investigated their prognostic implications in patients with early (eBC) and metastatic (mBC) breast cancer. Methods: Blood samples were obtained before treatment from 140 and 64 patients with eBC and mBC, respectively. Plasma miRNA expression levels were assessed by qRT-PCR and expression was classified as high or low according to the median values. Results: A panel of four miRNAs (miR-19a, miR-20a, miR-126 and miR-155) could discriminate eBC from mBC (AUC 0.802, p < 0.001). In early disease, miR-10b (p = 0.022) and miR-155 (p = 0.005) expression was lower in relapsed (n = 46) compared to non-relapsed (n = 94) patients; miR-155 expression along with lymph node infiltration and tumor grade had increased ability to predict relapse (AUC = 0.775; p = 0.003). In addition, miR-10b (p = 0.015), miR-19a (p = 0.003), miR-20a (p = 0.012), miR-126 (p = 0.001) and miR-155 (p < 0.001) expression levels were lower in patients with early relapse (relapse at ≤2 years). MiR-155 and miR-19a had the highest performance in discriminating early relapse (AUC 0.855; p < 0.001 and AUC = 0.729; p = 0.003, respectively), whereas, combined mir-155 and miR-19a expression further increased the accuracy of prediction (AUC = 0.867; p < 0.001). In eBC, the number of infiltrated lymph nodes and low miR-10b independently predicted for shorter DFS (p = 0.001 and p = 0.03, respectively) and axillary lymph nodes for shorter OS (p = 0.003). In the triple negative subgroup, low miR-155 strongly predicted for shorter DFS (p = 0.037). In mBC, recurrent disease and low miR-10b expression independently predicted for shorter PFS (p = 0.001 and p = 0.017, respectively), whereas performance status of 2 independently predicted for shorter OS (p = 0.03). Conclusions: Deregulated expression of circulating miRNAs involved in tumor-immune interactions can discriminate disease status in BC and independently predicts for patients’ outcome in early and mBC. Our results further support the notion that circulating miRNAs represent a useful prognostic tool in patients with BC.
Liquid biopsy of lung cancer by deep learning and spectroscopic analysis of circulating exosomes.
Authors: Shin Hyunku , Oh Seunghyun , Hong Soonwoo , Kang Minsung , Kang Daehyeon , Ji Yong-gu , Choi Byeong Hyeon , Kang Ka Won , Jeong Hyesun , Park Yong , Hong Sunghoi , Kim Hyun Koo , Choi Yeonho
Published: May 2020

e15532

Background: Lung cancer has a high mortality rate because of belated diagnosis at advanced stages beyond the treatable condition. Early detection of lung cancer can improve the survival rate. A liquid biopsy that detects tumor-related biomarkers in body fluids has a great potential for the purpose. Particularly, tumor-derived exosomes in blood have been proposed as a promising biomarker. The tumor-derived exosomes carry molecules of their parental cells; thus, they provide information about the tumor in the body. Unfortunately, exosomal markers conducive to the early detection of lung cancer are still obscure. Therefore, using the molecular fingerprint of exosomes markers can be useful to detect the tumor exosomes. Raman spectroscopy is one of the representative methods for the purpose. However, because the exosomes have a heterogeneous composition in blood, interpreting their spectroscopic signals is hard. Thus, we utilized a deep learning approach to analyze the spectroscopic signal of the exosomes for liquid biopsy of lung cancer. Methods: The basic concept was to evaluate how much the exosomes in human plasma resemble cancer cell exosomes. As a proof of concept, exosomes of 43 non-small cell lung cancer (NSCLC) adenocarcinoma patients and 20 healthy controls were isolated from plasma of peripheral blood. Also, cell exosomes were isolated from culture media of adenocarcinoma cell lines and a human pulmonary alveolar epithelial cell line. Then, the spectroscopic signals were detected using surface-enhanced Raman spectroscopy (SERS). Further, the deep learning algorithm was employed to classify the signals. Then, we calculated the relative similarity to cancerous exosomes against human plasma exosomes. Results: Our method was able to classify cancer and normal cell exosomes with 95% accuracy. Also, Raman signals of cancer patients’ exosomes were more similar to the cancer cell exosomes than those of healthy controls. Notably, the similarity was proportional to cancer stages. Importantly, our method even detected stage I patients. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was 0.912 for stage I and II, and 0.910 for stage I. Conclusions: We reported a novel diagnostic method using deep learning analysis against spectroscopic signals of circulating exosomes. Our method that evaluates the similarity to cancer exosomes accurately identified lung cancer patients, even stage I with high accuracy.
Biomarkers for predicting cancer in women with a suspicious mammogram.
Authors: Espina Virginia A. , Vuong Ngoc , Luchini Alessandra , Mueller Claudius , Mack Denitra S , Smith Michael , May Jennifer , Liotta Lance A. , Hoefer Richard A.
Published: May 2020

e15533

Background: Biomarker identification for early breast cancer diagnosis is confounded by comparing healthy control patients to patients undergoing surgical procedures and stress of a potential cancer diagnosis. We implemented a clinical research protocol that combines biomarker harvesting and identification with Breast Imaging-Reporting and Data System (BIRADS) results, within a cohort of women with a suspicious mammogram who donated samples prior to biopsy. The primary goals were to discover candidate novel plasma markers for stage I breast cancer versus benign lesions, and validate the markers by mass spectrometry and immunohistochemistry. Methods: 150 women found on screening mammography to have a BIRADS IV or V mammographic abnormality were enrolled in the IRB approved study, with one year follow-up. After informed consent, serum, plasma, and saliva specimens were obtained and frozen. The patient underwent image guided biopsy of the mammographic abnormality. Hydrogel nanoparticles were used to harvest and concentrate low abundance protein biomarkers from plasma. Proteins were identified by mass spectrometry. The BIRADS score and biopsy outcome were blinded to the laboratory researchers. Results: 37/150 women (median age 64, 73% ER+, 70% PR+) were diagnosed with biopsy-proven breast cancer. 15/37 had a family history of breast cancer. Within the context of stress of an abnormal mammogram and invasive biopsy, we identified 5478 plasma peptides. A model to predict endpoints that discriminate cancer vs no cancer was developed using cross-validation and lasso shrinkage method. The best fit multi-analyte ROC/AUC model of peptide spectral matches revealed 10 candidate peptides, including PLAA, TRAPPC9, PROS1, DDX41, ANKRD63, EGFLAM (AUC = 0.81), that discriminated cancer versus no cancer. The functional mechanisms of these proteins are calcium metabolism, GPI anchor biosynthesis, neural-immune crosstalk, DNA repair, and ubiquitin-mediated protein trafficking. Conclusions: Molecular profiling of blood can potentially complement imaging to improve diagnostic specificity in the setting of a suspicious mammogram. This unique trial design, enhanced by nanotechnology protein harvesting, identified potential novel cancer biomarkers in the presence of a suspicious mammogram. A confirmation trial is underway.
Potential utility of methylation levels detected from circulating tumor DNA (ctDNA) in predicting molecular residual disease (MRD) in patients with resected non-small cell lung cancer (NSCLC).
Authors: HU Hong , Li Hang , Ma Zelin , Xiang Jiaqing , Zhang Yawei , Lizaso Analyn , Hou Ting , Guan Xiaoqian
Published: May 2020

e15535

Background: To improve the prognosis of resected lung cancer patients, growing efforts are being invested in finding the most optimal approach to detect MRD and predict relapse. In our study, we aimed to investigate the utility of ctDNA methylation profiling in detecting MRD from patients with resected early-stage NSCLC. Methods: Surgically-resected tumor tissues were obtained from 65 patients diagnosed with resectable stage IA-III NSCLC with various histological subtypes. Matched blood samples were also collected before surgery (baseline) and during regular follow-up after 2-8 weeks of surgery. Comprehensive somatic mutation and methylation level profile from circulating tumor DNA (ctDNA) were performed using unique molecular identifier-based targeted sequencing and targeted bisulfite sequencing, respectively. A tumor-informed MRD prediction model was constructed based on methylation levels obtained from the patient’s resected tumor tissue to calculate the corresponding methylation signal intensity from the matched plasma sample of the patient at baseline or other time points during follow-up, which is reflected as MRD score. Results: Of the 28 patients with baseline ctDNA methylation data, 28.6% (8/28) of the patients had elevated ctDNA methylation levels at first post-operative follow-up (F1) as compared to baseline, indicating the possibility of MRD. Meanwhile, of the 20 patients (71.4%, 20/28) who had reduced ctDNA methylation levels at F1, elevation of ctDNA methylation level was detected from 3 and 1 patients at second (F2) and third (F3) follow-up, respectively. Based on the MRD prediction model, 17.9% (5/28) of the patients had higher MRD scores at F1. Of the 23 patients with lower MRD scores at F1, 5, 1 and 1 patients had an elevation in MRD scores at F2, F3, and F4, respectively, which indicates a possible MRD. Disease relapse was radiologically confirmed after 10-16 months post-surgery in three patients with concomitant elevation of ctDNA methylation level and MRD score during follow-up between 2-9 months prior to radiologic relapse. Conclusions: Our results demonstrate that post-operative ctDNA methylation levels could be used to detect MRD in patients with resected NSCLC. Moreover, ctDNA methylation-based prediction model of MRD could serve as a potential model to predict relapse in early-stage as well as disease progression in advanced-stage NSCLC.
Detection of pancreatic cancer using a novel blood-based DNA methylation signature.
Authors: Munugala Nishant , Hammad Hytham , Vrba Lukas , Oshiro Marc , Wertheim Betsy , Roe Denise , Gavini Hemanth , Latura Leah , Pennington Daniel , Dalgai Shelby , Babiker Hani M. , Elquza Emad , Scott Aaron James , Nelson Mark , Futscher Bernard W. , Shroff Rachna T.
Published: May 2020

e15546

Background: Pancreatic cancer (PC) is a high mortality malignancy typically found when curative surgery is not an option. Liquid biopsies are a minimally invasive option that may allow earlier detection of PC. Using a bioinformatic analysis of TCGA and GEO we identified a novel set of blood-based biomarkers to detect PC with high sensitivity and specificity (AUROC = 0.999) (Vrba et al, Epigenetics 2019). Here we tested the ability of this DNA methylation signature (MS) to distinguish metastatic PC from pancreatic cysts (Cy) and healthy controls (HC) through quantitative DNA methylation analysis of cell free DNA (cfDNA). Methods: A 10 gene DNA methylation marker set that identifies PC was used to evaluate the cfDNA component of the plasma of PC patients, Cy patients and HC. The ROC analysis and AUC calculations were performed using the R library pROC (Robin et al, BMC Bioinformatics 2011). Plasma samples were collected in cfDNA collection tubes from patients with benign Cy, PC, and HC. Clinical information was collected including age, gender, smoking history, CA 19-9 level, cyst size, presence of liver metastases, and survival status. After processing and cfDNA extraction, qRT-PCR methylation analysis was performed on these prospectively collected samples. The difference in median DNA MS per marker of the full 10-gene marker set between the above groups was analyzed by Wilcoxon rank sum test and ROC analysis. A Cox proportional hazard regression model was used to test the association between MS and survival. Results: A total of 66 samples were collected (PC = 10, Cy = 12, HC = 44). Clinical information was collected on all patients included in the analysis. Using liquid biopsies for cfDNA analysis and a novel set of DNA methylation biomarkers, MS was substantially stronger in PC (median, 13.2) patients than Cy (median, 3.5) patients (p = 0.0001). Importantly, while there was no clear association between MS and age, gender, smoking status, CA 19-9 levels, or presence of liver metastases, higher methylation levels were non-significantly associated with worse overall survival in PC patients (HR, 1.29; 95% CI, 0.97–1.72; p = 0.082). Conclusions: While this is a small sample size, early findings suggest that a DNA MS may reliably distinguish PC from benign Cy. The strength of signal may be a prognostic marker that could play a role in determining treatment approaches. Given its utility in predicting PC, future directions include testing this tool as a blood-based early detection mechanism as well as a predictor for recurrence or response to PC treatment.
Analytical validation of a tissue agnostic ctDNA MRD assay using tumor specific methylation and somatic variant profiles in early-stage CRC.
Authors: Artieri Carlo , Axelrod Haley , Baca Arthur , Burke Jordan , Chudova Darya , Dahdouli Mahmoud , Ghadiri Farsheed , Hartwig Anna , He Yupeng , Hite Dustin , Jaimovich Ariel , Juntilla Marisa , Kurata Jessica , Kong Yu , Nance Tracy , Ohshima Saiyou , Sharma Sweta , Westesson Oscar , Talasaz AmirAli
Published: May 2020

e15549

Background: Liquid biopsy is a candidate for detection of minimal residual disease (MRD) in early colorectal cancer. Detection of circulating tumor (ct)DNA in early stage cancer is challenging given the low abundance of ctDNA molecules. We developed a tissue agnostic MRD Test to optimize sensitivity and specificity of ctDNA detection by integrating tumor-specific genomic mutation and DNA methylation signatures coupled to noise-reduction of non-tumor background cfDNA signals. Methods: This MRD Test was developed following CLIA, Nex-StoCT Working Group, and AMP/CAP guidance and validation principles. Using a single input plasma sample, data from somatic and epigenomic targeted capture panels are integrated to generate a final test result: ctDNA detected or ctDNA not detected. ctDNA detected is defined by the presence of mutation or epigenomic classifier inputs exceeding a defined threshold. Quality control (QC) measures were implemented in both process and final sequencing results. Analytical specificity, sensitivity, and accuracy were determined using 130 samples. Results: The MRD Test specificity was 95% (70/74) using colonoscopy screened negative samples.A retrospective review of the clinical histories of these false positive samples showed high risk clinical features such as smoking, family history of CRC, and sub-optimal bowel prep prior to the colonoscopy. Analytical sensitivity (LoD) was established by diluting late stage CRC samples at two clinically relevant cfDNA inputs at three dilutions (30ng or 10ng, ranging 0.1% to 0.75% tumor fraction). The 95% LoD was determined to be 0.3% for 10ng and < 0.1% for 30ng, the lowest level tested for each input. Accuracy was determined by diluting advanced CRC samples to 0.5-0.75% MAF tested at 30ng or 10ng cfDNA with a detection result of 100% (38/38). Conclusions: We present a CLIA validated assay with performance characteristics sufficient to detect residual disease in Stage II/III CRC post-curative intent therapy. This assay is currently being used in multiple interventional clinical trials.
Lung cancer exosome specific protein 1(LESP-1) as a potential factor for diagnosis and treatment of non-small cell lung cancer.
Authors: Kim Hyun Koo , Jeong Hyesun , Choi Byeong Hyeon , Quan Yu Hua , Rho Jiyun , Park Ji-Ho , Park Yong , Choi Yeonho , Han Kook Nam , Choi Young Ho , Hong Sunghoi
Published: May 2020

e15550

Background: Exosomes are endosome-derived nano size (30-150 nm) extracellular microvesicles released from many cell types including cancer cells and encapsulated by cell membrane play a key role for cell to cell communication. Use of exosomes as a biomarkers in lung cancer is a rising nanotechnology in a liquid biopsy. We explored a role of an exosome specific marker and the relationship between pathological stage of lung cancer patients who underwent surgery and lung cancer specific exosome markers. Methods: Conditioned medium from non-small cell lung cancer (NSCLC) cells and human pulmonary alveolar endothelial cell (HPAEpiC) was collected and exosomes were isolated by size exclusion chromatography. Proteomics analysis was performed to investigate lung cancer specific proteins. Written informed consent was obtained from all human subjects (17 controls and 54 patients), approved by the Korea University Guro Hospital Institutional Review Bored. Plasma exosomes were isolated using dual size exclusion chromatography. We validated proteomics results with lung cancer exosome-specific protein 1 (LESP-1) ELISA assay and western blot assay in lung cancer patients with healthy control. Cancer cell lines with pCMV-CD63-GFP were transduced by lentiviral vectors containing LESP-1 shRNA. The distribution of GFP+ exosomes and cell migration were examined by immunocytochemistry (ICC) and cell migration assay. Results: We identified LESP1 by the proteomics analysis of exosomes from NSCLC cell lines, but not in HPAEpiC cell. Level of LESP1 was dramatically increased in exosomes from NSCLC cell lines and from NSCLC patients. LESP-1 concentration increased in lung cancer patients than healthy controls ( p < 0.0001) and increased according to the grade of lung cancer stage in peripheral blood ( p < 0.0001). Western blot results confirmed the presence of the LESP1 with higher intensity band at each grade of lung cancer stage than healthy controls. Interestingly, we found that the number of GFP+ exosomes was decreased and cell migration was inhibited when the LESP1 was suppressed in NSCLC cells. Conclusions: The LESP-1 in exosomes was highly expressed in blood plasma of lung cancer patients, and the exosome release and cancer cell migration was inhibited by the LESP-1, which suggest that LESP-1 could be a feasible factor for diagnosis and treatment of non-small cell lung cancer.
Natural killer cells and their activity as a potential biomarker for predicting response to checkpoint inhibitors in non-small cell lung cancer.
Authors: Song Paul Y. , Cho Yong-Hee , Choi Myeong Geun , Kim Dong Ha , Choi Yun Jung , Kim Seon Ye , Sung Ki Jung , Lee Jae Cheol , Kim Sang-Yeob , Rho Jin Kyung , Choi Chang-Min
Published: May 2020

e15559

Background: Despite the increased promise of checkpoint inhibitors in the treatment of non-small cell lung cancer, the majority of patients do not respond and the potential for severe toxicity is significant. Unfortunately, the predictive value of PD-L1 expression has not always proven to be definitive and the overall need to find better biomarkers that will optimize the selection of patients who will best respond to therapy remains. Recent evidence has pointed to Natural Killer (NK) cells as an essential mediator in overall response to checkpoint inhibitors. We explore whether NK cell populations and/or their activity have any correlation with response. Methods: Peripheral blood mononuclear cells (PBMCs) from nine patients with Stage IV non-small cell lung cancer (four adenocarcinoma, five squamous cell carcinoma) were collected pre and post six-weeks of checkpoint immunotherapy (six received pembrolizumab, and three received nivolumab). Overall, four of nine (44.5%) were diagnosed as SD or PR via RECIST 1.1. The immune cell composition of the PBMCs of all nine patients was analyzed using multi-variated single-cell analysis using mass cytometry (CyTOF) with an optimized 32-marker panel. Natural Killer (NK) cell activity was measured with the NKVue kit which detects NK-secreted IFN-γ levels using a quantitative sandwich ELISA from NK cells exposed to a specific recombinant cytokine. Results: The overall percentages of NK cell populations found in the immune cells of PBMCs were prominently elevated in patients who responded (SD or PR) to checkpoint therapy compared to those who had progressive disease (non-responders). While there were no significant differences in the population of other immune cells between these two groups, the overall NK cell activity in patients who responded was highly elevated compared to the NK cell activity in non-responders. From the analysis of NK subsets, there were no differences in the population of early NK cells between the two groups, but the functionally differentiated late NK cells were prominently high in the responder group. Conclusions: Natural Killer (NK) cells have been recently implicated to play a key role in anti-tumor response to checkpoint inhibitors and our results suggest that the overall number of NK cells and their activity could prove to be an independent and reliable predictive tool/biomarker in patients with NSCLC.