Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution

Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. Video Abstract [Figure presented] Development of the bioinformatics tool LOHHLA allows precise measurement of allele-specific HLA copy number, improves the accuracy in neoantigen prediction, and uncovers insights into how immune escape contributes to tumor evolution in non-small-cell lung cancer

Fc-Optimized Anti-CD25 Depletes Tumor-Infiltrating Regulatory T Cells and Synergizes with PD-1 Blockade to Eradicate Established Tumors

CD25 is expressed at high levels on regulatory T (Treg) cells and was initially proposed as a target for cancer immunotherapy. However, anti-CD25 antibodies have displayed limited activity against established tumors. We demonstrated that CD25 expression is largely restricted to tumor-infiltrating Treg cells in mice and humans. While existing anti-CD25 antibodies were observed to deplete Treg cells in the periphery, upregulation of the inhibitory Fc gamma receptor (FcγR) IIb at the tumor site prevented intra-tumoral Treg cell depletion, which may underlie the lack of anti-tumor activity previously observed in pre-clinical models. Use of an anti-CD25 antibody with enhanced binding to activating FcγRs led to effective depletion of tumor-infiltrating Treg cells, increased effector to Treg cell ratios, and improved control of established tumors. Combination with anti-programmed cell death protein-1 antibodies promoted complete tumor rejection, demonstrating the relevance of CD25 as a therapeutic target and promising substrate for future combination approaches in immune-oncology

Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution.

The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Anaerobic granular sludge and zero valent scrap iron (ZVSI) pre-treated with green tea as a sustainable system for conversion of CO2 to CH4

Global climate change is a worldwide concern that requires the dramatic reduction of greenhouse gases. Innovation in sustainable technologies for CO2 utilization to other high value products is therefore an emerging and rising field. It was found that CO2 as a sole carbon source can be converted to CH4 (higher than 97%) in a system consisted of anaerobic granular sludge, water and zero valent scrap iron. In this system (zero valent scrap iron, anaerobic granular sludge and CO2), pre-exposure of zero valent scrap iron to green tea resulted in a higher conversion rate and total CH4 production compared to bare zero valent scrap iron response. At the abiotic system (no anaerobic sludge) the zero valent scrap iron pre-treated with green tea also showed around 10% higher H2 production as well as higher final pH compared to zero valent scrap iron. The dominant crystalline product of the process both at the abiotic and at the biological system was siderite (FeCO3). It is likely that the formation of siderite on the zero valent scrap iron outer surface have reduced the amount of H2 release from zero valent scrap iron, shielding the substrate for methanogens. The presence of green tea compounds can form an iron-tannate complex with iron ions and this can possibly decrease or slow down the siderite layer, as a result more H2 was released when zero valent scrap iron was pre-exposed to green tea. Anaerobic spent filtered media was independently added to zero valent scrap iron to examine the effect of extracellular enzymes, however slight increase of the H2 production rate was found. The examined system combines potential solutions for two environmental problems; production of siderite derived from zero valent scrap iron for environmental applications and utilization of CH4 generated from CO2 for energy purposes

Polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) genotyping assay for detection of genes associated with rheumatoid arthritis and multiple sclerosis

Abstract-In this paper an assay for the detection of genes associated with rheumatoid arthritis (RA) and multiple sclerosis, using polymerase chain reaction (PCR) and sequence specific oligonucleotide probes (SSOP) is presented, in order to be further applied in a portable Lab-On-Chip (LOC) device. A substantial part of these reagents were based on the literature (11 th International Histocompatibility Workshop, IHW), bearing the advantage of proven successful implementation in genotyping, while others were designed for this study. More precisely, our methodology discriminates HLA-DRB1 as DRB1*01, *04 and *10, which include shared epitope (SE) alleles associated with RA and additionally DRB1*15 allele, including DRB1*1501 associated with MS (broad genotyping method). To further present the basic elements of the assay for high resolution genotyping of SE DRB1 alleles, we provide as an example the case of HLA-DRB1*10 alleles (HLA-DRB1*100101, *100102, *100103, *1002 and *1003). Regarding the methodology for developing a detection assay, for SNPs associated with RA or MS the basic steps are presented. DNA sequence data are obtained from IMGT/HLA and SNP database. Online software tools are used to define hybridization specificity of primers and probes towards human DNA, leading to hybridization patterns that uniquely designate a target allele and evaluate parameters influencing PCR efficiency. Respecting current technological limitations of autonomous molecular-based LOC systems the approach of broad genotyping of HLA-DRB1*01/*04/*10/*15 genes, is intended to be initially used, leaving, high resolution genotyping of SE alleles for future implementations. This method is easy to be updated and extended to detect additional associated loci with RA or MS

Microbial Electrosynthesis Inoculated with Anaerobic Granular Sludge and Carbon Cloth Electrodes Functionalized with Copper Nanoparticles for Conversion of CO2 to CH4

Microbial electrosynthesis (MES) can sustainably convert CO2 to products and significant research is currently being conducted towards this end, mainly in laboratory-scale studies. The high-cost ion exchange membrane, however, is one of the main reasons hindering the industrialization of MES. This study investigates the conversion of CO2 (as a sole external carbon source) to CH4 using membraneless MES inoculated with anaerobic granular sludge. Three types of electrodes were tested: carbon cloth (CC) and CC functionalized with Cu NPs, where Cu NPs were deposited for 15 and 45 min, respectively. During the MES experiment, which lasted for 144 days (six cycles), methane was consistently higher in the serum bottles with CC electrodes and applied voltage. The highest CH4 (around 46%) was found in the second cycle after 16 days. The system's performance declined during the following cycles; nevertheless, the CH4 composition was twice as high compared to the serum bottles without voltage. The MES with Cu NPs functionalized CC electrodes had a higher performance than the MES with plain CC electrodes. Microbial profile analysis showed that the Methanobacterium was the most dominant genus in all samples and it was found in higher abundance on the cathodes, followed by the anodes, and then in the suspended biomass. The genus Geobacter was identified only on the anodes regarding relative bacterial abundance at around 6-10%. Desulfovibrio was the most dominant genus in the cathodes; however, its relative abundance was significantly higher for the cathodes with Cu NPs

Biophysical Evaluation and In Vitro Controlled Release of Two Isomeric Adamantane Phenylalkylamines with Antiproliferative/Anticancer and Analgesic Activity

The aqueous dissolution profile of the isomeric synthetic adamantane phenylalkylamine hydrochlorides I and II was probed. These adducts have shown significant antiproliferative/anticancer activity associated with an analgesic profile against neuropathic pain. They are both devoid of toxic effects and show appreciable enzymatic human plasma stability. The structures of these two compounds have been elucidated using 2D NMR experiments, which were used to study their predominant conformations. Compound II’s scaffold appeared more flexible, as shown by the NOE spatial interactions between the alkyl bridge chain, the aromatic rings, and the adamantane nucleus. Conversely, compound I appeared very rigid, as it did not share significant NOEs between the aforementioned structural segments. MD simulations confirmed the NOE results. The aqueous dissolution profile of both molecules fits well with their minimum energy conformers’ features, which stem from the NOE data; this was nicely demonstrated, especially in the case of compound II