Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) supports blood-based genomic profiling but is not yet routinely implemented in the setting of a phase I trials clinic. TARGET is a ...molecular profiling program with the primary aim to match patients with a broad range of advanced cancers to early phase clinical trials on the basis of analysis of both somatic mutations and copy number alterations (CNA) across a 641 cancer-associated-gene panel in a single ctDNA assay. For the first 100 TARGET patients, ctDNA data showed good concordance with matched tumor and results were turned round within a clinically acceptable timeframe for Molecular Tumor Board (MTB) review. When a 2.5% variant allele frequency (VAF) threshold was applied, actionable mutations were identified in 41 of 100 patients, and 11 of these patients received a matched therapy. These data support the application of ctDNA in this early phase trial setting where broad genomic profiling of contemporaneous tumor material enhances patient stratification to novel therapies and provides a practical template for bringing routinely applied blood-based analyses to the clinic.
Cancers of Unknown Primary (CUP) remains a diagnostic and therapeutic challenge due to biological heterogeneity and poor responses to standard chemotherapy. Predicting tissue-of-origin (TOO) ...molecularly could help refine this diagnosis, with tissue acquisition barriers mitigated via liquid biopsies. However, TOO liquid biopsies are unexplored in CUP cohorts. Here we describe CUPiD, a machine learning classifier for accurate TOO predictions across 29 tumour classes using circulating cell-free DNA (cfDNA) methylation patterns. We tested CUPiD on 143 cfDNA samples from patients with 13 cancer types alongside 27 non-cancer controls, with overall sensitivity of 84.6% and TOO accuracy of 96.8%. In an additional cohort of 41 patients with CUP CUPiD predictions were made in 32/41 (78.0%) cases, with 88.5% of the predictions clinically consistent with a subsequent or suspected primary tumour diagnosis, when available (23/26 patients). Combining CUPiD with cfDNA mutation data demonstrated potential diagnosis re-classification and/or treatment change in this hard-to-treat cancer group.
Parkinson's disease (PD) is caused by loss in nigrostriatal dopaminergic neurons and is ranked as the second most common neurodegenerative disorder. Dopamine receptor D3 is considered as a potential ...target in drug development against PD because of its lesser side effects and higher degree of neuro-protection. One of the prominent therapies currently available for PD is the use of dopamine agonists which mimic the natural action of dopamine in the brain and stimulate dopamine receptors directly. Unfortunately, use of these pharmacological therapies such as bromocriptine, apomorphine, and ropinirole provides only temporary relief of the disease symptoms and is frequently linked with insomnia, anxiety, depression, and agitation. Thus, there is a need for an alternative treatment that not only hinders neurodegeneration, but also has few or no side effects. Since the past decade, much attention has been given to exploitation of phytochemicals and their use in alternative medicine research. This is because plants are a cheap, indispensable, and never ending resource of active compounds that are beneficial against various diseases. In the current study, 40 active phytochemicals against PD were selected through literature survey. These ligands were docked with dopamine receptor D3 using AutoDock and AutoDockVina. Binding energies were compared to docking results of drugs approved by the US Food and Drug Administration against PD. The compounds were further analyzed for their absorption, distribution, metabolism, and excretion-toxicity profile. From the study it is concluded that glycyrrhetinic acid and E.resveratroloside are potent compounds having high binding energies which should be considered as potential lead compounds for drug development against PD.
Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years
. Detection of circulating tumor cells (CTCs) at ...the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study
, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.
•A comprehensive analysis of epitope discontinuity from 488 B-cell epitopes.•Regions defined as ≥3 antibody-contacting residues with gaps of ≤3 residues.•Fragments defined as antibody-contacting ...residues that are not in regions.•14% of epitopes have only one region; only 4% are truly linear, having one region and no fragments.•39.6% of regions are straight, 48.8% are curved and 11.5% are folded.
Peptide vaccines have many potential advantages over conventional ones including low cost, lack of need for cold-chain storage, safety and specificity. However, it is well known that approximately 90% of B-cell epitopes (BCEs) are discontinuous in nature making it difficult to mimic them for creating vaccines. In this study, the degree of discontinuity in B-cell epitopes and their conformational nature is examined. The discontinuity of B-cell epitopes is analyzed by defining ‘regions’ (consisting of at least three antibody-contacting residues each separated by ≤3 residues) and small fragments (antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm has been developed that classifies each region's shape as straight, curved or folded on the basis that straight and folded regions are more likely to retain their native conformation as isolated peptides. We have investigated the structures of 488 B-cell epitopes from which 1282 regions and 1018 fragments have been identified. 90% of epitopes have five or fewer regions and five or fewer fragments with 14% containing only one region and 4% being truly linear (i.e. having one region and no fragments). Of the 1282 regions, 508 are straight in shape, 626 are curved and 148 are folded.
Peptide vaccines have many potential advantages including low cost, lack of need for cold-chain storage and safety. However, it is well known that approximately 90% of B-cell Epitopes (BCEs) are ...discontinuous in nature making it difficult to mimic them for creating vaccines. To perform a detailed structural analysis of these epitopes, they needs to be mapped onto antigen structures that are complexed with antibody. In order to obtain a clean dataset of antibody-antigen complex crystal structures, a pipeline was designed to process automatically and clean the antibody related structures from the PDB. To store this processed antibody structural data, a database (AbDb) was built and made available online. The degree of discontinuity in B-cell epitopes and their conformational nature was studied by mapping epitopes in the antibody-antigen dataset. The discontinuity of B-cell epitopes was analysed by defining extended ‘regions’ (R, consisting of at least 3 antibody-contacting residues each separated by ≤ 3 residues) and small fragments (F, antibody-contacting residues that do not satisfy the requirements for a region). Secondly, an algorithm was developed to classify region shape as linear, curved or folded. Molecular dynamics simulations were carried out on isolated epitope regions (wild type and mutant peptides). The mutant peptides have been designed by mutating non-contacting and hydrophobic residues in epitopes. Two types of mutations (hy- drophobic to alanine and hydrophobic to glutamine) have been studied using molec- ular dynamics simulations. Furthermore, the effect of end-capping on wild type and mutant epitope regions has been studied. Simulation studies were carried out on 5 linear and 5 folded shape regions. Out of these, 2 epitopes (one linear and one folded), along with their mutants and derivatives, were tested experimentally for conformational stability by CD spectroscopy and NMR. The binding of isolated epitopes with antibody was also validated by ELISA and SPR.
Abstract
Cancers of Unknown Primary (CUP) comprise a heterogeneous group of metastatic cancers where the primary site is undetectable. It accounts for 2% of all new UK cancer diagnoses and is the 6th ...leading cause of cancer death. Favourable subsets of CUP exist, based on clinico-pathological features, and although these patients have improved clinical outcomes, they make up only 20% of patients with CUP. The evolution of molecular profiling and tissue-of-origin (TOO) classifiers has the potential to transform diagnosis and management of patients with CUP. Reliable molecular profiling requires good quality and quantity tumour tissue material, often scarce in patients with CUP. Liquid biopsies offer an alternative, minimally invasive approach. We developed a novel cell-free DNA (cfDNA)-based multi-omics assay combining methylation profiling with mutation and copy number alterations (CNA) detection, to enable TOO prediction and biomarker discovery to aid treatment stratification.
Methods: Two novel library preparation methods were applied to 41 CUP cfDNA samples, 80 non-cancer controls and 80 cfDNA samples from known tumour types (breast, lung, colorectal cancer): 1) Targeted (641 gene panel) next generation sequencing (NGS); 2) A multiplex genome-wide methylation capture assay (T7-MBDSeq). Both sequencing outputs can estimate CNA. Cancer Genome Interpreter (CGI) was used to evaluate the potential actionability of molecular alterations detected. A novel TOO classifier, generated utilising in silico dilution mixture sets derived from The Cancer Genome Atlas (TCGA) methylation data, demonstrated an AUROC of 0.96 across 15 different cancer groups. This was applied to cfDNA methylation data from known tumour types to test performance accuracy and subsequently applied to CUP cfDNA samples.
Results: Multi-omics profiling from cfDNA was completed for 34/41 patients with CUP. cfDNA mutation profiling and estimated CNA analysis so far revealed >350 alterations across 225 genes. 17/34 (50%) of patients harboured potentially actionable alterations by CGI and, where performed, 68% of tumour mutations were concordant in the cfDNA. cfDNA methylation profiling revealed tumour-specific methylation changes in breast, lung and colorectal cancers. Our preliminary TOO classifier performance in these tumour types had an overall sensitivity and specificity of 85% and 98%, respectively. In patients with CUP, a predicted TOO was made in 26/41 (63%) patients and for 6 patients, where a subsequent primary tumour diagnosis was made, the TOO prediction was correct.
Conclusions: cfDNA multi-omics profiling revealed potential therapeutic benefit in 77% of this retrospective CUP cohort and illustrates the potential to re-classify patients to primary tumour types where alternative therapies may improve clinical outcomes. Further multi-omics cfDNA profiling of the CUP cohort and validation in known primary cancer types are ongoing.
Citation Format: Alicia-Marie Conway, Alexandra Clipson, Francesca Chemi, Simon Pearce, Katarzyna Kamieniecka, Saba Ferdous, Sophie Richardson, Elaine Kilgour, Matthew G. Krebs, Alastair Kerr, Dominic Rothwell, Natalie Cook, Caroline Dive. cfDNA multi-omics profiling and tissue of origin predictions in cancers of unknown primary abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2808.
Reptin functions in a wide range of biological processes including chromatin remodelling, nucleolar organization and transcriptional regulation of WNT signalling. As β-catenin dependent ...transcriptional repression and activation events involve binding of Reptin and histone deacetylase 1 to APPL endocytic proteins, this complex has become an important target to identify molecules governing endocytic processes and WNT signalling. Here, we describe the structural basis of APPL binding to Reptin to explore their mode of binding in context with APPL1/APPL2 dimerization. There is an evidence that both PH and BAR domains of APPL proteins exhibit alternately conserved regions involved in hetero-dimerization process and our in-silico data also corroborate this fact. Moreover, APPL2(PH) domain binds to the BAR domain region encompassing a nuclear localization signal. We conclude that APPL(PH) binding to BAR domain and Reptin is mutually exclusive which regulates the nucleocytoplasmic shuttling of Reptin. Furthermore, Reptin is unable to bind with membrane-associated APPL proteins. These observations were further expanded by experimental approaches where we identified a novel point mutation D316N lying in the APPL1(PH) domain which resulted in a significantly reduced binding with Reptin. By luciferase assays, we observed that overexpression of APPL1(D316N) and APPL1(WT) stimulated β-catenin/TCF dependent transcriptional activity in a similar manner which suggested that binding of Reptin to APPL1 is not necessary for β-catenin dependent target gene expression. Overall, our data attempt to highlight a comparative role of APPL proteins in controlling β-catenin dependent transcription mechanism which may improve our understanding of gene regulation.
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