Phase I clinical trial evaluating the safety and efficacy of ADP-A2M10 SPEAR T cells in patients with MAGE-A10+ advanced non-small cell lung cancer.

BACKGROUND: ADP-A2M10 specific peptide enhanced affinity receptor (SPEAR) T cells (ADP-A2M10) are genetically engineered autologous T cells that express a high-affinity melanoma-associated antigen A10 (MAGE-A10)-specific T-cell receptor (TCR) targeting MAGE-A10+ tumors in the context of human leukocyte antigen (HLA)-A*02. ADP-0022-003 was a phase I dose-escalation trial that aimed to evaluate the safety and antitumor activity of ADP-A2M10 in non-small cell lung cancer (NSCLC) (NCT02592577). METHODS: Eligible patients were HLA-A*02 positive with advanced NSCLC expressing MAGE-A10. Patients underwent apheresis; T cells were isolated, transduced with a lentiviral vector containing the TCR targeting MAGE-A10, and expanded. Patients underwent lymphodepletion with varying doses/schedules of fludarabine and cyclophosphamide prior to receiving ADP-A2M10. ADP-A2M10 were administered at 0.08-0.12×109 (dose group 1), 0.5-1.2×109 (dose group 2), and 1.2-15×109 (dose group 3/expansion) transduced cells. RESULTS: Eleven patients (male, n=6; female, n=5) with NSCLC (adenocarcinoma, n=8; squamous cell carcinoma, n=3) were treated. Five, three, and three patients received cells in dose group 1, dose group 2, and dose group 3/expansion, respectively. The most frequently reported grade ≥3 adverse events were lymphopenia (n=11), leukopenia (n=10), neutropenia (n=8), anemia (n=6), thrombocytopenia (n=5), and hyponatremia (n=5). Three patients presented with cytokine release syndrome (grades 1, 2, and 4, respectively). One patient received the highest dose of lymphodepletion (fludarabine 30 mg/m2 on days -5 to -2 and cyclophosphamide 1800 mg/m2 on days -5 to -4) prior to a second infusion of ADP-A2M10 and had a partial response, subsequently complicated by aplastic anemia and death. Responses included: partial response (after second infusion; one patient), stable disease (four patients), clinical or radiographic progressive disease (five patients), and not evaluable (one patient). ADP-A2M10 were detectable in peripheral blood and in tumor tissue. Peak persistence was higher in patients who received higher doses of ADP-A2M10. CONCLUSIONS: ADP-A2M10 demonstrated an acceptable safety profile and no evidence of toxicity related to off-target binding or alloreactivity. There was persistence of ADP-A2M10 in peripheral blood as well as ADP-A2M10 trafficking into the tumor. Given the discovery that MAGE-A10 and MAGE-A4 expression frequently overlap, this clinical program closed as trials with SPEAR T cells targeting MAGE-A4 are ongoing.

The complete costs of genome sequencing: a microcosting study in cancer and rare diseases from a single center in the United Kingdom.

PURPOSE: The translation of genome sequencing into routine health care has been slow, partly because of concerns about affordability. The aspirational cost of sequencing a genome is $1000, but there is little evidence to support this estimate. We estimate the cost of using genome sequencing in routine clinical care in patients with cancer or rare diseases. METHODS: We performed a microcosting study of Illumina-based genome sequencing in a UK National Health Service laboratory processing 399 samples/year. Cost data were collected for all steps in the sequencing pathway, including bioinformatics analysis and reporting of results. Sensitivity analysis identified key cost drivers. RESULTS: Genome sequencing costs £6841 per cancer case (comprising matched tumor and germline samples) and £7050 per rare disease case (three samples). The consumables used during sequencing are the most expensive component of testing (68-72% of the total cost). Equipment costs are higher for rare disease cases, whereas consumable and staff costs are slightly higher for cancer cases. CONCLUSION: The cost of genome sequencing is underestimated if only sequencing costs are considered, and likely surpasses $1000/genome in a single laboratory. This aspirational sequencing cost will likely only be achieved if consumable costs are considerably reduced and sequencing is performed at scale.

Identification of Circulating Genomic and Metabolic Biomarkers in Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer arising from the bile ducts with a need for earlier diagnosis and a greater range of treatment options. KRAS/NRAS mutations are common in ICC tumours and 6-32% of patients also have isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutations associated with metabolic changes. This feasibility study investigated sequencing circulating tumour DNA (ctDNA) combined with metabolite profiling of plasma as a method for biomarker discovery in ICC patients. Plasma was collected from four ICC patients receiving radio-embolisation and healthy controls at multiple time points. ctDNA was sequenced using Ampliseq cancer hotspot panel-v2 on Ion Torrent PGM for single nucleotide variants (SNV) detection and with Illumina whole genome sequencing for copy number variants (CNV) and further targeted examination for SNVs. Untargeted analysis of metabolites from patient and control plasma was performed using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS). Metabolite identification was performed using multi-parameter comparisons with analysis of authentic standards, and univariate statistical analysis was performed to identify differences in metabolite abundance between patient and control samples. Recurrent somatic SNVs and CNVs were identified in ctDNA from three out of four patients that included both NRAS and IDH1 mutations linked to ICC. Plasma metabolite analysis revealed biomarker metabolites associated with ICC and in particular 2-hydroxyglutarate (2-HG) levels were elevated in both samples from the only patient showing a variant allele in IDH1. A reduction in the number of CNVs was observed with treatment. This study demonstrates that ctDNA and metabolite levels can be identified and correlated in ICC patient blood samples and differentiated from healthy controls. We conclude that combining genomic and metabolic analysis of plasma offers an effective approach to biomarker identification with potential for disease stratification and early detection studies.

Mutation burden and other molecular markers of prognosis in colorectal cancer treated with curative intent: results from the QUASAR 2 clinical trial and an Australian community-based series.

BACKGROUND: Molecular indicators of colorectal cancer prognosis have been assessed in several studies, but most analyses have been restricted to a handful of markers. We aimed to identify prognostic biomarkers for colorectal cancer by sequencing panels of multiple driver genes. METHODS: In stage II or III colorectal cancers from the QUASAR 2 open-label randomised phase 3 clinical trial and an Australian community-based series, we used targeted next-generation sequencing of 82 and 113 genes, respectively, including the main colorectal cancer drivers. We investigated molecular pathways of tumorigenesis, and analysed individual driver gene mutations, combinations of mutations, or global measures such as microsatellite instability (MSI) and mutation burden (total number of non-synonymous mutations and coding indels) for associations with relapse-free survival in univariable and multivariable models, principally Cox proportional hazards models. FINDINGS: In QUASAR 2 (511 tumours), TP53, KRAS, BRAF, and GNAS mutations were independently associated with shorter relapse-free survival (p<0·035 in all cases), and total somatic mutation burden with longer survival (hazard ratio [HR] 0·81 [95% CI 0·68-0·96]; p=0·014). MSI was not independently associated with survival (HR 1·12 [95% CI 0·57-2·19]; p=0·75). We successfully validated these associations in the Australian sample set (296 tumours). In a combined analysis of both the QUASAR 2 and the Australian sample sets, mutation burden was also associated with longer survival (HR 0·84 [95% CI 0·74-0·94]; p=0·004) after exclusion of MSI-positive and POLE mutant tumours. In an extended analysis of 1732 QUASAR 2 and Australian colorectal cancers for which KRAS, BRAF, and MSI status were available, KRAS and BRAF mutations were specifically associated with poor prognosis in MSI-negative cancers. MSI-positive cancers with KRAS or BRAF mutations had better prognosis than MSI-negative cancers that were wild-type for KRAS or BRAF. Mutations in the genes NF1 and NRAS from the MAPK pathway co-occurred, and mutations in the DNA damage-response genes TP53 and ATM were mutually exclusive. We compared a prognostic model based on the gold standard of clinicopathological variables and MSI with our new model incorporating clinicopathological variables, mutation burden, and driver mutations in KRAS, BRAF, and TP53. In both QUASAR 2 and the Australian cohort, our new model was significantly better (p=0·00004 and p=0·0057, respectively, based on a likelihood ratio test). INTERPRETATION: Multigene panels identified two previously unreported prognostic associations in colorectal cancer involving TP53 mutation and total mutation burden, and confirmed associations with KRAS and BRAF. Even a modest-sized gene panel can provide important information for use in clinical practice and outperform MSI-based prognostic models. FUNDING: UK Technology Strategy Board, National Institute for Health Research Oxford Biomedical Research Centre, Cancer Australia Project, Cancer Council Victoria, Ludwig Institute for Cancer Research, Victorian Government.

Clinically actionable mutation profiles in patients with cancer identified by whole-genome sequencing.

Next-generation sequencing (NGS) efforts have established catalogs of mutations relevant to cancer development. However, the clinical utility of this information remains largely unexplored. Here, we present the results of the first eight patients recruited into a clinical whole-genome sequencing (WGS) program in the United Kingdom. We performed PCR-free WGS of fresh frozen tumors and germline DNA at 75× and 30×, respectively, using the HiSeq2500 HTv4. Subtracted tumor VCFs and paired germlines were subjected to comprehensive analysis of coding and noncoding regions, integration of germline with somatically acquired variants, and global mutation signatures and pathway analyses. Results were classified into tiers and presented to a multidisciplinary tumor board. WGS results helped to clarify an uncertain histopathological diagnosis in one case, led to informed or supported prognosis in two cases, leading to de-escalation of therapy in one, and indicated potential treatments in all eight. Overall 26 different tier 1 potentially clinically actionable findings were identified using WGS compared with six SNVs/indels using routine targeted NGS. These initial results demonstrate the potential of WGS to inform future diagnosis, prognosis, and treatment choice in cancer and justify the systematic evaluation of the clinical utility of WGS in larger cohorts of patients with cancer.

Targeted Next-Generation Sequencing of Plasma DNA from Cancer Patients: Factors Influencing Consistency with Tumour DNA and Prospective Investigation of Its Utility for Diagnosis.

Use of circulating tumour DNA (ctDNA) as a liquid biopsy has been proposed for potential identification and monitoring of solid tumours. We investigate a next-generation sequencing approach for mutation detection in ctDNA in two related studies using a targeted panel. The first study was retrospective, using blood samples taken from melanoma patients at diverse timepoints before or after treatment, aiming to evaluate correlation between mutations identified in biopsy and ctDNA, and to acquire a first impression of influencing factors. We found good concordance between ctDNA and tumour mutations of melanoma patients when blood samples were collected within one year of biopsy or before treatment. In contrast, when ctDNA was sequenced after targeted treatment in melanoma, mutations were no longer found in 9 out of 10 patients, suggesting the method might be useful for detecting treatment response. Building on these findings, we focused the second study on ctDNA obtained before biopsy in lung patients, i.e. when a tentative diagnosis of lung cancer had been made, but no treatment had started. The main objective of this prospective study was to evaluate use of ctDNA in diagnosis, investigating the concordance of biopsy and ctDNA-derived mutation detection. Here we also found positive correlation between diagnostic lung biopsy results and pre-biopsy ctDNA sequencing, providing support for using ctDNA as a cost-effective, non-invasive solution when the tumour is inaccessible or when biopsy poses significant risk to the patient.

Possible loss of the chloroplast genome in the parasitic flowering plant Rafflesia lagascae (Rafflesiaceae).

Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ∼350× sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth (<2×) and could not recover any substantial portion of a chloroplast genome. The gene fragments we identified included photosynthesis and energy production genes (atp, ndh, pet, psa, psb, rbcL), ribosomal RNA genes (rrn16, rrn23), ribosomal protein genes (rps7, rps11, rps16), transfer RNA genes, as well as matK, accD, ycf2, and multiple nongenic regions from the inverted repeats. None of the identified plastid gene sequences had intact reading frames. Phylogenetic analysis suggests that ∼33% of these remnant plastid genes may have been horizontally transferred from the host plant genus Tetrastigma with the rest having ambiguous phylogenetic positions (<50% bootstrap support), except for psaB that was strongly allied with the plastid homolog in Nicotiana. Our inability to identify substantial plastid genome sequences from R. lagascae using multiple approaches--despite success in identifying and developing a draft assembly of the much larger mitochondrial genome--suggests that the parasitic plant genus Rafflesia may be the first plant group for which there is no recognizable plastid genome, or if present is found in cryptic form at very low levels.

Different evolutionary histories of two cation/proton exchanger gene families in plants.

BACKGROUND: Gene duplication events have been proposed to be involved in the adaptation of plants to stress conditions; precisely how is unclear. To address this question, we studied the evolution of two families of antiporters. Cation/proton exchangers are important for normal cell function and in plants, Na+,K+/H+ antiporters have also been implicated in salt tolerance. Two well-known plant cation/proton antiporters are NHX1 and SOS1, which perform Na+ and K+ compartmentalization into the vacuole and Na+ efflux from the cell, respectively. However, our knowledge about the evolution of NHX and SOS1 stress responsive gene families is still limited. RESULTS: In this study we performed a comprehensive molecular evolutionary analysis of the NHX and SOS1 families. Using available sequences from a total of 33 plant species, we estimated gene family phylogenies and gene duplication histories, as well as examined heterogeneous selection pressure on amino acid sites. Our results show that, while the NHX family expanded and specialized, the SOS1 family remained a low copy gene family that appears to have undergone neofunctionalization during its evolutionary history. Additionally, we found that both families are under purifying selection although SOS1 is less constrained. CONCLUSIONS: We propose that the different evolution histories are related with the proteins' function and localization, and that the NHX and SOS1 families are examples of two different evolutionary paths through which duplication events may result in adaptive evolution of stress tolerance.

Modularity of intrinsic disorder in the human proteome.

Predicting regions of disorder has become of increasing interest when determining protein structure and function. With approximately 33% of eukaryotic proteins having significant disordered regions, and an increasing occurrence of disorder in higher organisms, an analysis of the importance of disorder from an evolutionary perspective was clearly warranted. Focusing on the human proteome, we have studied how abundant disorder is and its relevance to protein function and structure. We have shown that disordered regions frequently appear to be independent functional units, and judged by complete association to certain protein domains, may be evolutionarily conserved. Our work also supports previous analyses on association between disorder and alternate splicing and provides support for the modularity of disorder by showing that with respect to splicing events, disordered regions frequently appear to be spliced as whole units.

Computational resources for the prediction and analysis of native disorder in proteins.

Proteomics attempts to characterise the gene products expressed in a cell or tissue via a range of biophysical techniques including crystallography and NMR and, more relevantly to this volume, chromatography and mass spectrometry. It is becoming increasingly clear that the native states of segments of many of the cellular proteins are not stable, folded structures, and much of the proteome is in an unfolded, disordered state. These proteins and their disordered segments have functionally interesting properties and provide novel challenges for the biophysical techniques that are used to study them. This chapter focuses on computational approaches to predicting such regions and analyzing the functions linked to them, and has implications for protein scientists who wish to study such properties as molecular recognition and post-translational modifications. We also discuss resources where the results of predictions have been collated, making them publicly available to the wider biological community.

Insights into the regulation of intrinsically disordered proteins in the human proteome by analyzing sequence and gene expression data.

BACKGROUND: Disordered proteins need to be expressed to carry out specified functions; however, their accumulation in the cell can potentially cause major problems through protein misfolding and aggregation. Gene expression levels, mRNA decay rates, microRNA (miRNA) targeting and ubiquitination have critical roles in the degradation and disposal of human proteins and transcripts. Here, we describe a study examining these features to gain insights into the regulation of disordered proteins. RESULTS: In comparison with ordered proteins, disordered proteins have a greater proportion of predicted ubiquitination sites. The transcripts encoding disordered proteins also have higher proportions of predicted miRNA target sites and higher mRNA decay rates, both of which are indicative of the observed lower gene expression levels. The results suggest that the disordered proteins and their transcripts are present in the cell at low levels and/or for a short time before being targeted for disposal. Surprisingly, we find that for a significant proportion of highly disordered proteins, all four of these trends are reversed. Predicted estimates for miRNA targets, ubiquitination and mRNA decay rate are low in the highly disordered proteins that are constitutively and/or highly expressed. CONCLUSIONS: Mechanisms are in place to protect the cell from these potentially dangerous proteins. The evidence suggests that the enrichment of signals for miRNA targeting and ubiquitination may help prevent the accumulation of disordered proteins in the cell. Our data also provide evidence for a mechanism by which a significant proportion of highly disordered proteins (with high expression levels) can escape rapid degradation to allow them to successfully carry out their function.

ISPIDER Central: an integrated database web-server for proteomics.

Despite the growing volumes of proteomic data, integration of the underlying results remains problematic owing to differences in formats, data captured, protein accessions and services available from the individual repositories. To address this, we present the ISPIDER Central Proteomic Database search (http://www.ispider.manchester.ac.uk/cgi-bin/ProteomicSearch.pl), an integration service offering novel search capabilities over leading, mature, proteomic repositories including PRoteomics IDEntifications database (PRIDE), PepSeeker, PeptideAtlas and the Global Proteome Machine. It enables users to search for proteins and peptides that have been characterised in mass spectrometry-based proteomics experiments from different groups, stored in different databases, and view the collated results with specialist viewers/clients. In order to overcome limitations imposed by the great variability in protein accessions used by individual laboratories, the European Bioinformatics Institute's Protein Identifier Cross-Reference (PICR) service is used to resolve accessions from different sequence repositories. Custom-built clients allow users to view peptide/protein identifications in different contexts from multiple experiments and repositories, as well as integration with the Dasty2 client supporting any annotations available from Distributed Annotation System servers. Further information on the protein hits may also be added via external web services able to take a protein as input. This web server offers the first truly integrated access to proteomics repositories and provides a unique service to biologists interested in mass spectrometry-based proteomics.

Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified.

BACKGROUND: In recent years, model based approaches such as maximum likelihood have become the methods of choice for constructing phylogenies. A number of authors have shown the importance of using adequate substitution models in order to produce accurate phylogenies. In the past, many empirical models of amino acid substitution have been derived using a variety of different methods and protein datasets. These matrices are normally used as surrogates, rather than deriving the maximum likelihood model from the dataset being examined. With few exceptions, selection between alternative matrices has been carried out in an ad hoc manner. RESULTS: We start by highlighting the potential dangers of arbitrarily choosing protein models by demonstrating an empirical example where a single alignment can produce two topologically different and strongly supported phylogenies using two different arbitrarily-chosen amino acid substitution models. We demonstrate that in simple simulations, statistical methods of model selection are indeed robust and likely to be useful for protein model selection. We have investigated patterns of amino acid substitution among homologous sequences from the three Domains of life and our results show that no single amino acid matrix is optimal for any of the datasets. Perhaps most interestingly, we demonstrate that for two large datasets derived from the proteobacteria and archaea, one of the most favored models in both datasets is a model that was originally derived from retroviral Pol proteins. CONCLUSION: This demonstrates that choosing protein models based on their source or method of construction may not be appropriate.

Does a tree-like phylogeny only exist at the tips in the prokaryotes?

The extent to which prokaryotic evolution has been influenced by horizontal gene transfer (HGT) and therefore might be more of a network than a tree is unclear. Here we use supertree methods to ask whether a definitive prokaryotic phylogenetic tree exists and whether it can be confidently inferred using orthologous genes. We analysed an 11-taxon dataset spanning the deepest divisions of prokaryotic relationships, a 10-taxon dataset spanning the relatively recent gamma-proteobacteria and a 61-taxon dataset spanning both, using species for which complete genomes are available. Congruence among gene trees spanning deep relationships is not better than random. By contrast, a strong, almost perfect phylogenetic signal exists in gamma-proteobacterial genes. Deep-level prokaryotic relationships are difficult to infer because of signal erosion, systematic bias, hidden paralogy and/or HGT. Our results do not preclude levels of HGT that would be inconsistent with the notion of a prokaryotic phylogeny. This approach will help decide the extent to which we can say that there is a prokaryotic phylogeny and where in the phylogeny a cohesive genomic signal exists.