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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.

Serial biopsy of pancreatic ductal adenocarcinoma (PDAC), to chart tumour evolution presents a significant challenge. We examined the utility of circulating free DNA (cfDNA) as a minimally invasive approach across a cohort of 55 treatment-naïve patients with PDAC; 31 with metastatic and 24 with locally advanced disease. Somatic mutations in cfDNA were detected using next generation sequencing in 15/24 (62.5%) and 27/31 (87%) of patients with locally advanced and metastatic disease, respectively. Copy number changes were detected in cfDNA of 10 patients of whom 7 exhibited gain of chromosome 12p harbouring KRAS as well as a canonical KRAS codon 12 mutation. In multivariable Cox Regression analysis, we show for the first time that patients with KRAS copy number gain and KRAS mutation have significantly worse outcomes, suggesting that this may be linked to PDAC progression. The simple cfDNA assay we describe will enable determination of the presence of KRAS copy number gain and KRAS mutations in larger studies and clinical trials.

Circulating miRNA stability suggests potential utility of miRNA based biomarkers to monitor tumor burden and/or progression, particularly in cancer types where serial biopsy is impractical. Assessment of miRNA specificity and sensitivity is challenging within the clinical setting. To address this, circulating miRNAs were examined in mice bearing human SCLC tumor xenografts and SCLC patient derived circulating tumor cell explant models (CDX). We identified 49 miRNAs using human TaqMan Low Density Arrays readily detectable in 10 μl tail vein plasma from mice carrying H526 SCLC xenografts that were low or undetectable in non‐tumor bearing controls. Circulating miR‐95 measured serially in mice bearing CDX was detected with tumor volumes as low as 10 mm3 and faithfully reported subsequent tumor growth. Having established assay sensitivity in mouse models, we identified 26 miRNAs that were elevated in a stage dependent manner in a pilot study of plasma from SCLC patients (n = 16) compared to healthy controls (n = 11) that were also elevated in the mouse models. We selected a smaller panel of 10 previously reported miRNAs (miRs 95, 141, 200a, 200b, 200c, 210, 335#, 375, 429) that were consistently elevated in SCLC, some of which are reported to be elevated in other cancer types. Using a multiplex qPCR assay, elevated levels of miRNAs across the panel were also observed in a further 66 patients with non‐small cell lung, colorectal or pancreatic cancers. The utility of this circulating miRNA panel as an early warning of tumor progression across several tumor types merits further evaluation in larger studies., Highlights Simple miRNA assay developed suitable for plasma volumes as low as 10 μl.Assay suitable for serial weekly monitoring of human xenograft growth in mice.Can detect xenograft tumor volumes as low as 10 mm3 and correlates with tumor growth.Selected miRNAs elevated in SCLC, NSCLC, colorectal and pancreatic cancers.

IntroductionSCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring.MethodsWe applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran’s I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample.ResultsGenome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival.ConclusionsWe demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.

Background: 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO) is a water-soluble mitochondrial toxin that binds to adenine nucleotide translocase in the inner mitochondrial membrane, thereby targeting cell proliferation. This phase 1 study investigated safety, dose-limiting toxicities (DLTs), maximum tolerated dose (MTD) and pharmacokinetics (PK) of GSAO as a daily 1-h infusion for 5 days a week for 2 weeks in every three. Pharmacodynamics of GSAO was evaluated by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and circulating markers of angiogenesis. Methods: Patients with advanced solid tumours received GSAO in a dose-escalation trial according to a standard '3 + 3' design that was guided by toxicity and, for the final dose escalation, by arsenic PK data. Results: A total of 34 patients were treated with GSAO across 9 dose levels (1.3-44.0 mg/m2). Treatment was well tolerated with few adverse events. An additional three patients were enrolled at the 12.4 mg/m 2 dose level following a DLT of derangement of liver function tests (grade 4). At the 44.0 mg/m2 dose level, two out of three patients had DLTs (reversible encephalopathy; paroxysmal atrial fibrillation). Conclusions: The MTD of GSAO was 22.0 mg/m2/day. There was no biomarker evidence from DCE-MRI or circulating markers of angiogenesis of an anti-vascular effect of GSAO. © 2013 Springer-Verlag Berlin Heidelberg.

Molecular information obtained from cancer patients' blood is an emerging and powerful research tool with immense potential as a companion diagnostic for patient stratification and monitoring. Blood, which can be sampled routinely, provides a means of inferring the current genetic status of patients' tumours via analysis of circulating tumour cells (CTCs) or circulating tumour DNA (ctDNA). However, accurate assessment of both CTCs and ctDNA requires all blood cells to be maintained intact until samples are processed. This dictates for ctDNA analysis EDTA blood samples must be processed with 4 h of draw, severely limiting the use of ctDNA in multi‐site trials. Here we describe a blood collection protocol that is amenable for analysis of both CTCs and ctDNA up to four days after blood collection. We demonstrate that yields of circulating free DNA (cfDNA) obtained from whole blood CellSave samples are equivalent to those obtained from conventional EDTA plasma processed within 4 h of blood draw. Targeted and genome‐wide NGS revealed comparable DNA quality and resultant sequence information from cfDNA within CellSave and EDTA samples. We also demonstrate that CTCs and ctDNA can be isolated from the same patient blood sample, and give the same patterns of CNA enabling direct analysis of the genetic status of patients' tumours. In summary, our results demonstrate the utility of a simple approach that enabling robust molecular analysis of CTCs and cfDNA for genotype‐directed therapies in multi‐site clinical trials and represent a significant methodological improvement for clinical benefit., Highlights Demonstrated collection of blood in CellSave tubes preserves cfDNA up to 96 h post draw.No significant difference between standard EDTA cfDNA and preserved cfDNA.Preserved cfDNA applicable to both targeted and genome wide NGS analysis.CTCs and cfDNA can be isolated from same CellSave blood sample.Comparable copy number aberrations seen in both CTCs and ctDNA from same blood sample.

Background: OXi4503 is a tubulin-binding vascular disrupting agent that has recently completed a Cancer Research UK-sponsored phase I trial. Preclinical studies demonstrated early drug-induced apoptosis in tumour endothelial cells at 1-3 h and secondary tumour cell necrosis between 6 and 72 h. Methods: To capture both possible outcomes of OXi4503 treatment on cell death, plasma samples for analysis by M30 and M65 ELISAs, which measure different circulating forms of cytokeratin 18 as biomarkers of apoptosis and necrosis, respectively, were collected from patients entered into the trial at early (4/6 h) and later time points (24 h, day 8 and day 15). Results: OXi4503 induced a selective dose-dependent elevation in M30 antigen levels (apoptosis) at 4/6 h and a similar elevation in M65 antigen levels at 24 h (necrosis) consistent with its preclinical cell death profile. For the purposes of investigating potential biomarker relationships to patient characteristics, the trial population was divided into three groups based on radiological and clinical response: (a) early progression, (b) progressive disease and (c) stable disease (SD)/partial response. A significant increase in antigen concentrations was measured by M65 at 24 h in the SD group compared with the two other groups (P=0.015, mean increase 30.9%). Conclusion: These results provide pharmacodynamic evidence of drug mechanism of action in cancer patients and highlight the M65 ELISA as a potentially useful biomarker assay of response to OXi4503. © 2012 Cancer Research UK.

Introduction: Tumor genomes can be reconstructed from the molecular information obtained from circulating cell-free DNA (cfDNA) and circulating tumor cells (CTCs) obtained from the peripheral blood of patients with cancer. The analysis of cfDNA and CTCs is a minimally invasive approach and represents a powerful research tool, with potential as a companion diagnostic for both patient stratification and monitoring. Here, we use cfDNA next-generation sequencing (NGS) analysis to assess and compare ctDNA profiles in patients with SCLC. Methods: Whole genome sequencing (WGS) libraries were prepared from cfDNA isolated from the pre-treatment peripheral blood samples from 69 patients and 32 cancer-free controls. Libraries were subjected to WGS to establish genome wide copy number aberrations (CNA) as well as targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated from CNA such as Percent Genome Altered (PGA; percentage of genomic regions altered), Z-score (measure of standard deviation) and Moran's I (measure of spatial autocorrelation). In addition CellSearch®, an epitope dependent enrichment platform was used to enumerate CTCs from a parallel blood sample. Results: An examination of CNA patterns revealed SCLC associated changes such as losses on chromosomes 3p, 5q and 17p and gains on chromosome 3q and 5p as well as amplification of MYC in 21/69 (30%) and SOX2 in 36/69 (52%) as well losses on FHIT in 40/69 (58%), RASSFI in 38/69(55%) and RB1 in 24/69 (35%) patients. A combination of three CNA metrics enabled detection of tumor associated changes in 58/64 (84%) patients, with Moran's I emerging as the most sensitive CNA metric for detecting ctDNA. Targeted NGS detected tumor associated mutations in 60/64 (94%) with TP53 mutations detected in 50 patients (83% of patients with any detectable ctDNA). Somatic mutations and CNA were detected in both limited stage SCLC (LS-SCLC, confined to 1 hemithorax) and extensive stage (ES-SCLC, with distant metastases) with statistically significant differences seen for CNA metrics and variant allele frequencies of mutations consistent with higher levels of ctDNA in ES-SCLC. However, no significant differences between ES and LS were observed in the mutation patterns with respect to DNA damage repair, RAS and PI3K and transcriptional regulation pathways between the two stages of SCLC. There was considerable overlap between the detection of ctDNA and CTC counts, with cfDNA NGS readouts detecting tumor related changes in 96% of patients and CTCs were detected only in 76% of patients. Conclusion: We have established sensitive methods for detecting ctDNA in cfDNA and combined with CTC enumeration we have an effective liquid biopsy for 98% of patients (96% in LS and 100% in ES) in this cohort. Future work will involve utilising this optimised NGS approach in an independent cohort of patients to correlate cfDNA metrics with clinical outcome. Citation Format: Sumitra Mohan, Victoria Foy, Hui Sun Leong, Pietà G. Schofield, Mahmood Ayub, Nigel K. Smith, Sudhakar Sahoo, Chang Sik-Kim, Lynsey Priest, Mathew Carter, Hedley T. Carr, Crispin Miller, Corinne Finn-Faivre, Fiona Blackhall, Dominic G. Rothwell, Caroline Dive, Gerard Brady. Detection of circulating cell-free tumor DNA (ctDNA) in patients with small cell lung cancer (SCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5602.

The radiosynthesis of a new [18F]fluoroalkylating agent, [18F]fluoroacetaldehyde, is described. It was produced using the Kornblum method by oxidation with dimethyl sulphoxide of 2-[18F]fluoroethyl p-toluenesulphonate ([18F]FETos). In these conditions the oxidation proceeds smoothly and rapidly to the selective conversion of tosyl esters of primary alcohols to aldehydes with no carboxylic acids being produced. The chemical identity of [18F]fluoroacetaldehyde was determined by comparing its chromatographic properties as well as those of its 2,4-dinitrophenylhydrazone (2,4-DNPH) derivative with those of, respectively, the standard fluoroacetaldehyde and its 2,4-DNPH derivative. Standard fluoroacetaldehyde was prepared by oxidation of fluoroethanol with pyridinium dichromate and characterized as its 2,4-DNPH derivative by mass spectrometry. To test its reactivity with amines under reductive alkylation conditions, [18F]fluoroacetaldehyde was reacted with benzylamine used as model substrate. The chemical identity of the resulting radiolabelled product was determined to be [18F]N-(2-fluoroethyl)-benzylamine by comparing its chromatographic properties with those of the synthesized standard N-(2-fluoroethyl)-benzylamine characterized by 19F and 1H NMR spectroscopy and mass spectrometry. This new fluorine-18 labelled synthon may find applications in radiolabelling peptide, protein and antibody fragments as well as in aldol condensation or in the Mannich reaction. Copyright © 2008 John Wiley & Sons, Ltd.

A potentially biomimetic synthesis of the guanidine-containing marine natural product crambescidin 359 via a double Michael addition of guanidine to a suitably functionalised bis-enone is reported.

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc.

A potentially biomimetic synthesis of the guanidine-containing marine natural product crambescidin 359 via a double Michael addition of guanidine to a suitably functionalised bis-enone is reported.

The isolation, structure determination, and solution conformation of two conformers of the cyclic heptapeptide phakellistatin 2 (cyclo-[Phe1-cis-Pro2-Ile3-Ile4-cis-Pro5-Tyr6-cis-Pro7]) isolated from the Fijian marine sponge Stylotella aurantium are reported. The conformers can be isolated separately by HPLC and are stable in methanol solution over a period of weeks as determined by NMR. Their NMR spectra and mass spectral fragmentation patterns differ significantly. Their solution conformations were determined by NOE-restrained molecular dynamics calculations and indicated that the two conformers had different folds, hydrogen bonding patterns, and solvent accessible surfaces. These factors may contribute to the independent stability of the two conformers, and may explain the variable biological activity previously reported for phakellistatin 2.

Benzo-fused model compounds 21a and 21b, resembling in structure the marine metabolite ptilomycalin A, were prepared and were shown to display significant activity against a series of cancer cell lines and to also possess a significant activity against the DNA polymerase activity of the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT). © 2001 Elsevier Science Ltd. All rights reserved. Ptilomycalin A 1 and related natural products have been the subject of considerable synthetic interest owing to their high levels of diverse biological activity. This includes cytotoxicity towards several cancer cell lines as well as antifungal and antiviral activities. 1 Despite this range of activity, only one report of the synthesis of a structural analogue of ptilomycalin A has been reported, by Hart and Grillot, which detailed the prepa- ration of the racemic bicyclic guanidine 2 2 (Scheme 1). As can be seen, ptilomycalin A can be considered as four separate components, a guanidine-containing poly- cyclic core, an ester linkage to an -hydroxyacid spacer unit and a terminal spermidine residue. In common

Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.

The isolation, structure determination, and solution conformation of two conformers of the cyclic heptapeptide phakellistatin 2 (cyclo-[Phe1-cis-Pro2-Ile3-Ile4-cis-Pro5-Tyr6-cis-Pro7]) isolated from the Fijian marine sponge Stylotella aurantium are reported. The conformers can be isolated separately by HPLC and are stable in methanol solution over a period of weeks as determined by NMR. Their NMR spectra and mass spectral fragmentation patterns differ significantly. Their solution conformations were determined by NOE-restrained molecular dynamics calculations and indicated that the two conformers had different folds, hydrogen bonding patterns, and solvent accessible surfaces. These factors may contribute to the independent stability of the two conformers, and may explain the variable biological activity previously reported for phakellistatin 2.

A potentially biomimetic synthesis of the guanidine-containing marine natural product crambescidin 359 via a double Michael addition of guanidine to a suitably functionalised bis-enone is reported.

The majority of squamous cell carcinomas of the cervix (60 to 80%) contain human papillomavirus (HPV) 16 or 18 DNA, and infection with these viruses is believed to be a major factor in the pathogenesis of the disease. These tumours tend to retain and actively transcribe HPV early region open reading frames (ORFs). The E6 and E7 proteins have been implicated in tumorigenesis owing to their prevalence in cervical. tumours and cell lines derived from them (Smotkin and Wettstein, 1986; Seedorf et al., 1987), transforming activity in vitro(DiMaio, 1991), and their ability to bind to the product of the tumour suppressor genes p53 and Rb, respectively (Dyson et al., 1989; Werness et al., 1990).

Sera from patients with cervical cancer, cervical intraepithelial neoplasia (CIN) and non-genital cancers, and from healthy individuals, were investigated for antibodies to human papilloma virus (HPV) early proteins E4, E6 and E7 and the major capsid protein LI by Western blot analysis of recombinant HPV proteins. There was a significantly higher prevalence of sera with antibodies to E6 in cervical cancer patients than in healthy individuals or in CIN or non-genital-cancer patients. Antibodies to E7 were detected in 25% of cervical-cancer patients, which is significantly higher than in HPV-associated cervical lesions or in control populations, but not significantly different from the incidence in patients with non-genital cancers. Antibodies to LI were found more frequently in CIN, while antibodies to E4 had a similar prevalence in cervical-cancer, cervical-dysplasia and non-genital-cancer groups, with 24% in the controls. The inability to detect antibodies to E6 and E7 in the majority of cervical-cancer patients limits the application of this methodology to the monitoring of HPV infection and the development of cervical cancer. However, the latter approach may be useful in combination with other assay systems which allow detection of different, including conformational, epitopes of HPV E6 and/or E7 recombinant proteins.