A locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer

British Journal of Cancer (2013) 109, 2579–2586. doi:10.1038/bjc.2013.619 www.bjcancer.com
Published online 29 October 2013
First-in-human Phase I study of EZN-4176, a locked nucleic acid antisense oligonucleotide to exon 4 of the androgen receptor mRNA in patients with castration-resistant prostate cancer

D Bianchini1,2, A Omlin1,2, C Pezaro1,2, D Lorente1,2, R Ferraldeschi1,2, D Mukherji1,2, M Crespo1,2, I Figueiredo1,2, S Miranda1,2, R Riisnaes1,2, A Zivi1,2, A Buchbinder1,2, D E Rathkopf1,2, G Attard1,2, H I Scher1,2, J de Bono1,2,3,4 and D C Danila1,2,3,4

1Prostate Cancer Targeted Therapy Group and Drug Development Unit, Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, Downs Road, Sutton, Surrey, UK
2Memorial Sloan-Kettering Cancer Center (MSKCC) and Weill Cornell Medical College, Center for Prostate and Urologic Cancers, New York, NY, USA
3ENZON Pharmaceuticals Inc.; Bridgewater, NJ, USA

Correspondence: Professor J de Bono, E-mail: johann.de-bono@icr.ac.uk

Abstract
Background:

Prostate cancer remains dependent of androgen receptor (AR) signalling, even after emergence of castration resistance. EZN-4176 is a third-generation antisense oligonucleotide that binds to the hinge region (exon 4) of AR mRNA resulting in full-length AR mRNA degradation and decreased AR protein expression. This Phase I study aimed to evaluate EZN-4176 in men with castration-resistant prostate cancer (CRPC).
Methods:

Patients with progressing CRPC were eligible; prior abiraterone and enzalutamide treatment were allowed. EZN-4176 was administered as a weekly (QW) 1-h intravenous infusion. The starting dose was 0.5 mg kg−1 with a 4-week dose-limiting toxicity (DLT) period and a 3+3 modified Fibonacci dose escalation design. After determination of the DLT for weekly administration, an every 2 weeks schedule was initiated.
Results:

A total of 22 patients were treated with EZN-4176. At 10 mg kg−1 QW, two DLTs were observed due to grade 3–4 ALT or AST elevation. No confirmed biochemical or soft tissue responses were observed. Of eight patients with greater than or equal to5 circulating tumour cells at baseline, a conversion to <5 was observed in three (38%) patients. The most common EZN-4176-related toxicities (all grades) were fatigue (59%), reversible abnormalities in liver function tests ALT (41%) and AST (41%) and infusion-related reactions including chills (36%) and pyrexia (14%).
Conclusion:

Activity of EZN-4176 at the doses and schedules explored was minimal. The highest dose of 10 mg kg−1 QW was associated with significant but reversible transaminase elevation.

Keywords:

castration-resistant prostate cancer; EZN-4176; antisense oligonucleotide; phase I clinical trial

Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA

Article

European Journal of Human Genetics advance online publication 13 January 2016; doi: 10.1038/ejhg.2015.281
Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA
EJHGOpen

Fiona S Togneri1, Douglas G Ward2, Joseph M Foster3, Adam J Devall2, Paula Wojtowicz1, Sofia Alyas1, Fabiana Ramos Vasques1, Assa Oumie3, Nicholas D James4, K K Cheng5, Maurice P Zeegers6, Nayneeta Deshmukh2, Brendan O'Sullivan7, Philippe Taniere7, Karen G Spink3, Dominic J McMullan1, Mike Griffiths1 and Richard T Bryan2

1West Midland Regional Genetics Laboratory, Birmingham Women’s NHS Foundation Trust, Birmingham, UK
2Institute of Cancer & Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK
3Affymetrix UK Ltd, High Wycombe, UK
4Cancer Research Unit, University of Warwick, Coventry, UK
5School of Health and Population Sciences, University of Birmingham, Birmingham, UK
6Department of Complex Genetics, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, The Netherlands
7Department of Histopathology, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

Abstract

Urothelial bladder cancers (UBCs) have heterogeneous clinical characteristics that are mirrored in their diverse genomic profiles. Genomic profiling of UBCs has the potential to benefit routine clinical practice by providing prognostic utility above and beyond conventional clinicopathological factors, and allowing for prediction and surveillance of treatment responses. Urinary DNAs representative of the tumour genome provide a promising resource as a liquid biopsy for non-invasive genomic profiling of UBCs. We compared the genomic profiles of urinary cellular DNA and cell-free DNA (cfDNA) from the urine with matched diagnostic formalin-fixed paraffin-embedded tumour DNAs for 23 well-characterised UBC patients. Our data show urinary DNAs to be highly representative of patient tumours, allowing for detection of recurrent clinically actionable genomic aberrations. Furthermore, a greater aberrant load (indicative of tumour genome) was observed in cfDNA over cellular DNA (P<0.001), resulting in a higher analytical sensitivity for detection of clinically actionable genomic aberrations (P<0.04) when using cfDNA. Thus, cfDNA extracted from the urine of UBC patients has a higher tumour genome burden and allows greater detection of key genomic biomarkers (90%) than cellular DNA from urine (61%) and provides a promising resource for robust whole-genome tumour profiling of UBC with potential to influence clinical decisions without invasive patient interventions. Introduction

Urothelial bladder cancer (UBC) is the seventh most common cancer in Western societies with a rising global incidence.

1 Disease management poses numerous challenges because of the following:
(i) the propensity for non-muscle-invasive bladder cancer (NMIBC) to recur, necessitating long-term surveillance;
(ii) a variable risk of NMIBC progression, associated with poor 5-year survival;2, 3
(iii) a lack of proven biomarker prognosticators to identify those subsets of patients who will suffer tumour recurrence, progression and death; and (iv) the radical therapies required to treat muscle-invasive disease (MIBC).4 UBCs are thus highly heterogeneous in their clinical characteristics and this is mirrored in their genomics, characteristics of which traverse conventional grade and stage groupings.5

Typically, genomic aberrations in tumours have been characterised using formalin-fixed paraffin-embedded (FFPE) or fresh-frozen tumour tissue, with such analyses elucidating promising biomarkers and suggesting genomic signatures with potential to influence future therapeutic interventions.6, 7, 8 Identifying such genomic complexity in a non-invasive manner could be highly advantageous for facilitating the diagnosis, treatment and surveillance of patients with NMIBC or MIBC.9, 10

Genetic changes in UBCs have previously been investigated non-invasively using genetic material present in the urine. Both genetic material from exfoliated cells (which pellet upon centrifugation) and cell-free DNA (cfDNA; which remains in the supernatant following centrifugation) have been studied. Most studies to date have focused on exfoliated cells, with data giving a specific read out, for example, the presence or absence of UBC.11 Urine tests looking at genomic copy number (CN) include the FISH-based UroVysion test (Abbott, Des Plaines, IL, USA; FDA-approved UBC diagnosis),12 which uses individual exfoliated tumour cells isolated from urine, and the CGH-based BCA-1 test, which uses DNA extracted from these exfoliated cells. BCA-1 has been used to examine more detailed CN data in bladder cancer patients than that provided by UroVysion, and shows some promise.13, 14 Unfortunately, obtaining sufficient cellular material for analysis is not always possible, hindering the clinical applicability of such tests. A small number of studies have therefore also investigated urinary cfDNA for UBC analysis with mixed results, and it has previously been suggested that due to its origin, cfDNA may be enriched for tumour-specific biomarkers with reduced contamination from germline DNA of non-cancerous cells.15

cfDNA in blood plasma, arising through cancer cell death (necrotic or apoptotic cells) and actively released DNA,16, 17 has been well studied as a liquid biopsy for various solid tumours. cfDNA in urine of bladder cancer patients has also been studied in this setting.15 This nucleic acid resource has been proposed to be predominantly necrotic in origin and quantitative changes in necrotic-specific cfDNA levels have been studied to discriminate between cancer and non-cancer patients.18

In this study, we report the utilisation of Affymetrix’s OncoScan FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA) for detailed genomic profiling of UBC using matched FFPE tumour-derived DNA, cellular DNA from urine cell pellets and cfDNA from urine supernatant. We demonstrate that the complex genomics and important clinically actionable aberrations that are evident in FFPE tumour material (currently the predominant diagnostic biospecimen for solid tumours) are echoed in urinary DNAs, and that the tumour genome is enriched in cfDNA compared with cellular DNA. These data illustrate that urinary cfDNA may represent a reliable resource for non-invasive genomic profiling of bladder cancer.

Immunotherapy Two antigens are better than one

Nature Reviews Cancer | Research Highlight

Immunotherapy
Two antigens are better than one

Sarah Seton-Rogers

Nature Reviews Cancer 16,128–129(2016)doi:10.1038/nrc.2016.17

Published online
19 February 2016


T cells carrying chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) have shown remarkable efficacy against some tumour types, primarily B cell malignancies. However, the use of these engineered T cells is limited to tumours that express highly specific antigens; in most cases tumour antigens are also expressed in normal 'bystander' tissues, and T cells that target them can cause lethal side effects.

Jennie Vallis/NPG

One solution to increase the specificity of engineered T cells might be to design a new receptor that is independent of CAR and TCR pathways, but that can reliably activate a CAR for a second antigen.
Morsut, Roybal et al. first designed a modular receptor based on Notch (SynNotch receptor) that binds to a target antigen and then triggers receptor cleavage to release a transcriptional activation domain that can activate a target gene(s) of choice.
Roybal et al. then used this platform to design a system in which one tumour antigen activates SynNotch in T cells, thereby activating transcription of a CAR that recognizes a second tumour antigen, ultimately leading to T cell activation.
The authors designed a proof-of-concept experiment in Jurkat T cells using a SynNotch receptor that recognized CD19 and contained a tetracycline-transactivator (tTa) domain; the tTa domain then activated transcription of a tetracycline response element-driven CAR against mesothelin.
In culture, these Jurkat cells were only activated by tumour cells that expressed both CD19 and mesothelin, and not those expressing only one of these antigens.

They then designed a similar system in primary human CD4+ or CD8+ T cells. In these cells, they found that a SynNotch receptor containing the Gal4-VP64 transcriptional activation domain had low basal transcriptional activity, which is required to prevent the induction of CAR expression in the absence of the first antigen.

These cells expressed a SynNotch–Gal4-VP64 that bound green fluorescent protein (GFP) and a CAR against CD19. K562 leukaemia cells that expressed both GFP and CD19 activated these primary T cells effectively in vitro, but K562 cells lacking either antigen did not; only K562 cells expressing both antigens were killed by these engineered T cells. Similar results were observed with two different SynNotch–CAR systems in primary T cells that responded to tumour cell CD19 and mesothelin, or to GFP and mesothelin.

Primary human T cells with the GFP SynNotch and CD19 CAR also exhibited localized activation in tumours in vivo. Immunocompromised mice were injected with CD19+ Daudi B cell lymphoblastoid cells; cells injected in one flank were also GFP+. Following tumour development, engineered T cells were injected; the CD19 CAR was expressed only in T cells within GFP+ tumours. Furthermore, in a similar bilateral tumour model, the engineered T cells promoted clearance of established K562 xenografts expressing both antigens but not of bystander cells that lacked GFP. In addition, when bilateral models were established with K562 cells each expressing only CD19 or GFP, engineered SynNotch T cells were not activated, indicating that SynNotch activation by one cell and then CAR activation by another following T cell migration (which could lead to off-target side effects) does not occur.

“in principle it is possible to use this flexible, modular system to design more effective and safer T cell therapies”

Although much additional work is required before these types of T cell could be tested clinically, these results demonstrate that in principle it is possible to use this flexible, modular system to design more effective and safer T cell therapies for a wider range of tumour types.