Suzanne E. Brooks

Clinical evaluation of cellular immunotherapy in acute myeloid leukaemia

Immunotherapy is currently under active investigation as an adjuvant therapy to improve the overall survival of patients with acute myeloid leukaemia (AML) by eliminating residual leukaemic cells following standard therapy. The graft-versus-leukaemia effect observed following allogeneic haematopoietic stem cell transplantation has already demonstrated the significant role of immune cells in controlling AML, paving the way to further exploitation of this effect in optimized immunotherapy protocols. In this review, we discuss the current state of cellular immunotherapy as adjuvant therapy for AML, with a particular focus on new strategies and recently published results of preclinical and clinical studies. Therapeutic vaccines that are being tested in AML include whole tumour cells as an autologous source of multiple leukaemia-associated antigens (LAA) and autologous dendritic cells loaded with LAA as effective antigen-presenting cells. Furthermore, adoptive transfer of cytotoxic T cells or natural killer cells is under active investigation. Results from phase I and II trials are promising and support further investigation into the potential of cellular immunotherapeutic strategies to prevent or fight relapse in AML patients.

New targets for the immunotherapy of colon cancer—does reactive disease hold the answer?

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in both men and women, posing a serious demographic and economic burden worldwide. In the United Kingdom, CRC affects 1 in every 20 people and it is often detected once well established and after it has spread beyond the bowel (Stage IIA–C and Stage IIIA–C). A diagnosis at such advanced stages is associated with poor treatment response and survival. However, studies have identified two sub-groups of post-treatment CRC patients—those with good outcome (reactive disease) and those with poor outcome (non-reactive disease). We aim to review the state-of-the-art for CRC with respect to the expression of cancer-testis antigens (CTAs) and their identification, evaluation and correlation with disease progression, treatment response and survival. We will also discuss the relationship between CTA expression and regulatory T-cell (Treg) activity to tumorigenesis and tumor immune evasion in CRC and how this could account for the clinical presentation of CRC. Understanding the molecular basis of reactive CRC may help us identify more potent novel immunotherapeutic targets to aid the effective treatment of this disease. In this review, based on our presentation at the 2012 International Society for the Cell and Gene Therapy of Cancer annual meeting, we will summarize some of the most current advances in CTA and CRC research and their influence on the development of novel immunotherapeutic approaches for this common and at times difficult to treat disease.

Optical propulsion of mammalian eukaryotic cells on an integrated channel waveguide

The optical propulsion of mammalian eukaryotic cells along the surface of an integrated channel waveguide is demonstrated. 10μm diameter polymethylmethacrylate (PMMA) spherical particles and similarly sized mammalian eukaryotic cells in aqueous medium are deposited in a reservoir over a caesium ion-exchanged channel waveguide. Light from a fibre laser at 1064nm was coupled into the waveguide, causing the polymer particles or cells to be propelled along the waveguide at a velocity which is dependent upon the laser power. A theoretical model was used to predict the propulsion velocity as a function of the refractive index of the particle. The experimental results obtained for the PMMA particles and the mammalian cells show that for input powers greater than 50mW the propulsion velocity is approximately that obtained by the theoretical model. For input powers of less than ~50mW neither particles nor cells were propelled; this is considered to be a result of surface forces (which are not considered in the theoretical model). The results are discussed in light of the potential application of optical channel waveguides for bioanalytical applications, namely in the identification and sorting of mammalian cells from mixed populations without the need for fluorescence or antibody labels.

CD66a (CEACAM1) is the only CD66 variant expressed on the surface of plasma cells in multiple myeloma: a refined target for radiotherapy trials?

The membrane protein CD66 is a member of the carcinoembryonic antigen (CEA) and immunoglobulin superfamily. CD66 proteins are expressed as a number of isoforms, also termed CEACAMs (CarcinoEmbryonic Antigen Cellular Adhesion Molecule), which have a wide range of biologically important functions including cell adhesion, cellular migration, pathogen binding and activation of signalling pathways. The CD66 isoforms are derived as mRNA splice and transcriptional variants from the CEACAM gene cluster on chromosome 19q13.1-13.2. CD66 isoforms a-d have been reported to be present on myeloid lineage cells with increasing density of expression from the promyelocyte through to mature granulocytes (Hammarstrom, 1999; Nair & Zingde, 2001) with CD66a, b, c and d equivalent to CEACAM1, 8, 6 and 3, respectively. The expression of CD66 isoforms on myeloid lineage cells in the bone marrow can be exploited as targets for therapy. Phase I and II clinical trials at our centre (Orchard et al, 2006) and others (Ringhoffer et al, 2005; Zhang & Gopal, 2008) utilize this property for the delivery of targeted radiotherapy to the bone marrow as part of the conditioning regimen for transplantation in acute leukaemias and multiple myeloma. Previous studies revealed the abnormal expression of CD66 isoforms on leukaemia and solid tumour cells (Luo et al, 1999; Kang et al, 2007; Lasa et al, 2008; Ratei et al, 2008). Indeed, we have shown the presence of CD66 antigen on normal and malignant plasma cells (Richardson et al, 2005). However, little is known about the differential expression pattern of the CD66 variants on plasma cells from patients with multiple myeloma. We performed flow cytometry on two human myeloma cell lines (U266 and ARH77) and on plasma cells from patients with multiple myeloma (Table I). Fresh bone marrow samples were obtained from patients attending Southampton General Hospital between 2007 and 2008. Mononuclear cells expressing CD138, a specific plasma cell marker, were either positively selected (EasySep magnetic separation kit) or mononuclear cells were isolated and incubated with saturating amounts of CD138 antibody (Dako UK Ltd., Cambridgeshire, UK) and a single CD66 monoclonal antibody (either CD66a: R&D Systems Europe Ltd., Abingdon, UK, CD66b:GENOVAC GmbH, Freiburg, Germany, CD66c: Santa Cruz Biotechnology Inc., Heidelberg, Germany CD66d: R&D or CD66e: AbD Serotec, Oxford, UK) to examine the expression of each CD66 isoform in every patient sample. Appropriate isotype-matched controls (AbD Serotec) were used in all experiments. Data acquisition and analysis were performed using the FACScalibur cellquest Software (Becton Dickinson UK Ltd., Oxford, UK). Expression of CD66a, but not CD66b, c, d or e, was identified on both myeloma cell lines analysed (Fig 1A) and in all five clinical bone marrow specimens (purified plasma cells or CD138 gated cells)(Figs 1B, C). Positive expression of CD66a ranged from 69% to 100% of the plasma cells with a median fluorescence ranging from 54 to 673, with no detectable expression of any of the other isoforms of CD66 (Fig 1C). Positive but weaker expression was seen with utilization of the pan CD66 antibody (CD66a/b/c/e) in primary samples (Fig 1C) and the cell lines (Fig 1A). We have shown for the first time the expression of CD66a but no other CD66 isoforms on multiple myeloma. These findings may help in the optimization of future radioimmunotherapeutic strategies by supporting the use of a monoclonal CD66a antibody for targeted radiotherapy in patients with multiple myeloma.

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

Infrequent Expression of the Cancer-Testis Antigen, PASD1, in Ovarian Cancer:

Ovarian cancer is very treatable in the early stages of disease; however, it is usually detected in the later stages, at which time, treatment is no longer as effective. If discovered early (Stage I), there is a 90% chance of five-year survival. Therefore, it is imperative that early-stage biomarkers are identified to enhance the early detection of ovarian cancer. Cancer-testis antigens (CTAs), such as Per ARNT SIM (PAS) domain containing 1 (PASD1), are unique in that their expression is restricted to immunologically restricted sites, such as the testis and placenta, which do not express MHC class I, and cancer, making them ideally positioned to act as targets for immunotherapy as well as potential biomarkers for cancer detection where expressed. We examined the expression of PASD1a and b in a number of cell lines, as well as eight healthy ovary samples, eight normal adjacent ovarian tissues, and 191 ovarian cancer tissues, which were predominantly stage I (n = 164) and stage II (n = 14) disease. We found that despite the positive staining of skin cancer, only one stage Ic ovarian cancer patient tissue expressed PASD1a and b at detectable levels. This may reflect the predominantly stage I ovarian cancer samples examined. To examine the restriction of PASD1 expression, we examined endometrial tissue arrays and found no expression in 30 malignant tumor tissues, 23 cases of hyperplasia, or 16 normal endometrial tissues. Our study suggests that the search for a single cancer-testes antigen/biomarker that can detect early ovarian cancer must continue.

Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside

The link between immune responses and cancer is evident from findings such as, a com‐ promised immune system resulting in an increased tumour incidence [1] and cancer pa‐ tient sera evidencing recognition of autologous cancer antigens [2]. The identification of tumour associated antigens (TAAs) plays a central role in our understanding of how can‐ cer cells can inhibit the immune system and how we can overcome this tumour immune suppression to break tolerance and achieve cancer destruction [3]. Antibodies reacting with TAAs on the surface of cancer cells provoke an extremely effective immune re‐ sponse [4] which can be exquisitely specific to the tumour cells present in the body. However not many surface proteins are present on tumour cells and limited otherwise in expression to healthy non-essential tissues.

Endocrine Disruption in Fish. David E. Kime

David Kime’s book Endocrine Disruption in Fish provides a clear and concise overview of the complex science surrounding one of the most reported-upon environmental news stories of the 1990s, that of ‘gender-bending’ chemicals and their effects on fish. After an introduction to environmental pollution and a simple description of endocrine disruption, the author discusses the importance of biomonitors and gives us his extremely convincing arguments as to why fish, in this situation, make the best biomarkers. The background information provided in this book is detailed and wide-ranging, something which is very helpful to the casual reader. There is a chapter summarzing the sources of aquatic pollution, describing both the origin of the pollutants and the different types of pollutants, although this section is not fully exhaustive: that would be difficult to achieve, considering there are a very large number of classes of chemicals which remain to be classified for their endocrine disrupting capability. The next chapter details modes of uptake of pollutants by fish together with the importance of bioconcentration of these substances in fish tissues. The chapters describing fish reproduction are extremely comprehensive. The author starts with a very informative introduction, describing the effects which the pollutants have upon the endocrine system and the role played by the endocrine system within the reproductive process. He moves on to discuss in more detail the disruption of hypothalamic and pituitary function, disruption of male and female reproduction, together with the effects that endocrinedisrupting chemicals have on the early life stages of fish. The book then focuses upon certain important target tissues; there is a chapter on disruption of liver function by oestrogen mimics and another chapter about how these chemicals affect thyroid and interrenal function, all important aspects of the action of these pollutants, and topics relevant to the reader which need to be covered independently from the sections on reproduction. David Kime’s book finishes with a wide-ranging discussion of the connections between fish, other wildlife and humans themselves, emphasizing the fact that fish are not isolated from other animal communities and are an important food resource (not just for humans). Important data on the chemicals tested, the fish species studied, and the doses they received are provided in an appendix and there is a large reference section followed by a species key and clear, easy-to-use index. I do feel, however, that this book would be enhanced by the addition of a glossary containing definitions of some of the terms used. I have no doubtsEndocrine Disruption in Fish will prove a valuable addition to the pollution section of many libraries, providing an excellent source of information for anyone interested in this field. This is essentially a reference text; the book takes a scholarly approach, containing reasoned arguments well supported by scientific studies. In no way does this book sensationalize or over-dramatize the facts surrounding the effects of oestrogenic chemicals on fish, something which is supported by the book’s appearance. The publication has a very plain cover; there are no glossy colour photographs, the text is simply laid out and interspersed with black and white photographs, diagrams and line drawings, all of which are relevant and helpful to the reader. Clearly this is supposed to be a ‘serious’ and not a populist guide to endocrine disruption in fish, aimed at readers wanting facts rather than supposition. The publishers say that the book is intended to “assist toxicologists having a limited background in either mammalian or fish endocrinology”; I think that the simplicity of the text coupled with its comprehensive nature and clear explanations makes it accessible to a wider audience. In my opinion, anyone interested in the scientific background to ‘gender-bending’ chemicals and their effect on the endocrine systems of wildlife and humans will find this an informative and useful reference book.