Cindy Lee

Weighted Frequent Gene Co-expression Network Mining to Identify Genes Involved in Genome Stability

Gene co-expression network analysis is an effective method for predicting gene functions and disease biomarkers. However, few studies have systematically identified co-expressed genes involved in the molecular origin and development of various types of tumors. In this study, we used a network mining algorithm to identify tightly connected gene co-expression networks that are frequently present in microarray datasets from 33 types of cancer which were derived from 16 organs/tissues. We compared the results with networks found in multiple normal tissue types and discovered 18 tightly connected frequent networks in cancers, with highly enriched functions on cancer-related activities. Most networks identified also formed physically interacting networks. In contrast, only 6 networks were found in normal tissues, which were highly enriched for housekeeping functions. The largest cancer network contained many genes with genome stability maintenance functions. We tested 13 selected genes from this network for their involvement in genome maintenance using two cell-based assays. Among them, 10 were shown to be involved in either homology-directed DNA repair or centrosome duplication control including the well- known cancer marker MKI67. Our results suggest that the commonly recognized characteristics of cancers are supported by highly coordinated transcriptomic activities. This study also demonstrated that the co-expression network directed approach provides a powerful tool for understanding cancer physiology, predicting new gene functions, as well as providing new target candidates for cancer therapeutics.

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.

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.

Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair of DNA Double Strand Breaks

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology‐directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty‐three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N‐ and C‐ termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc‐coordinating amino acids and to the BRCA1‐BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.

Ran Binding Protein 9 (RanBP9) is a novel mediator of cellular DNA damage response in lung cancer cells

Ran Binding Protein 9 (RanBP9, also known as RanBPM) is an evolutionary conserved scaffold protein present both in the nucleus and the cytoplasm of cells whose biological functions remain elusive. We show that active ATM phosphorylates RanBP9 on at least two different residues (S181 and S603). In response to IR, RanBP9 rapidly accumulates into the nucleus of lung cancer cells, but this nuclear accumulation is prevented by ATM inhibition. RanBP9 stable silencing in three different lung cancer cell lines significantly affects the DNA Damage Response (DDR), resulting in delayed activation of key components of the cellular response to IR such as ATM itself, Chk2, γH2AX, and p53. Accordingly, abrogation of RanBP9 expression reduces homologous recombination-dependent DNA repair efficiency, causing an abnormal activation of IR-induced senescence and apoptosis. In summary, here we report that RanBP9 is a novel mediator of the cellular DDR, whose accumulation into the nucleus upon IR is dependent on ATM kinase activity. RanBP9 absence hampers the molecular mechanisms leading to efficient repair of damaged DNA, resulting in enhanced sensitivity to genotoxic stress. These findings suggest that targeting RanBP9 might enhance lung cancer cell sensitivity to genotoxic anti-neoplastic treatment.

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.

Expression of CD66 in non-Hodgkin lymphomas and multiple myeloma

To the Editor: Josef et al. (1) recently described the analysis of CD66 expression in a number of lymphoproliferative malignancies. CD66 is a membrane protein and a member of the carcinoembryonic antigen (CEA) and immunoglobulin superfamily. CD66 proteins are expressed as a number of isoforms also termed carcinoembryonic antigen cellular adhesion molecule (CEACAMs) and have a wide range of biologically important functions. Previous studies have revealed the expression of specific CD66 isoforms on leukaemic blasts, solid tumours and multiple myeloma (MM) plasma cells (2–8). We examined the expression of each isoform of CD66 (a, b, c, d and e) using flow cytometry on CD138 cells from patients with MM (positively isolated using beads or gated following antibody staining) (4). We showed that CD66a was the sole isoform expressed on patient samples, although CD66b was expressed on one of the MM cell lines, U266. Josef et al. (1) extended these findings showing (‡20%) CD66 expression in >76% of non-Hodgkin lymphoma and patients with MM using the polyspecific anti-CD66 clone Kat4c (Dako) that binds CD66abce. They also examined CD66 expression with the BW 250 ⁄ 183 clone (Scintec Diagnostics GmBH, Zug, Switzerland) taken from a commercially available diagnostic kit containing a partially reduced form of the native antibody, intended for labelling with technetium. They mistakenly believed that the decreased staining of CD66 by BW 2501 ⁄ 183, when compared with Kat4c staining, reflected the sole recognition of CD66b by the BW 250 ⁄ 183 antibody. However, the clone BW 250 ⁄ 183 is a pan-CD66 antibody (Scintec Diagnostics registration file; submitted to the European Medicines Agency) and is not a mouse IgG1 monoclonal antibody specific for CD66b as suggested by Josef et al. (1) Hence, the lower levels of CD66 expression detected, compared with the other polyclonal antibody Kat4c, may reflect the use of the reduced rather than the native form of the BW 250 ⁄ 183 antibody, which alters its affinity for CD66. In addition, we have found that the native BW 250 ⁄ 183 binds more efficiently at 37 C rather than room temperature or 4 C, which may also explain the lower levels of CD66 expression found by Josef et al. (1) as they performed their analyses at room temperature. In extensive analysis of the original BW 250 ⁄ 183 native antibody, strong binding was demonstrated against CD66b (CEACAM6) and CD66e (CEA or CEACAM5) and weaker binding to CD66a (CEACAM1) and CD66c (CEACAM6) (Scintec Diagnostics registration file; submitted to the European Medicines Agency), indicating a wide specificity for the CD66 isoforms. The exact epitope recognised by the BW250 ⁄ 183 antibody has yet to be identified. Josef et al.’s (1) study offers an important insight into the expression of CD66 on a wide range of lymphoid malignancies including MM but inaccurately suggests their investigation, in part, of CD66b expression alone. The conclusion from their paper was that BW250 ⁄183 would seem inferior to an alternative antibody that had a wider specificity for members of the CD66 family, when used in the context of targeted radiotherapy. This conclusion was based on the incorrect assumption of the specificity of the BW250 ⁄ 183 antibody and failed to take into account several other factors that are important in determining the utility of the vector in targeted radiotherapy. These include the in vivo biodistribution of the labelled antibody.

Identification of survivin as a promising target for the immunotherapy of adult B-cell acute lymphoblastic leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is a rare heterogeneous disease characterized by a block in lymphoid differentiation and a rapid clonal expansion of immature, non-functioning B cells. Adult B-ALL patients have a poor prognosis with less than 50% chance of survival after five years and a high relapse rate after allogeneic haematopoietic stem cell transplantation. Novel treatment approaches are required to improve the outcome for patients and the identification of B-ALL specific antigens are essential for the development of targeted immunotherapeutic treatments. We examined twelve potential target antigens for the immunotherapy of adult B-ALL. RT-PCR indicated that only survivin and WT1 were expressed in B-ALL patient samples (7/11 and 6/11, respectively) but not normal donor control samples (0/8). Real-time quantitative (RQ)-PCR showed that survivin was the only antigen whose transcript exhibited significantly higher expression in the B-ALL samples (n = 10) compared with healthy controls (n = 4)(p = 0.015). Immunolabelling detected SSX2, SSX2IP, survivin and WT1 protein expression in all ten B-ALL samples examined, but survivin was not detectable in healthy volunteer samples. To determine whether these findings were supported by the analyses of a larger cohort of patient samples, we performed metadata analysis on an already published microarray dataset. We found that only survivin was significantly over-expressed in B-ALL patients (n = 215) compared to healthy B-cell controls (n = 12)(p = 0.013). We have shown that survivin is frequently transcribed and translated in adult B-ALL, but not healthy donor samples, suggesting this may be a promising target patient group for survivin-mediated immunotherapy.