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Characterising tumour-associated antigens (TAAs) not only represents an important approach to the identification of new diagnostic/prognostic markers, but can also provide information on disease processes and additional potential therapeutic targets. Preliminary screening of a protein macroarray, containing more than 12,000 different proteins, with sera from anaplastic lymphoma kinase (ALK)-negative and ALK-positive anaplastic large cell lymphoma (ALCL) patients identified ribonuclease and tumour suppressor protein Ribonuclease T2 (RNASET2), phosphatase lipid phosphate phosphatase-related protein type 3 (LPPR3) and apoptotic adaptor molecule Fas-associating protein (FADD) as ALK-negative ALCL-associated TAAs. Further validation of these observations was confirmed using the ALCL sera in reverse ELISAs. The circulating anti-RNASET2 autoantibodies present in ALCL patients' sera also recognised eukaryotically expressed RNASET2 protein. RNASET2 expression was then investigated in normal tissues and in lymphomas to explore its clinical potential. RNASET2 protein and mRNA levels showed highest expression in the spleen, leucocytes and pancreas. RNASET2 protein expression was not restricted to ALK-negative ALCL (81%), being expressed in ALK-positive ALCL (65%) as well as in a number of other lymphomas. The immunological recognition of RNASET2, its expression in ALCL and other lymphomas together with its known tumourigenic properties suggest that further studies on this autoantigen are warranted. © 2012.

Background: The Notch pathway is an evolutionarily conserved cell-signaling system that plays an important role in both physiologic and pathologic conditions such as embryonic development and cancer. In the latter, Notch signaling has been reported to promote tumor growth by regulating different aspects of tumor biology such as cell survival, proliferation, angiogenesis, and stemness. Because of its importance, targeting of this pathway has been attempted via both small molecules (γ-secretase inhibitors; GSIs) and neutralizing antibodies (against individual Notch receptors or DLL4 ligand) but limitations still hamper the clinical use of such therapeutics, generally due to pathway complexity and toxicities caused by complete pathway inhibition (GSIs). Targeting Notch ligands such as Jagged1 (JAG1), offers the opportunity to selectively block specific elements of the pathway important in tumor biology thus avoiding normal tissue toxicities. Methods: Structural studies defined a region of JAG1 (DSL + EGF1-3) that bound Notch1, which was used as an immunogen. Using classical hybridoma technology we have generated and subsequently characterised a panel of monoclonal antibodies (mAbs) against the JAG1 ligand, both in vitro (cell signaling and cell biology assays) and in vivo (tumor xenografts in both mice and rats). Results: Four functional blocking mAbs recognised a unique JAG1 epitope within its DSL domain binding interface with Notch1, effectively blocking ligand-receptor interaction in vitro. Importantly, our mAbs were cross-reactive with rat Jag1 but not the murine orthologue. These were able to inhibit endogenous JAG1-induced signaling in tumor and stromal cells (eg. vascular smooth muscle cells). JAG1 mAb treatment reduced in vitro breast cancer 3D growth, exhibiting reduced expression of important genes such as HES1, IL6 and decreased numbers of cancer stem cells. Treatment did not affect JAG2-induced growth, or signaling mediated by other Notch ligands (eg. DLL4), confirming mAb specificity. Importantly, JAG1 mAb treatment in vivo inhibited Notch signaling and tumor growth in cancer xenograft models in two different host animals. Host body weight, blood tests and histological analysis detected no toxicity. Conclusions: We generated neutralizing mAbs able to inhibit JAG1-induced signaling both in vitro and in vivo. The ability of JAG1 blockade to impair tumor growth without toxicity indicates it has the potential to make a contribution to the current arsenal of cancer therapeutics. This mAb treatment will be subjected to further in vivo testing, both alone and in combination with other therapeutic approaches, to further expand our knowledge of its clinical potential and mechanism of action. Funding and conflicts of interest This work is supported by Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We have filed a patent application, WO/2014/111704, claiming therapeutic use of the JAG1 antibodies. Citation Format: Massimo Masiero, Demin Li, Pat Whiteman, Carol Bentley, Jenny Greig, Tasneem Hassanali, Sarah Watts, Stephen Stribbling, Jenna Yates, Ji-Liang Li, Susan Mary Lea, Penny Ann Handford, Adrian Llewellyn Harris, Alison Hilary Banham. Development of therapeutic anti-Jagged1 monoclonal antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C128.

BACKGROUND: Vaccine development targeting the novel immunogenic Per ARNT Sim Domain containing 1 (PASD1) cancer testis antigen represents an attractive therapeutic approach for the significant number of patients with diffuse large B-cell lymphoma who are refractory to conventional treatment. Since CD4-positive T helper cells have crucial roles in promoting and maintaining immune responses to tumor antigens, the presence of a CD4-positive T-helper immune response to the PASD1 antigen in patients with diffuse large B-cell lymphoma was investigated in the current study. DESIGN AND METHODS: Thirty-one patients with diffuse large B-cell lymphoma (25 with de novo, five with transformed and one with T-cell-rich B-cell lymphoma) were studied. Five immunogenic PASD1 peptides predicted to bind to several major histocompatibiliy complex, class II DR beta 1 alleles were identified using web-based algorithms. Peripheral blood mononuclear cells from patients were used to investigate the immunogenicity of these DR beta 1-restricted peptides in vitro using both gamma-interferon release enzyme-linked immunospot and cytolytic assays. RESULTS: Two of the five PASD1 peptides, PASD1(6) and PASD1(7), were shown to be immunogenic in 14 out of 32 patients studied in a gamma-interferon release assay. CD4-positive T-helper cell lines from two patients raised against PASD1 peptides were able to lyse cell lines derived from hematologic malignancies expressing endogenous PASD1 protein. CONCLUSIONS: This is the first report of a CD4-positive T-helper response to the PASD1 protein in patients with lymphoma. The immunogenic peptides described here represent valuable additional candidates for inclusion in a vaccine to treat patients with PASD1-positive diffuse large B-cell lymphoma whose disease is refractory to conventional therapies.

We report the characterization of vaccinia virus gene B12R which is predicted to encode a 33K protein with 36% amino acid identity to the serine/threonine protein kinase encoded by vaccinia virus gene B1R. S1 nuclease protection experiments showed that gene B12R is transcribed early during infection from an initiation site 11 bp upstream of the open reading frame (ORF). The gene encodes a 33K polypeptide that is not required for virus replication in tissue culture nor for virus virulence in a murine intranasal model. Expression of the B12R gene in Escherichia coli produced an abundant 33K polypeptide which lacked protein kinase activity under conditions in which the protein kinases encoded by vaccinia virus gene B1R and African swine fever virus gene j9L are active.

We previously described PASD1 as a new cancer testis antigen in multiple myeloma (MM) that is retained post-therapy, suggesting the use of vaccination strategies to induce anti-PASD1 immunity in a setting of minimal residual disease. We have focused on DNA fusion gene vaccines, coupling fragment C domain (DOM) of tetanus toxin with PASD1 sequence, and examined efficacy in Human Leukocyte Antigen (HLA)-A2 (HHD) transgenic mice using a human MM cell line expressing PASD1 protein and chimeric HLA-A2 class I molecules as target. DNA vaccines encoded two HLA-A2-restricted epitopes (p.DOM-PASD1(1), p.DOM-PASD1(2)) and full-length PASD1 (p.DOM-PASD1FL). p.DOM-PASD1(1) proved superior to p.DOM-PASD1(2) in generating T-cell responses in HHD mice, able to lyse the chimeric murine RMA-HHD cells. Boosting by electroporation significantly enhanced p.DOM-PASD1(1). Only p.DOM-PASD1(1) induced cytotoxic T-lymphocytes (CTLs) were able to lyse human MM target cells expressing endogenous antigen. The p.DOM-PASD1FL vaccine predominantly induced strong PASD1(1) over PASD1(2) T-cell immune responses, indicative of immunodominance. Importantly, p.DOM-PASD1FL generated immune-mediating killing of native chimeric MM cells, in the absence of exogenous added peptide, implicating PASD1(1) specific CTLs. These data demonstrate that PASD1-derived epitopes are both efficiently and selectively processed and presented by native human MM cells. Notably, they permit the use of PASD1-encoding DNA vaccine therapy in a clinical setting.

CD4(+)CD25(+) regulatory T (T(reg)) cells can control the allergic response to allergen, airway eosinophilia and airway hypersensitivity. We speculated that chronic inflammation persisting in asthma airways is dependent on abnormalities of these T(reg) cells. There are differences in the pathology of asthma in adults and children, and the airways of pediatric asthma are considered to be more naive than those of adults. Therefore, we analyzed the functionality of T(reg) cells in pediatric asthma and the relationship between T(reg) function and asthma symptoms.The anergic state, which is one of the defining properties of T(reg), was analyzed by measuring intracellular Ca(2+) influx following T cell receptor (TCR) stimulation. FOXP3-positive cells and FOXP3 mRNA expression were measured by flow analysis and real-time PCR with the SYBR method, respectively.CD45RO(+) cells make up approximately 99% of CD4(+)CD25(high) T cells and 89% of CD4(+)CD25(low) T cells in human adult blood. The proportion of CD45RO(+) cells in CD4(+)CD25(+) (high + low) T cells from pediatric asthma was much smaller (about 56%). Interestingly, our data indicated that CD45RO(+) T(reg) cells from pediatric asthma aberrantly increased intracellular Ca(2+) concentrations following TCR activation compared with pediatric nonasthma controls.These impaired CD45RO(+) T(reg) cell functions were correlated with asthma symptoms. The correlation was observed in the group with a highly expressed atopic phenotype and longer duration of asthma. We suggest that chronic inflammation in pediatric asthma airways may be the result of impaired regulatory functions of CD45RO(+) T(reg) cells.

FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients' samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM-derived cell lines (n = 4). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66.7% of patients with the pre-neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138(+) plasma cells was significantly higher in MGUS (P = 0.0005; mean 46.4%) and MM patients (P < or = 0.0001; mean 57.3%) than in reactive marrows (mean 2.5%). FOXP2 (>10% nuclear positivity) was detectable in 90.2% of MM (55/61) and 90.9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56-negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.

Purpose To evaluate the prognostic value of cell of origin immunohistochemical markers and BCL2, BCL6, and c-MYC translocations in a homogeneous cohort of patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Patients and Methods Patients with CD20+ DLBCL were enrolled in the randomized LNH98-5 and 01-5B Groupe d'Etude des Lymphomes de l'Adulte trials. Paraffin-embedded tumor samples of 119 patients treated with R-CHOP were analyzed by immunohistochemistry for CD10, BCL6, MUM1/IRF4, LMO2, and forkhead box protein P1 (FOXP1) expression and for BCL2, BCL6, and c-MYC breakpoints by fluorescence in situ hybridization (FISH) on tissue microarray. Results LMO2 expression and BCL2 breakpoint were associated with the germinal center (GC) subtype defined by Hans' algorithm, respectively (P < .0001; P = .0002) whereas FOXP1 expression and BCL6 breakpoint were associated with the non-germinal center (non-GC) subtype (P = .008 and P = .0001, respectively). The immunohistochemical markers analyzed independently, GC/non-GC phenotype and BCL2 breakpoint did not predict overall survival (OS). BCL6 breakpoint was significantly associated with an unfavorable impact on OS (P = .04). Interestingly, an immunoFISH index, defined by positivity for at least two of three non-GC markers (FOXP1, MUM1/IRF4, BCL6 breakpoint) was significantly associated with a shorter 5-year OS rate (44%; 95% CI, 28 to 60 v 78%; 95% CI, 59 to 89; P = .01) which was independent (P = .04) of the age-adjusted International Prognostic Index (P = .04) in multivariate analysis. Conclusion Our study demonstrates that combining immunohistochemistry with FISH allows construction of an immunoFISH index that significantly predicts survival in elderly DLBCL patients treated with R-CHOP.

The identification of immunogenic cancer testis antigens (CTAs) as immunotherapeutic targets represents one approach to improve treatment options for diffuse large B-cell lymphoma (DLBCL). We previously identified PASD1 [PAS (Per ARNT Sim) domain containing 1 (PASD1)], a DLBCL-associated CTA that was expressed in a range of hematopoietic malignancies. The aim of the present study was to investigate the presence of a cytotoxic T-cell (CTL) response to PASD1 in DLBCL patients. A significant gamma-interferon (IFN) release was detected in 21/29 HLA-A*0201-positive DLBCL patients (18 de novo DLBCL, two transformed DLBCL and one T-cell rich B-cell lymphoma) following short-term culture of their peripheral blood mononuclear cells stimulated with five HLA-A*0201-restricted PASD1 peptides. No significant responses were detected in 21 HLA-A*0201-negative DLBCL patients (12 de novo DLBCL, seven transformed DLBCL, two T-cell rich B-cell lymphoma) or six normal subjects. CTL cell lines were able to lyse PASD1-positive tumour cells in a major histocompatibility complex-Class I dependent manner. The presence of a gamma-IFN response correlated with PASD1 protein expression in the tumour cells in 12/15 cases studied. This is the first report of a CTL response to a CTA in DLBCL. Our results provide additional valuable evidence supporting PASD1 as a potential immunotherapeutic target for the treatment of DLBCL and other malignancies.

Gene expression profiling studies initially enabled diffuse large B-cell lymphoma to be divided into germinal center and activated B-cell-like subtypes, which define high- and low-risk patient groups when treated with chemotherapy. Attempts to reproduce the prognostic classification immunohistochemically have, however, provided inconsistent results. The aim of this study was to determine whether modified immunohistochemical classification of cell of origin focusing on activated B-cell-like markers could be used to predict the outcome of immunochemotherapy-treated diffuse large B-cell lymphoma patients. The expression of CD10, Bcl-6, MUM1/IRF4, Bcl-2, and FOXP1 was determined immunohistochemically from 88 samples of diffuse large B-cell lymphoma patients treated uniformly with R-CHOP. When the modified classification using MUM1/IRF4 and FOXP1 positivities as activated B-cell-like markers was applied to distinguish the patients between the activated B-cell-like and other diffuse large B-cell lymphoma subtypes, a significantly worse outcome was seen for the patients with the activated B-cell-like phenotype (3-year failure-free survival 63 vs 82%, P=0.048, overall survival 69 vs 85%, P=0.110). Similarly, according to the Muris algorithm, the group 2 patients representing Bcl-2-positive post-germinal center patients showed an inferior outcome in comparison to the group 1 patients (failure-free survival 59 vs 81%, P=0.041, overall survival 67 vs 82%, P=0.159). In contrast, when the classification of the same cohort was performed according to the Hans algorithm, no significant difference in survival was observed between the germinal center and non-germinal center patients. In conclusion, the data suggest that both the modified activated B-cell-like and Muris classifications define the non-germinal center phenotype as an adverse risk factor in R-CHOP-treated diffuse large B-cell lymphoma patients.

BACKGROUND: CD4(+)CD25(+) regulatory T cells (Tregs) regulate immunotolerance. Treg depletion causes autoimmune disease, whereas Treg expansion can prevent effective immunosurveillance of 'non-self' antigens in many clinical settings, including cancer. OBJECTIVE: Patent WO2008081581 presents an invention to target Tregs therapeutically via vaccination with immunogenic peptides from the forkhead box P3 (FOXP3) transcription factor. Our objective was the scientific evaluation of this patent and advantages versus potential risks of this therapeutic strategy. METHODS: There are 432 patents on Tregs and a diverse array of approaches for their therapeutic targeting; these have already been reviewed elsewhere. This article focuses on the utility of the selected peptides for specifically targeting FOXP3, whether FOXP3 expression is Treg specific and the likely effectiveness versus risk of autoimmunity relating to FOXP3-targeted immunotherapy. RESULTS/CONCLUSIONS: Our analysis of the immunogenic FOXP3 peptides found many (10/21) with significant (i.e. only one or two amino-acid differences) or complete identity with other proteins; these are not suitable for specific FOXP3 targeting. Some peptides have the ability to target specific FOXP3 isoforms. FOXP3 vaccination might be an effective method for reducing Treg numbers in cancer patients. However, this must be balanced against the risks of developing autoimmunity and targeting effector T cells.

Summary Background Regulatory T cells (Tregs) have an essential role in tolerance and immune regulation. However, few and controversial data have been published to date on the role and number of these cells in atopic dermatitis (AD). Objectives To investigate the number of CD4+CD25+FOXP3+ Tregs and interleukin 10-producing T regulatory type 1 (Tr1) cells in patients with AD. Methods Peripheral blood and skin biopsy samples from atopy patch test (APT)-positive patients with acute- and chronic-phase AD were investigated. Immunohistochemistry was applied to identify CD4+CD25+FOXP3+ Tregs in the skin, while flow cytometry was used to detect CD4+CD25highFOXP3+ Tregs and Tr1 cells in the peripheral blood. Results In the peripheral blood samples of patients with AD significantly elevated numbers of Tr1 cells were found. Although neither the absolute number nor the percentage of CD4+CD25highFOXP3+ Tregs showed significant alteration in the peripheral blood of patients, increased numbers of FOXP3+ Tregs were detected in skin biopsy specimens. All of the APT-positive skin samples showed epidermal dendritic cell aggregates, morphologically consistent with so-called Langerhans cell microgranulomas, which also contained intermingled FOXP3+ Tregs. Conclusions Tr1 cell numbers were elevated in the peripheral blood and increased numbers of CD4+CD25highFOXP3+ Tregs were detected in the skin of patients with AD. The epidermal dendritic cell clusters in APT-positive lesional skin showed a close connection to the FOXP3+ Tregs.

We screened lymphoma patients’ serum samples against protein/antigen arrays, containing over 10,000 different human proteins, to identify disease-associated auto-IgG antibody interactions. This approach identified a humoral immune response to the HIP1R antigen in 40% of patients’ sera (4/6 follicular lymphomas, 3/5 germinal centre [GC]-derived diffuse large B-cell lymphomas [DLBCL], 2/4 transformed DLBCL, 1/5 mantle cell lymphomas, 1/5 non-GC DLBCL and 1/5 peripheral T-cell lymphomas). HIP1R has also previously been identified as an autoantigen in colon cancer using the SEREX technique. HIP1R is one of two members of the Huntingtin interacting protein 1 family. Both HIP1R and HIP1 share significant sequence homology and are cytoplasmic proteins that interact with inositol lipids, clathrin and actin. Interestingly, both proteins can stabilize pools of receptor tyrosine kinases by inhibiting their trafficking to the lysosome for degradation, which may mediate affects on cell growth and transformation. HIP1 has been more extensively characterized in cancer and was originally implicated in hematological malignancy when it was identified as a platelet-derived growth factor β receptor fusion partner in chronic myelomonocytic leukemia. HIP1 expression is elevated in lymphomas and can be induced by activated NF-κB while transgenic overexpression of HIP1 is associated with the development of lymphoid neoplasms. Interestingly, the increased severity of a murine Hip1/Hip1r double-knockout phenotype indicates that Hip1r can partially functionally compensate for the loss of Hip1, suggesting they have overlapping roles in vivo. Autoantibodies to HIP1 have been reported in 59% of human lymphoma sera, compared to 31% of healthy controls and showed higher titre in patients who had undergone remission than those that relapsed (Bradley et al. 2007. Cancer Res 67:8923). However, the region of HIP1 tested for this serum reactivity shares high sequence homology with HIP1R, and thus this previous study is likely to have identified autoantibodies to both HIP1 and HIP1R. In contrast the region of HIP1R on our antigen array does not share homology with HIP1, suggesting that HIP1R expression is also altered in lymphomas. Quantitative RT-PCR was performed for both HIP1 and HIP1R on a panel of lymphoma cell lines, normal tissues and purified B- and T-cell populations. Overall HIP1R was more widely expressed and at higher levels than HIP1 in both normal lymphocytes and lymphoma cell lines. The particularly high level expression of HIP1R in Burkitt lymphoma cell lines and increased expression in GC- compared to non-GC DLBCL is consistent with the high frequency of autoantibodies in the sera from patients with GC-derived lymphomas. Two commercial monoclonal antibodies were successfully validated for the detection of HIP1R, although only the BD Transduction Laboratories’ antibody is raised to a region that lacks any significant stretches of homology with HIP1. Western blotting experiments have confirmed the expression of the HIP1R protein in lymphoma cell lines and showed a good correlation with mRNA expression levels. The BD Transduction Laboratories anti-HIP1R antibody recognises a formalin resistant epitope and studies are already underway to elucidate HIP1R expression patterns in lymphoma biopsies.

Deregulation of FOXP1 expression plays an important role in lymphoma development although the underlying molecular mechanism is poorly understood. FOXP1 is targeted by chromosome translocations in MALT lymphoma and diffuse large B-cell lymphoma, where high-level protein expression is associated with poor prognosis. Nonetheless, the incidence and nature of FOXP1 abnormalities at both the genetic and protein levels, and their correlation in these lymphomas are not well established. We investigated FOXP1 translocation, copy number change and protein expression in MALT lymphoma (n=321), MALT lymphoma with a diffuse large B-cell lymphoma component (59), nodal diffuse large B-cell lymphoma (64) and extranodal diffuse large B-cell lymphoma (151) by interphase fluorescence in situ hybridization and immunohistochemistry. FOXP1 translocation was found in eight MALT lymphomas and three MALT lymphomas with diffuse large B-cell lymphoma, with all positive cases originating in the stomach. In diffuse large B-cell lymphoma, the translocation was seen in 5 cases originating in the stomach (2), tonsil (1), large intestine (1) and lymph node (1). Immunoglobulin heavy chain gene was the translocation partner in 11 of the 16 positive cases. Fluorescence in situ hybridization mapping revealed FOXP1 breakpoints within the 5′ untranslated region of the gene (upstream of exon 6, the first coding exon of full-length FOXP1) in 14 cases, but downstream of exon 6 (most likely upstream of exon 8) in the remaining 2 cases. Three copies of the FOXP1 gene were observed in MALT lymphoma (17%), MALT lymphoma with diffuse large B-cell lymphoma (12%) and diffuse large B-cell lymphoma (32%), including cases with FOXP1 translocation (19%). Immunohistochemistry showed strong/moderate FOXP1 staining in all the cases with FOXP1 translocation. However, FOXP1 expression was independent of FOXP1 translocation or copy number changes. Our findings suggest that (1) FOXP1 translocation may disrupt the full-length FOXP1 transcript and lead to expression of FOXP1 transcript variants with alternate 5′ ends and (2) mechanisms other than translocation and copy number changes are also responsible for FOXP1 overexpression in lymphoma. © 2008 USCAP, Inc All rights reserved.

Circulating nucleic acids have been shown to have potential as non-invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour-associated MIRN155 (miR-155), MIRN210 (miR-210) and MIRN21 (miR-21) in serum from diffuse large B-cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (P = 0.009, 0.02 and 0.04 respectively). Moreover, high MIRN21 expression was associated with relapse-free survival (P = 0.05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non-invasive diagnostic markers for DLBCL and possibly other cancers.

To the editor: Three related articles in Blood have recently highlighted an issue with a significant impact on the study of regulatory T cells (Tregs), namely the specificity and affinity of the antibodies used to detect forkhead box P3 (FOXP3).[1][1][⇓][2]–[3][3] There is phenomenal interest

The FOXP1 forkhead transcription factor is targeted by recurrent chromosome translocations in several subtypes of B-cell non-Hodgkin lymphomas, where high-level FOXP1 protein expression has been linked to a poor prognosis. Western blotting studies of diffuse large B-cell lymphoma (DLBCL) cell lines unexpectedly identified the atypical high-level expression of 2 smaller, 60 to 65 kDa, FOXP1 isoforms in all 5 of those with the activated B cell (ABC)–like DLBCL subtype and in a subgroup of primary DLBCL. The anti-FOXP1 (JC12) monoclonal antibody cannot distinguish FOXP1 isoforms by immunohistochemistry, a finding that may be clinically relevant as high-level expression of the full-length FOXP1 protein was observed in some germinal center–derived DLBCLs. ABC-like DLBCL-derived cell lines were observed to express 2 novel, alternatively spliced FOXP1 mRNA isoforms, encoding N-terminally truncated proteins. These transcripts and the smaller protein isoforms were induced as a consequence of normal B-cell activation, which thus represents an additional mechanism for up-regulating FOXP1 expression in lymphomas. The expression of potentially oncogenic smaller FOXP1 isoforms may resolve the previously contradictory findings that FOXP1 represents a favorable prognostic marker in breast cancer and an adverse risk factor in B-cell lymphomas.

We previously identified a correlation between estrogen receptor alpha (ERalpha) and the candidate tumour suppressor gene Forkhead Box P1 (FOXP1), whose nuclear protein expression in breast tumours was associated with improved patient survival. However, the expression pattern of FOXP1 in normal breast tissue is more reminiscent of the second receptor, ERbeta, which has an emerging role as a tumour suppressor in breast cancer and critically may underlie the ability of some ERalpha-negative tumours to respond to tamoxifen. In a series of 283 breast cancers, in which ERalpha-positive tumours were treated with tamoxifen, the nuclear expression of ERbeta correlated significantly with ERalpha (p = 0.004), low-tumour grade (p = 0.008) and nuclear FOXP1 (p = 0.01). High-grade tumours exhibited significantly more cytoplasmic ERbeta than the low-grade tumours (p = 0.006). Regression analysis demonstrated that FOXP1 expression was most closely related to nuclear ERbeta (p = 0.021). Neither, nuclear or cytoplasmic ERbeta expression demonstrated prognostic significance. FOXP1 is not estrogen regulated and silencing FOXP1 expression, using siRNA, did not affect ERalpha, ERbeta or progesterone receptor expression, suggesting ER and FOXP1 co-expression may reflect a common regulatory mechanism.

Background and Aims: There is a need for genetic biomarkers to guide prognosis and management of gastric mucosa-associated lymphoid tissue (MALT) lymphomas. We assessed the incidence and clinical significance of the MALT lymphoma-associated genetic abnormalities t(11;18)/ API2-MALT1 , t(1;14)/ BCL10-IGH , t(14;18)/ IGH-MALT1 and t(3;14)/ FOXP1-IGH , in gastric MALT lymphomas from Japan. Methods: The presence of translocations and copy number changes involving MALT1 , IGH and FOXP1 were assessed in 90 cases of gastric MALT lymphoma using interphase fluorescence in situ hybridisation (FISH). In cases carrying a MALT1 translocation, FISH for API2-MALT1 was performed, while in those carrying an IGH translocation, FISH was performed for BCL10 , BCL6 , BCL2 , c-MYC and/or CCND1 . MALT1 and FOXP1 copy number was also evaluated. Results: t(11;18)/ API2-MALT1 was detected in 18 (21%) of 87 cases, and was significantly associated with Helicobacter pylori -negativity, resistance to H pylori eradication, and Bcl10 nuclear expression. Four (6%) of 68 cases carried a translocation involving IGH and FOXP1 (n=1), BCL2 (n=1) or an unknown partner (n=2). Neither t(1;14)/ BCL10-IGH nor t(14;18)/ IGH-MALT1 was detected. Extra copies of MALT1 and FOXP1 were detected in 18 of 71 cases (25%) and 10 of 59 cases (17%), respectively. The presence of extra copies of MALT1 was significantly associated with progression or relapse of lymphoma, and was an independent adverse prognostic factor for event-free survival as determined by multivariate analysis. Conclusions: t(11;18)/ API2-MALT1 is frequent, while IGH -involved translocations are rare in gastric MALT lymphoma in Japan. The presence of extra copies of MALT1 , often suggestive of partial or complete trisomy 18, is a frequent genetic aberration in gastric MALT lymphoma, which appears to predict adverse clinical behaviour.

We have found that FOXP3 is an oligomeric component of a large supramolecular complex. Certain FOXP3 mutants with single amino acid deletions in the leucine zipper domain of FOXP3 are associated with the X-linked autoimmunity-allergic dysregulation (XLAAD) and immunodysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome in humans. We report that the single amino acid deletion found in human XLAAD/IPEX patients within the leucine zipper domain of FOXP3 does not disrupt its ability to join the larger protein complex, but eliminates FOXP3 homo-oligomerization as well as heteromerization with FOXP1. We found that the zinc finger-leucine zipper domain region of FOXP3 is sufficient to mediate both homodimerization and homotetramerization. However, the same domain region from XLAAD/IPEX FOXP3 containing an E251 deletion prevents oligomerizaton and the protein remains monomeric. We also found that wild-type FOXP3 directly binds to the human IL-2 promoter, but the E251 deletion in FOXP3 in XLAAD/IPEX patient's T cells disrupts its association with the IL-2 promoter in vivo and in vitro, and limits repression of IL-2 transcription after T-cell activation. Our results suggest that compromising FOXP3 homo-oligomerization and hetero-oligomerization with the FOXP1 protein impairs DNA-binding properties leading to distinct biochemical phenotypes in humans with the XLAAD/IPEX autoimmune syndrome. This study explains some features of the pathogenesis of a disease syndrome that arises as a consequence of specific assembly failure of a transcriptional repressor due to certain mutations within the FOXP3 leucine zipper.

BACKGROUND Regulatory T cells (TR) mediate peripheral immunological tolerance and are implicated in tumor progression. Because prostate cancer is being investigated for treatment by immunotherapy, we have assessed tumor TR in prostate cancers. METHODS TR cells were identified by FOXP3 in tissue microarrays (TMAs) from 146 radical prostatectomies and correlated with clinicopathological tumor parameters and prostatic specific antigen rise (PSA). RESULTS Twenty of 146 tumors contained no TR. The mean of the average for the remaining 146 patients was 7.24. There was a significant correlation between TR and androgen receptor (P = 0.003) and with hypoxia-inducible factor (HIF)-2α (P = 0.007) but not HIF-1α (P = 0.25). There was no significant correlation between TR numbers and stage, capsular invasion, urethral margins, vascular invasion, Gleason score, pre-operative PSA, or time to PSA recurrence (all P > 0.05). CONCLUSIONS TR in prostate tumors shows significant heterogeneity and may be the result of hormonal and hypoxic signaling. Targeting these may reduce TR in tumors allowing more successful immune therapies. Prostate 67: 623–629, 2007. © 2007 Wiley-Liss, Inc.

Recent results have shown a correlation between survival and frequency of tumour infiltrating T lymphocytes in colorectal cancer patients. However, it remains unclear whether the frequency of regulatory T cells is higher in colorectal cancer as compared to normal colon. To address this question we analysed the frequency and function of regulatory T cells in the peripheral blood and tumour infiltrating lymphocytes of colorectal cancer patients. The proportion of regulatory T cells in the peripheral blood of colorectal cancer patients (mean 8%) was significantly higher than that in normal controls (mean 2.2%). There were significantly more regulatory T cells in tumour infiltrating lymphocytes (mean 19.2%) compared to lymphocytes from an autologous non-malignant portion of the colon (mean 9%). Regulatory T cells from colorectal cancer patients were FOXP3 positive and suppressed the proliferation of autologous CD4+ CD25- cells. A higher density of tumour infiltrating regulatory T cells was found in patients with advanced as compared to early disease. These results support the hypothesis that increased numbers of regulatory T cells in the blood and tumours of colorectal cancer patients may influence the immune response against cancer and suggest that strategies to overcome regulatory T cell activity may be beneficial in the treatment of human colorectal cancer.

Fms interacting protein (FMIP) is a substrate for Fms tyrosine kinase, and a nuclear/cytoplasm shuttling protein with a leucine zipper. As the phosphorylation of FMIP is observed in insulin-stimulated preadipocytes, we examined the role of FMIP in adipocyte differentiation, using the mesenchymal multipotent stem cells, C2C12 cells, that can differentiate into adipocytes, muscle cells and osteoblasts. Ectopic expression of FMIP in C2C12 impairs the adipocyte differentiation induced by treatment with insulin, dexamethasone and 3-isobutyl-1-methylxanthine. These cells exhibit muscle phenotype with multinuclear morphology. Furthermore, knockdown of endogenous FMIP expression by small interfering RNA improves adipocytic lineage commitment of C2C12 cells, while impairing muscle differentiation. Upon stimulation with insulin, CCAAT/enhancer binding protein (C/EBP)beta, but not C/EBPalpha, is upregulated in cells expressing ectopic FMIP, whereas in FMIP knockdown cells, C/EBPalpha is constitutively expressed. Ectopic expression of C/EBPalpha counteracts the effects of FMIP, whereas C/EBPalpha knockdown partially mimics the effects of FMIP in this system. Northern blot analysis and reverse transcriptase-polymerase chain reaction study reveal that ectopic FMIP-expressing cells do not contain the polyadenylated C/EBPalpha mRNA, but contain the C/EBPalpha pre-mRNA, suggesting that FMIP plays a role in RNA processing and/or export. Indeed, a member of the THO complex that plays a role in mRNA export, THOC1, is co-precipitated with FMIP. The data we have acquired on FMIP suggest that it is a target for tyrosine kinase receptors that potentiate mRNA export.

In view of the certain anatomic site-dependent frequency of chromosomal translocations involved in extranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) pathogenesis, 17 salivary gland MALT lymphoma cases were analyzed for MALT1 and FOXP1 translocations. B cell CLL/lymphoma 10 (BCL10) and forkhead box PA (FOXP1) protein expression were studied by immunohistochemistry and translocations identified using fluorescence in situ hybridization (FISH)-specific probes FOXP1, t(11 ; 18)(q21 ; q21)/API2-MALT1 and t(14 ; 18)(q32 ; q21)/IgH-MALT1. None of the 11 analyzed cases showed FOXP1 rearrangement or amplification. The t(11 ; 18) was present in five of 13 cases and the t(14 ; 18) in three of 13 cases. MALT1 translocations were mostly mutually exclusive except in a single case. FOXP1 protein expression showed differences in the proportion of tumor cells with nuclear expression but not in their intensity, with the exception of one case where very intense nuclear staining was noted. BCL10 nuclear expression was present in four of 17 cases, two of which lacked t(11 ; 18). Our results suggest that MALT1-specific translocations and FOXP1 rearrangements are not commonly involved in pathogenesis. A case with strong FOXP1 protein expression indicates the possibility that the upregulation of FOXP1 expression is significant in a small subset of salivary gland MALT lymphomas. Also a single case in which both MALT1 translocations were present indicates that these are not always mutually exclusive.

FOXP3+ regulatory T (Treg) cells have crucial roles in maintaining self-tolerance and modulating adaptive immune responses. Functional studies of Treg cells have been hampered by a lack of suitable cell-surface markers that specifically enable their purification without contamination by non-regulatory CD25+ effector T cells. Two recent studies have demonstrated that downregulation of the interleukin-7 receptor (CD127) distinguishes Treg cells from activated T cells, facilitating both Treg-cell purification and their functional characterization in human diseases. CD127 uniquely enables the purification of FOXP3+ Treg cells and, potentially, also "adaptive" regulatory T-cell subsets from the CD4+CD25- T-cell population.

CD4+CD25high FOXP3-expressing regulatory T cells (Treg) can suppress immune responses to infections and tumors, thereby promoting microbial persistence and tumor progression. However, little is known about the phenotype and function of human mucosal Treg. Therefore, we analyzed the suppressive activity and homing phenotype of Treg in gastric mucosa of Helicobacter pylori-infected gastric adenocarcinoma patients. We found increased numbers of CD4+FOXP3+ Treg in the tumor compared to tumor-free gastric mucosa. Gastric Treg cells were able to suppress H. pylori-induced T cell proliferation and IFN-γ production. Furthermore, gastric Treg expressed increased levels of l -selectin and CCR4, compared to non-Treg cells, suggesting that these receptors contribute to Treg recruitment. The presence of functional antigen-specific Treg in H. pylori-infected gastric mucosa supports an important role for these cells in suppression of mucosal effector T cell responses, which probably contribute to bacterial persistence and possibly also to gastric tumor progression.

Purpose To assess the clinical significance of tumor-infiltrating FOXP3-positive regulatory T cells (TR) in breast cancer patients with long-term follow-up. Patients and Methods FOXP3-positive TR were detected by immunohistochemistry with our new, extensively characterized FOXP3 monoclonal antibody, 236A/E7. Numbers of FOXP3-positive lymphocytes in tissue microarray cores from pure ductal carcinoma in situ (DCIS; n = 62), invasive breast cancer (n = 237) or from comparable areas of normal terminal duct lobular breast tissue (n = 10) were determined. A median cutoff of ≥ 15 defined patients with high numbers of TR. Results TR numbers were significantly higher in in situ and invasive breast carcinomas than in normal breast; invasive tumors have significantly higher numbers than DCIS (P = .001). High numbers of FOXP3-positive TR identified patients with DCIS at increased risk of relapse (P = .04) and patients with invasive tumors with both shorter relapse-free (P = .004) and overall survival (P = .007). High TR numbers were present in high-grade tumors (P ≤ .001), in patients with lymph node involvement (P = .01), and in estrogen receptor (ER) –negative tumors (P = .001). Importantly, high numbers of TR within ER-positive tumors identified high-risk patients (P = .005). Unlike conventional clinicopathologic factors, high numbers of FOXP3-positive TR can identify patients at risk of relapse after 5 years. Conclusion These findings indicate that quantification of FOXP3-positive TR in breast tumors is valuable for assessing disease prognosis and progression, and that TR are an important therapeutic target for breast cancer. FOXP3-positive TR represent a novel marker for identifying late-relapse patients who may benefit from aromatase therapy after standard tamoxifen treatment.

Immunotherapy is an important treatment option in multiple myeloma (MM), with allotransplantation demonstrating an inducible graft-versus-myeloma effect.[1][1] This could be potentiated by vaccination, first requiring knowledge of tumor-associated antigens. The cancer testis antigens (CTAs) are

In this study, we investigated whether CD4+CD25high regulatory T cells (Treg) are increased in the tumor tissue and peripheral blood of early-stage prostate cancer patients undergoing prostatectomy. We show that the prevalence of CD4+CD25high T cells inside the prostate was significantly higher in the tumor compared with benign tissue from the same prostate. Furthermore, the frequency of CD4+CD25high T cells in peripheral blood was significantly higher in prostate cancer patients compared with normal donors. A proportion of the CD4+CD25high T cells was also shown to be glucocorticoid-induced TNF receptor, ICOS, and FOXP3 positive. Moreover, CD4+CD25+ T cells from blood and supernatants from cultured prostate tumor tissue samples exhibited immunosuppressive function in vitro. Furthermore, supernatants from cultured prostate tissue samples and prostate cancer ascites fluid induced migration of CD4+CD25+ T cells and were shown to contain the regulatory T cell chemokine CCL22 by ELISA. Our findings indicate that Tregs are an important cellular component of early-stage prostate tumors, and thus new therapeutic strategies aimed at inhibition or depletion of Tregs may improve prostate cancer immunotherapy.

Background Chromosomal aberrations of BCL11A at 2p16.1 have been reported in a variety of B-cell malignancies and its deficiency in mice leads to a profound block in B-cell development. Results Alternative pre-mRNA splicing of BCL11A produces multiple isoforms sharing a common N-terminus. The most abundant isoform we have identified in human lymphoid samples is BCL11A-XL, the longest transcript produced at this locus, and here we report the conservation of this major isoform and its functional characterization. We show that BCL11A-XL is a DNA-sequence-specific transcriptional repressor that associates with itself and with other BCL11A isoforms, as well as with the BCL6 proto-oncogene. Western blot data for BCL11A-XL expression coupled with data previously published for BCL6 indicates that these genes are expressed abundantly in germinal-center-derived B cells but that expression is extinguished upon terminal differentiation to the plasma cell stage. Although BCL11A-XL/BCL6 interaction can modulate BCL6 DNA binding in vitro, their heteromeric association does not alter the homomeric transcriptional properties of either on model reporter activity. BCL11A-XL partitions into the nuclear matrix and colocalizes with BCL6 in nuclear paraspeckles. Conclusion We propose that the conserved N-terminus of BCL11A defines a superfamily of C2HC zinc-finger transcription factors involved in hematopoietic malignancies.

Contains fulltext : 49283.pdf (Publisher’s version ) (Closed access) Strong expression of Forkhead box-P1 (FOXP1), a winged-helix transcription factor, has been identified as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, possible mechanisms of deregulation of this gene, on 3p14.1, have yet to be elucidated. We have identified a breakpoint at the IGA1 gene in the immunoglobulin heavy chain (IGH) locus at 14q32 that was juxtaposed to the FOXP1 gene locus in a gastric DLBCL that showed strong expression of FOXP1. This may be one possible mechanism of deregulating FOXP1 expression by placing it under the control of IGH enhancers.

Serological analysis of recombinant complementary deoxyribonucleic acid (cDNA) expression libraries (SEREX) is a powerful approach to identify immunogenic cancer-associated proteins using antibodies that are naturally present in the serum of cancer patients. This technique involves the screening of a relevant cDNA expression library with patient serum that has been cleaned to remove any antibodies that may recognize bacterial and/or viral proteins. Once antigens have been identified and their reactivity has been confirmed with a second round of screening, the gene encoding the protein can be sequenced and identified. Antigens can then be screened with a panel of allogenic sera from other patients and control individuals. This identifies disease-specific antigens, which may be useful diagnostic markers or, alternatively, targets for immunotherapy. This chapter describes the SEREX methodology in full.

Serological analysis of recombinant complementary deoxyribonucleic acid (cDNA) expression libraries (SEREX) is a powerful approach to identify immunogenic cancer-associated proteins using antibodies that are naturally present in the serum of cancer patients. This technique involves the screening of a relevant cDNA expression library with patient serum that has been cleaned to remove any antibodies that may recognize bacterial and/or viral proteins. Once antigens have been identified and their reactivity has been confirmed with a second round of screening, the gene encoding the protein can be sequenced and identified. Antigens can then be screened with a panel of allogenic sera from other patients and control individuals. This identifies disease-specific antigens, which may be useful diagnostic markers or, alternatively, targets for immunotherapy. This chapter describes the SEREX methodology in full.

The transcription factor FOXP3 plays a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. Despite its strong association with regulatory T cell function, in humans little is known about the frequency of CD4(+)CD25(+) cells that express FOXP3 protein nor the distribution of these cells in vivo. Here we report the characterization of seven anti-FOXP3 monoclonal antibodies enabling the detection of endogenous human FOXP3 protein by flow cytometry and immunohistochemistry. Flow-cytometric analysis showed that FOXP3 was expressed by the majority of CD4(+)CD25(high) T cells in peripheral blood. By contrast, less than half of the CD4(+)CD25(int) population were FOXP3(+), providing an explanation for observations in human T cells that regulatory activity is enriched within the CD4(+)CD25(high) pool. Although FOXP3 expression was primarily restricted to CD4(+)CD25(+) cells, it was induced following activation of both CD4(+) and CD8(+) T cell clones. These findings indicate that the frequency of FOXP3(+) cells correlates with the level of expression of CD25 in naturally arising regulatory T cells and that FOXP3 protein is expressed by some activated CD4(+) and CD8(+) T cell clones. These reagents represent valuable research tools to further investigate FOXP3 function and are applicable for routine clinical use.

Purpose: Recent studies of Hodgkin's lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients.Experimental Design: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome.Results: The combined influence of the presence of FOXP3+ and TIA-1+ cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3+ cells and a high proportion of TIA-1+ cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1+ cells and a lower proportion of FOXP3+ cells in the reactive background.Conclusions: These data suggest that low infiltration of FOXP3+ cells in conjunction with high infiltration of TIA-1+ cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

Gene expression profiling studies have reported up-regulated mRNA expression of the FOXP1 forkhead transcription factor in response to normal B-cell activation and high expression in a poor prognosis subtype of diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to investigate the prognostic importance of FOXP1 protein expression in an independent series of DLBCL.First, the specificity of our FOXP1 monoclonal antibody was verified by confirming that it did not recognize the closely related FOXP2, FOXP3, or FOXP4 proteins. FOXP1 protein expression was then analyzed by immunohistochemistry using a DLBCL tissue microarray constructed from 101 previously untreated de novo cases from the British Columbia Cancer Agency. FOXP1 expression was scored as either positive (30% positive nuclei) or negative (30% positive nuclei). The overall survival curves clearly showed that patients grouped as FOXP1-positive (40%) had a significantly decreased overall survival (P = 0.0001). FOXP1-positive patients had a median overall survival of 1.6 years compared with 12.2 years in FOXP1-negative cases. In addition, FOXP1-positive patients showed a clear trend to earlier progression in comparison to the FOXP1-negative patients. The analysis of FOXP1 expression within low, medium, and high International Prognostic Index groupings found that FOXP1-negative patients had better overall survival within each group indicating that FOXP1 expression has predictive value independent of the International Prognostic Index subgrouping, a finding that was confirmed in multivariate analysis. These initial results suggest that FOXP1 expression may be important in DLBCL pathogenesis.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of all adult non-Hodgkin's lymphomas, yet the understanding of its underlying genetic abnormalities remains poor. Our present study used the serological analysis of recombinant cDNA expression libraries (SEREX) technique to identify DLBCL-associated antigens. SEREX screening of testis libraries has previously identified cancer-testis antigens (CTAs) that may act as disease-specific targets for immunotherapy. Screening a testis cDNA expression library with serum from a DLBCL patient identified a total of 94 positive clones, representing 28 distinct antigens. Two of these antigens were novel, 8 were previously uncharacterised, and the remainder were proteins of known function. Screening of the antigens with sera from DLBCL (n = 10), acute myeloid leukaemia (AML, n = 10) and chronic myeloid leukaemia (CML, n = 10) patients, alongside normal healthy donor controls (n = 20), revealed that 7 of the antigens were recognised by DLBCL sera but not normal donor sera, whilst 2 of these antigens were also recognised by leukaemic sera. Some of the genes identified here were already known to be transcribed in DLBCL. The mRNA expression of the majority of the remaining antigens was confirmed in DLBCL cell lines using reverse-transcriptase PCR (RT-PCR). Our study identified a number of DLBCL associated antigens that may be suitable as prognostic/diagnostic markers and/or for the immunotherapy of haematologic malignancies.

Purpose: The FOXP1 protein belongs to a functionally diverse family of winged-helix or forkhead transcription factors that have diverse roles in cellular proliferation, differentiation, and neoplastic transformation. The FOXP1 gene, which maps to 3p14, shows common loss of heterozygosity in breast tumors and is a candidate tumor suppressor gene. However, its role in breast cancer is unknown. Experimental Design: We have therefore investigated the pattern of FOXP1 expression in whole sections from normal (n = 16) and neoplastic (n = 90) breast tissues and correlated the level of expression in 283 invasive breast carcinomas on tissue microarrays with clinicopathological factors and survival. Because a relationship with estrogen receptor (ER) was identified, estrogen (17β-estradiol) regulation and ER/FOXP1 colocalization was also investigated. Results: Expression of FOXP1 was significantly positively associated with ER (P = 0.03) and negatively with epidermal growth factor receptor (P = 0.01) but no association with age (P = 0.91), lymph node status (P = 0.94), size (P = 0.76), or grade (P = 0.22). In a multivariate analysis of survival, FOXP1 expression was associated with a significantly improved relapse-free (P = 0.03) and borderline overall (P = 0.09) survival. Unlike normal breast, there was common coexpression of FOXP1 and ER in cell lines and tumors, but no 17β-estradiol (10-9 m) regulation of FOXP1 in MCF-7 cells was demonstrated. Conclusions: Our findings support a role for FOXP1 as a potential ER coregulator in human breast carcinoma and suggest that it may also independently regulate additional important pathways that control the progression of breast cancer.

The expression of the FOXP1 forkhead/winged helix transcription factor has been investigated in normal and neoplastic lymphoid tissues using the FOXP1-specific monoclonal antibody, JC12. Using single and double immunoenzymatic staining, FOXP1 expression has been studied in a series of classical and lymphocyte predominant Hodgkin's lymphomas, follicle centre lymphomas and Hodgkin's lymphoma-derived cell lines. Neoplastic cells in the majority of classical and lymphocyte predominant Hodgkin's lymphoma were FOXP1-negative. In two cases of classical Hodgkin's lymphoma, the tumour cells showed mislocalisation of FOXP1 to the cytoplasm, while in one case of lymphocyte predominant Hodgkin's lymphoma, and in the Hodgkin's lymphoma cell line KMH2, scattered tumour cells showed nuclear expression of FOXP1. In contrast, the tumour cells in the majority of follicle centre lymphomas showed strong nuclear FOXP1 reactivity. Double labelling studies of lymphoid tissue indicated that a variable proportion of CD20-positive germinal centre B cells express FOXP1 while the vast majority of CD30-positive cells are FOXP1-negative. The heterogeneity of FOXP1 expression in germinal centre-derived lymphomas, may have more to do with the transforming events underlying these distinct types of lymphoma than with their common origin.

Diffuse large B-cell lymphoma (DLBCL) can be divided into prognostically important subgroups with germinal center B-cell-like (GCB), activated B-cell-like (ABC), and type 3 gene expression profiles using a cDNA microarray. Tissue microarray (TMA) blocks were created from 152 cases of DLBCL, 142 of which had been successfully evaluated by cDNA microarray (75 GCB, 41 ABC, and 26 type 3). Sections were stained with antibodies to CD10, bcl-6, MUM1, FOXP1, cyclin D2, and bcl-2. Expression of bcl-6 (P

The detection of chromosomal translocations by fluorescence in situ hybridization (FISH) is widely performed, but very few studies have attempted to apply this technique to paraffin-embedded routine biopsy samples. We report the analysis of paraffin sections from 36 B-cell lymphoma biopsies for MYC translocation breakpoints by FISH. The probes consisted of multi-YAC constructs that flanked the breakpoint region and that, therefore, separate upon a chromosomal translocation and generate split (or "segregated") signals (rather than a more ambiguous "co-localization" pattern, obtained when the two partners in a hybrid gene are detected). The results were assessed by a simple approach that avoids the counting of signal numbers per nucleus and so is appropriate for use in routine practice. A total of 19 of the 36 lymphomas were scored as positive for MYC translocation and this included 16 of the 20 patients in whom classic cytogenetics had shown the presence of the (8;14) translocation (or one of its two variants). We conclude that this two-colour "split-signal" technique based on breakpoint flanking probes can readily detect chromosomal translocations in paraffin sections. Furthermore, our results suggest that cases categorized as "atypical Burkitt's/Burkitt-like" lymphoma (at least for adult patients) are heterogeneous with respect to translocations involving the MYC oncogene, as well as immunophenotype and clinical features.

AIMS: To characterise the 64 kDa intracellular antigen present on normal and neoplastic plasma cells detected by monoclonal antibody VS38 and by another antibody, MC186, of similar reactivity. METHODS: The VS38 monoclonal antibody was used to screen a bacterially expressed peripheral blood cDNA library, and the immunocytochemical staining of the two antibodies was compared with those raised specifically to the protein identified as the VS38 antigen. RESULTS: A partial cDNA encoding the VS38 antigen was cloned and shown to be identical to the human p63 gene. p63 is a non-glycated, reversibly palmitoylated type II transmembrane protein which is found in rough endoplasmic reticulum. Antibody MC186 also recognised this protein and both VS38 and MC186 together with two antibodies raised to p63 gave identical immunostaining patterns. CONCLUSIONS: The VS38 antigen was identified as the rough endoplasmic reticulum protein p63. While it is not exclusively expressed on plasma cells, the presence of p63 distinguishes plasma cells from other lymphoid cells because of their high secretory activity.

Introduction: FOXP1 is frequently overexpressed in DLBCL independently to copy number variations and translocations (Goatly et. al. 2008). The prognostic significance of FOXP1 in DLBCL is controversial with some studies describing FOXP1 as a poor prognostic marker (Banham et. al. 2005) and others found no significance (Hans et. al. 2004). Truncated FOXP1 isoforms were identified in DLBCL and are associated with poor outcomes (Brown et. al. 2008). Over 10 isoforms are known, where some exhibit COOH- or NH2- truncations and absence of regulatory domains. Since truncated isoforms lack regulatory domains, we suggest that isoforms may aberrantly regulate FOXP1 target genes. We aim to determine FOXP1 function in DLBCL and specifically its target genes, and also investigate how FOXP1 isoforms may contribute to a more aggressive phenotype. Methods: Expression constructs of FOXP1 isoforms (isoforms 1, 2, 3 and 8) were kindly provided by Dr. Philip Brown and Dr. Alison Banham. A stable BJAB cell line expressing FOXP1 isoform 1 was established. Gene expression of BJAB cell lines and 4 DLBCL tumors was analysed using Illumina HT12v4 microarray. Genes with >2-fold changes in expression were investigated. VisANT analysis and GSEA was performed on these genes and candidates were validated by qRT-PCR. Promoter analysis using MatInspector identified forkhead-binding sites in candidate genes, and dual luciferase reporter gene assays were performed to ascertain promoter regulation by FOXP1 isoforms. Results: The overexpression of FOXP1 isoform 1 in BJAB cell line resulted in the differential expression of 271 genes >2-fold. A similar comparison of high/low FOXP1 expressing DLBCL patients identified 2472 genes with >2-fold level. Comparison of gene lists for both the cell line and patient samples revealed all 271 genes differentially expressed in the cell line overlapped with the patient genes, indicating these results are translatable to FOXP1 function in DLBCL tumors. The 271 differentially expressed genes were investigated by pathway analysis, though no canonical pathways were significant. Alternatively, an integrative analysis of biological network information and microarray data showed critical interactions that were not revealed by pathway analysis, and identified genes that are promising FOXP1 targets. The promoter regions of 5 genes were investigated using reporter gene assays, and FOXP1 isoforms 3 and 8 were significantly stronger repressors compared to isoforms 1 and 2. For example, protein O-fucosyltransferase 1 (POFUT1) was downregulated in the presence of isoform 1, however isoform 3 and 8 downregulated POFUT1 expression >2-fold. This gene has not previously been associated with DLBCL, though POFUT1 is implicated in notch signaling and lymphoid homeostasis (Yao et. al. 2011). We are currently investigating the other novel FOXP1 target genes, and how truncated isoforms are involved in the differential regulation of these genes. Conclusion: We have identified 271 genes that are differentially expressed by FOXP1 expression. These are novel genes for DLBCL pathogenesis, and are differentially regulated by FOXP1 isoforms, indicating truncated isoforms may have aberrant functions and may contribute to a more pathogenic phenotype. Citation Format: Emily Camilleri, Carlos Aya-Bonilla, Jamie Nourse, Philip J. Brown, Alison H. Banham, Paula Marlton, Maher K. Gandhi, Lyn R. Griffiths. FOXP1 truncated isoforms differentially regulate target genes in diffuse large B cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B38.

Two proteins of the 40S ribosomal subunit were shown to be phosphorylated in vitro by a vaccinia virus-encoded serine/threonine protein kinase. These were identified by two-dimensional gel electrophoresis as ribosomal proteins Sa and S2 and were shown by phosphoamino acid analysis to both be phosphorylated on serine and threonine residues. The three phosphorylated forms of S2 produced by the B1R protein kinase in vitro matched the phosphorylated forms of S2 previously observed in cells infected with vaccinia virus. These data strongly suggest that this enzyme is responsible for the phosphorylation of S2 and Sa which occurs early during vaccinia virus infection.

Abstract 4294 Multiple myeloma (MM) is a bone marrow localized plasma cell tumor comprising 1% of all cancers and 10–15% of hematological malignancies. Despite significant advances in treatment, such as bortezomib, patients currently only have a 5-year survival rate of approximately 35%. The identification of improved therapeutic options therefore remains a priority. There is increasing evidence for a role of the immune system in tumor progression. Examples include the remission of some leukemias and lymphomas in immunocompetent patients while allogeneic stem cell transplantation also has a graft-versus-tumor effect in MM. The immunotherapeutic targeting of tumor-associated antigens (TAAs) on MM cells therefore represents an important approach for improved treatment of this disease. MM cells express a number of TAAs, of which cancer testis antigens (CTAs) are of particular interest. Their restricted normal tissue distribution combined with widespread expression in tumors makes them attractive immunotherapeutic targets whilst minimizing potential problems with autoimmunity. Reports of cytotoxic T cells (CTLs, the major effector cells in tumor immunity) and CD4-positive T-helper cells recognizing the NY-ESO-1 and MAGE CTAs in patients with MM suggests the presence of a spontaneous immune response to these molecules, which can be boosted through vaccination with CTAs such as NY-ESO-1. We previously identified PAS (Per ARNT Sim) domain containing 1 (PASD1) protein as a novel diffuse large B-cell lymphoma (DLBCL)-associated CTA. Importantly, PASD1 has a restricted normal tissue distribution but is present in a range of hematological malignancies, including MM. Subsequent in vitro studies have identified immunogenic PASD1 peptides that elicit PASD1-driven CTL or CD4-positive T-helper responses in peripheral blood mononuclear cells from DLBCL patients. Studies using a pre-clinical in vivo murine model have confirmed the immunogenicity of the PASD1 CTL peptides. These critical steps support the use of PASD1 as a potential immunotherapeutic target in DLBCL. The current study was performed to ascertain whether the PASD1 CTL peptides were immunogenic in MM patients and thus have utility for immunotherapy in this disease. Blood samples were obtained from 9 post-treatment myeloma patients attending the John Radcliffe Hospital following informed consent. Peripheral blood mononuclear cells were incubated with the PASD1 CTL peptides PASD1(1)38-47 (QLLDGFMITL) and PASD1(2)167-175 (YLVGNVCIL). A gamma-interferon ELISPOT release assay was performed after 8–10 days. The results are summarized in Table 1.Table 1:Gamma-IFN response to PASD1 in patients with MM.PatientsMHC Class I*Gamma-IFN response to peptides/50,000 cellsPASD1(1)PASD1(2)HIV-1PHADSA*0201+2+/−112+/−41+/−1100+/−14MWA*0201+52+/−244+/−220+/−266+/−8DMA*0201− A*2601+28+/−246+/−68+/−298+/−10JBA*0201+0+/−00+/−02+/−088+/−12JYA*0201+–––72+/−10DAA*0201−2+/−20+/−00+/−082+/−10GGA*0201−58+/−268+/−244+/−288+/−10MDA*0201−52+/−228+/−242+/−292+/−12RSA*0201––––*Results were considered positive if the number of spots in the test wells were at least twice that found in the irrelevant HIV-1 cultures. A significant gamma-interferon response was detected to one or both of the PASD1 peptides in 2/4 A*0201-positive evaluable patients. Analysis of the SYPETHI web-based algorithm predicted the PASD1 peptides used here to be immunogenic in the context of A*2601 and this was confirmed in the one A*2601+ patient studied here. No significant response was detected in the 3 A*0201 and A*2601-negative patients. Double immunolabeling studies using antibodies to PASD1 and CD138 showed PASD1 to be present in a subset of tumor cells in all 7 patients with evaluable ELISPOT data. Our findings demonstrate the immunogenicity of both the PASD1(1) and PASD1(2) peptides in patients with MM. These ‘generic’ peptides therefore represent vaccine candidates for inclusion in a vaccine targeting multiple PASD1-positive hematological malignancies. Disclosures: Banham: University of Oxford: Patents & Royalties. Pulford:Leukaemia and Lymphoma Research: Patents & Royalties.

Vaccinia virus open reading frame B1R was expressed in E. coli and shown to encode a serine/threonine protein kinase which phosphorylated casein and calf thymus histones in vitro. A polyclonal rabbit antiserum was raised against a TrpE-B1R bacterial fusion protein and used to characterize the B1R gene product. Immunoprecipitation and immunoblotting analyses detected a 34-kDa polypeptide that was synthesized early during vaccinia virus infection and which was apparently stable since it was easily detectable 18 hr postinfection. Immunofluorescence demonstrated that this protein localizes in cytoplasmic virus factories, the sites of virus DNA replication. Immunoblotting of vaccinia virions showed that the enzyme is packaged into virus particles.

Chronic lymphocytic leukaemia (CLL) is a neoplastic proliferation of mature B-lymphocytes that has a marked variability of clinical outcome. Two distinct groups of CLL may be distinguished by the presence (M-CLL) or absence (UM-CLL) of somatic hypermutation of the immunoglobulin heavy-chain gene. UM-CLL has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we have found that M-CLL and UM-CLL are consistently distinguished according to protein expression pattern. Of those proteins, the most important predictor of CLL class was the protein nucleophosmin (NPM). A significant increase in expression of NPM was found in UM-CLL compared with M-CLL (3.2 fold p