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1: JAG1 antibody characterization. 2: antibody inhibition of JAG1 mediated Notch signalling in tumor cell lines. 3: antibody inhibition of JAG1 signalling in vascular cells. 4: antibody inhibition of JAG1 signalling in HUVEC-HUVSMC co-culture. 5: effect of anti-Notch pathway treatment on MDA-MB231 2D growth and J1-65D titration and effect on 3D growth and gene expression. 6: J1-65D treatment of established OVCAR3 xenograft. 7: JAG1 antibodies cross-reactivity with rat Jag1. 8: anti-JAG1 treatment effect in a rat brain metastasis model.

Supplementary material and methods. Supplementary table 1: cell lines and growth conditions. Supplementary table 2: Notch ligands and control protein for plate coating. Supplementary table 3: qPCR primers. Supplementary table 4: flow cytometry antibodies. Supplementary table 5: ICC/IHC antibodies. Supplementary table 6: titration of Notch1-JAG1 binding inhibition by JAG1 antibodies. Supplementary table 7: amino acid sequence of JAG1 antibodies. Supplementary table 8: blood analysis on antibody-treated tumor-bearing rats.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201–presented wild-type p53 T-cell epitope, p5365–73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitro. In vivo, the antibody targets p5365–73 peptide–expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699–711. ©2017 AACR.

Supplemental Materials and Methods from Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy

CCR Translation for the Article from A New Immunostain Algorithm Classifies Diffuse Large B-Cell Lymphoma into Molecular Subtypes with High Accuracy

Supplementary Data from Immunomodulation of FOXP3+ Regulatory T Cells by the Aromatase Inhibitor Letrozole in Breast Cancer Patients

Suppl Table 1: Quantitation of T1-116C binding sites per target cell; Suppl Figure 1: Validation of the HLA-A2/p53RMP tetramer; Suppl Figure 2: ELISA screening of HLA-A2/p53RMP tetramer-reactive TCRm hybridoma supernatants; Suppl Figure 3: T1-116C staining of HL-60 cells; Suppl Figure 4: TP53 transcripts levels in cancer cell lines; Suppl Figure 5: The proteasome actively turns over p53 in NCI-H1395 lung cancer cells; Suppl Figure 6: The p53 TCRm T1-116C does not stain normal human peripheral blood mononuclear cells (PBMCs).

Purpose: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-α signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival.Experimental Design: FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral “metronomic” cyclophosphamide (50 mg/d).Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03).Conclusion: This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-α-negative tumors in combination with immunotherapy approaches.

Purpose: Hans and coworkers previously developed an immunohistochemical algorithm with ∼80% concordance with the gene expression profiling (GEP) classification of diffuse large B-cell lymphoma (DLBCL) into the germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. Since then, new antibodies specific to germinal center B-cells have been developed, which might improve the performance of an immunostain algorithm.Experimental Design: We studied 84 cases of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP)–treated DLBCL (47 GCB, 37 ABC) with GCET1, CD10, BCL6, MUM1, FOXP1, BCL2, MTA3, and cyclin D2 immunostains, and compared different combinations of the immunostaining results with the GEP classification. A perturbation analysis was also applied to eliminate the possible effects of interobserver or intraobserver variations. A separate set of 63 DLBCL cases treated with rituximab plus CHOP (37 GCB, 26 ABC) was used to validate the new algorithm.Results: A new algorithm using GCET1, CD10, BCL6, MUM1, and FOXP1 was derived that closely approximated the GEP classification with 93% concordance. Perturbation analysis indicated that the algorithm was robust within the range of observer variance. The new algorithm predicted 3-year overall survival of the validation set [GCB (87%) versus ABC (44%); P < 0.001], simulating the predictive power of the GEP classification. For a group of seven primary mediastinal large B-cell lymphoma, the new algorithm is a better prognostic classifier (all “GCB”) than the Hans' algorithm (two GCB, five non-GCB).Conclusion: Our new algorithm is significantly more accurate than the Hans' algorithm and will facilitate risk stratification of DLBCL patients and future DLBCL research using archival materials. (Clin Cancer Res 2009;15(17):5494–502)

Background Low pH suppresses the proliferation and cytotoxic activity of CD8+ cytotoxic and natural killer lymphocytes. The hypoxia-regulated transmembrane protein, carbonic anhydrase CA9, converts carbon dioxide produced by the Krebs cycle to bicarbonate and protons that acidify the extracellular milieu. We examined whether CA9 is also involved in intratumoural immunosuppression pathways. Methods A series of 98 tissue samples of primary non-small-cell lung carcinomas (NSCLC) from patients treated with surgery were analysed for the expression of CA9 and programmed-death ligand PD-L1 by cancer cells, and of FOXP3 by tumour-infiltrating lymphocytes (TILs). Results There was no direct association of CA9 with PD-L1 expression or the density of TILs in the tumour stroma, but CA9 was directly related to the extent of FOXP3+ TIL density (p = 0.008). Double-stratification survival analysis showed that patients with high CA9 expression and low TIL score had significantly poorer survival compared with all other groups (p Conclusion The study provides a basis for testing CA9 as a marker of resistance to immune-checkpoint inhibitors and as a therapeutic target to enhance the efficacy of immunotherapy.

ELTD1 is expressed in endothelial and vascular smooth muscle cells and has a role in angiogenesis. It has been classified as an adhesion GPCR, but as yet, no ligand has been identified and its function remains unknown. To establish its role, ELTD1 was overexpressed in endothelial cells. Expression and consequently ligand independent activation of ELTD1 results in endothelial-mesenchymal transistion (EndMT) with a loss of cell-cell contact, formation of stress fibres and mature focal adhesions and an increased expression of smooth muscle actin. The effect was pro-angiogenic, increasing Matrigel network formation and endothelial sprouting. RNA-Seq analysis after the cells had undergone EndMT revealed large increases in chemokines and cytokines involved in regulating immune response. Gene set enrichment analysis of the data identified a number of pathways involved in myofibroblast biology suggesting that the endothelial cells had undergone a type II EMT. This type of EMT is involved in wound repair and is closely associated with inflammation implicating ELTD1 in these processes.

Transient receptor potential (TRP) proteins are a diverse family of ion channels present in multiple types of tissues. They function as gatekeepers for responses to sensory stimuli including temperature, vision, taste, and pain through their activities in conducting ion fluxes. The TRPM (melastatin) subfamily consists of eight members (i.e., TRPM1-8), which collectively regulate fluxes of various types of cations such as K+ , Na+ , Ca2+ , and Mg2+ . Growing evidence in the past two decades indicates that TRPM ion channels, their isoforms, or long noncoding RNAs encoded within the locus may be oncogenes involved in the regulation of cancer cell growth, proliferation, autophagy, invasion, and epithelial-mesenchymal transition, and their significant association with poor clinical outcomes of cancer patients. In this review, we describe and discuss recent findings implicating TRPM channels in different malignancies, their functions, mechanisms, and signaling pathways involved in cancers, as well as summarizing their normal physiological functions and the availability of ion channel pharmacological inhibitors.

Funder: Rhodes Scholarships; doi: http://dx.doi.org/10.13039/501100000697, Funder: Clarendon Scholarship, Funder: Ernest Oppenheimer Memorial Trust; doi: http://dx.doi.org/10.13039/501100009978, Funder: European Union (European Social Fund), Funder: Free State of Saxony, Funder: Cancer Research UK; doi: http://dx.doi.org/10.13039/501100000289, Funder: Breast Cancer Research Foundation; doi: http://dx.doi.org/10.13039/100001006, ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor–endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1–specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. Implications: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

Thymocyte selection-associated high-mobility group box (TOX) is a DNA-binding factor that is able to regulate transcription by modifying local chromatin structure and modulating the formation of multi-protein complexes. TOX has multiple roles in the development of the adaptive immune system including development of CD4 T cells, NK cells and lymph node organogenesis. However very few antibodies recognizing this molecule have been reported and no extensive study of the expression of TOX in reactive and neoplastic lymphoid tissue has been performed to date. In the present study, we have investigated TOX expression in normal and neoplastic lymphoid tissues using a novel rat monoclonal antibody that recognizes its target molecule in paraffin-embedded tissue sections. A large series of normal tissues and B- and T-cell lymphomas was studied, using whole sections and tissue microarrays. We found that the majority of precursor B/T lymphoblastic, follicular and diffuse large B-cell lymphomas, nodular lymphocyte-predominant Hodgkin lymphomas and angioimmunoblastic T-cell lymphomas strongly expressed the TOX protein. Burkitt and mantle cell lymphomas showed TOX expression in a small percentage of cases. TOX was not found in the majority of chronic lymphocytic leukemia, myelomas, marginal zone lymphomas and classical Hodgkin lymphomas. In conclusion, we describe for the first time the expression of TOX in normal and neoplastic lymphoid tissues. The co-expression of TOX and PD-1 identified in normal and neoplastic T cells is consistent with recent studies identifying TOX as a critical regulator of T-cell exhaustion and a potential immunotherapy target. Its differential expression may be of diagnostic relevance in the differential diagnosis of follicular lymphoma, the identification of the phenotype of diffuse large B-cell lymphoma and the recognition of peripheral T-cell lymphoma with a follicular helper T phenotype. This work was supported by grants from the Plan Nacional de I+D+I, co-financed by the ISCIII-Subdireccion General de Evaluacion and the Fondo Europeo de Desarrollo Regional (FEDER), CIBERONC -CB16/12/00291 (MAP), and Programs of R&D activities among research groups of the Community of Madrid in Biomedicine (B2017/BMD-3778) (MAP, GR, JFGG). All funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sí

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1′s full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1′s role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

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.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Purpose/Aim: Regulatory FOXP3+ T-cells control the cytotoxic activity of effector cells and may have an essential role in the development of immune tolerance in cancer patients. Programed death ligand 1 PD-L1, expressed on cancer cell membranes also blocks the cytotoxic activity of PD1+ cytotoxic lymphocytes. Materials and Methods: We assessed the immunohistochemical detection of these immune-tolerance related markers in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery. The Tumor Infiltration Lymphocyte TIL density (mean number per x400 optical field) and the percentage of FOXP3+ TILs were assessed. Results: PD-L1 expression was directly linked with the TIL density (p = 0.01) and with the extent of infiltration with FOXP3+ TILs, named as the FIL-score (p = 0.01). FIL-score was significantly higher in stage I disease (p = 0.04). IL6 expression was linked with high TIL-score. A low TIL-score, characterizing immune deficient tumors defined a significantly poorer prognosis subgroup of patients (p = 0.03). Stratification of these tumors according to the FIL-score showed that FOXP3 expression by TILs correlated with an even a poorer prognosis in univariate (p = 0.007; median survival 14 vs. 44 months, respectively) and in multivariate analysis (p = 0.01, hazard ratio 4.3). Conclusion: Tumor stroma infiltration by FOXP3+ Tregs is an early event in the progression of NSCLC. Low lymphocytic infiltration defines poor prognosis, which becomes worse when the small numbers of infiltrating lymphocytes characterizing these tumors contain FOXP3 + Tregs. Furthermore, the direct association of FOXP3+ Treg infiltration density with PD-L1 expression by cancer cells implies a co-ordinated immune-suppressive activity in NSCLC.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of mature B-cell lymphoma. While the majority of patients are cured with immunochemotherapy incorporating the anti-CD20 monoclonal antibody rituximab (R-CHOP), relapsed and refractory patients still have a dismal prognosis. DLBCL subtypes including an aggressive activated B-cell-like (ABC) and a more favorable prognosis germinal center-like (GCB) DLBCL have been identified by gene expression profiling and are characterized by distinct genetic abnormalities and oncogenic pathways. This identification of novel molecular targets is now enabling clinical trials to evaluate more effective personalized approaches to DLBCL therapy. The forkhead transcription factor FOXP1 is highly expressed in the ABC-DLBCL gene signature and has been extensively studied within the context of DLBCL for more than a decade. Here, we review the significance of FOXP1 in the pathogenesis of DLBCL, summarizing data supporting its utility as a prognostic and subtyping marker, its targeting by genetic aberrations, the importance of specific isoforms, and emerging data demonstrating a functional role in lymphoma biology. FOXP1 is one of the critical transcription factors whose deregulated expression makes important contributions to DLBCL pathogenesis. Thus, FOXP1 warrants further study as a potential theranostic in ABC-DLBCL.

Huntingtin-interacting protein 1-related (HIP1R) is an endocytic protein involved in receptor trafficking, including regulating cell surface expression of receptor tyrosine kinases. We have previously shown that low HIP1R protein expression was associated with poorer survival in diffuse large B-cell lymphoma (DLBCL) patients from Denmark treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In this multicenter study, we extend these findings and validate the prognostic and subtyping utility of HIP1R expression at both transcript and protein level. Using data mining on three independent transcriptomic datasets of DLBCL, HIP1R transcript was preferentially expressed in germinal center B-cell (GCB)-like DLBCL subtype (Phi/HIP1R lo phenotype) exhibited poorer OS (P=0.0038) and PFS (P=0.0134). Multivariate analysis showed that HIP1Rhi/HIP1R lo subgroup of patients exhibited inferior OS and PFS (P

Huntingtin-interacting protein 1 (HIP1R) is an endocytic protein involved in endocytosis of surface receptors by regulating actin polymerization. We have previously shown that HIP1R was expressed in lymphoid B cells and diffuse large B-cell lymphoma (DLBCL) associated with better survival. Herein, we examined the expression profile of HIP1R in different immune cell populations and its potential functions in DLBCL. By utilizing a validated anti-HIP1R monoclonal antibody (clone 44), we examined whether the following immune cells in human reactive tonsils expressed HIP1R through double immunostaining: T cells (CD3+), macrophages (CD68+), mantle zone (MZ) B cells (PAX5+), germinal centre (GC) B cells (BCL6+) and plasma cells (IRF4/MUM1+). HIP1R was strongly expressed in PAX5+ MZ B cells, moderately expressed in BCL6+ GC B cells, but absent in CD3+ T cells, CD68+ macrophages and IRF4/MUM1+ plasma cells. In particular, we observed that HIP1R was absent in IRF4/MUM1+ plasma cells residing within the GC or non-GC interfollicular regions, suggesting that IRF4/MUM1 might downregulate HIP1R expression in activated B cells. We have previously shown that HIP1R expression is directly suppressed by the transcription factor FOXP1 in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells, however FOXP1 is absent in normal plasma cells, suggesting the presence of other regulators. Our previous immunostaining results in a series of DLBCL patient cases (n=155) showed a significant inverse correlation between HIP1R and IRF4/MUM1 (Pearson r = −0.495; p < 0.001). Indeed, knockdown of IRF4/MUM1 expression in the ABC-DLBCL cell line OCI-LY3 by two independent IRF4 siRNA constructs increased HIP1R expression at both transcript and protein levels. In terms of functional relevance, the bioinformatics approach Gene Set Enrichment Analysis (GSEA) was adopted to examine gene sets positively-associated with HIP1R transcript expression profile in three independent gene expression profiling (GEP) datasets of DLBCL patient cases derived from Gene Expression Omnibus database i.e. GSE10846 (n=233), GSE23501 (n=63), and GSE19246 (n=59). Our GSEA results showed that the gene set �Rho GTPase Activator Activity� (GO ID:0005100) was significantly positively-associated with HIP1R expression profile across all three GEP datasets GSE10846 (p = 0.0016), GSE23501 (p < 0.0001) and GSE19246 (p = 0.0167). These results suggest that HIP1R is involved in the activation of Rho GTPase signaling pathway, which has been documented to inhibit migration of DLBCL cells, and HIP1R expression is suppressed by transcription factors involved in B-cell activation including FOXP1 and IRF4/MUM1.

The FOXP1 (forkhead box P1) transcription factor is a marker of poor prognosis in diffuse large B-cell lymphoma (DLBCL). Here microarray analysis of FOXP1-silenced DLBCL cell lines identified differential regulation of immune response signatures and major histocompatibility complex class II (MHC II) genes as some of the most significant differences between germinal center B-cell (GCB)-like DLBCL with full-length FOXP1 protein expression versus activated B-cell (ABC)-like DLBCL expressing predominantly short FOXP1 isoforms. In an independent primary DLBCL microarray data set, multiple MHC II genes, including human leukocyte antigen DR alpha chain (HLA-DRA), were inversely correlated with FOXP1 transcript expression (P

Contains fulltext : 171662.pdf (Publisher’s version ) (Open Access) Antibodies are widely exploited as research/diagnostic tools and therapeutics. Despite providing exciting research opportunities, the multitude of available antibodies also offers a bewildering array of choice. Importantly, not all companies comply with the highest standards, and thus many reagents fail basic validation tests. The responsibility for antibodies being fit for purpose rests, surprisingly, with their user. This paper condenses the extensive experience of the European Monoclonal Antibody Network to help researchers identify antibodies specific for their target antigen. A stepwise strategy is provided for prioritising antibodies and making informed decisions regarding further essential validation requirements. Web-based antibody validation guides provide practical approaches for testing antibody activity and specificity. We aim to enable researchers with little or no prior experience of antibody characterization to understand how to determine the suitability of their antibody for its intended purpose, enabling both time and cost effective generation of high quality antibody-based data fit for publication.

To increase our knowledge of leukaemia‐associated antigens, especially in acute myeloid leukaemia (AML) M4, we prepared a phage display cDNA library using mRNA from the bone marrow cells of a patient with AML M4 at diagnosis. We immunoscreened 106 pfu with autologous sera and identified an antigen which we named GKT‐AML8. The cDNA showed more than 99% similarity to a sequence on 2q21.2 and 95% sequence similarity to a sequence on 19q13.3. These genes were named ZNF465 and ZNF466, respectively, following HUGO Gene Nomenclature Committee (HGNC) guidelines. Expressed sequence tag data suggests that both genes are transcriptionally active. ZNF465 and ZNF466 encode a 5′ kruppel associated box domain typical of negative regulators of gene transcription. We have confirmed the translational start site in the +1 frame in a near‐Kozak sequence that produces a 102 amino acid polypeptide from ZNF465. The high level of sequence similarity between ZNF465 and ZNF466 makes their transcripts almost indistinguishable by real‐time polymerase chain reaction (RT‐PCR). However, GKT‐AML8 showed most sequence similarity to ZNF465 and no transcript matching the 3′ ZNF466 sequence could be detected in patient samples or healthy volunteers. ZNF465/466 expression was detectable in 12/13 AML and 10/14 chronic myeloid leukaemia patients' samples but not in normal donor peripheral blood (0/8) or 0/3 bone marrow samples which had been separated into CD34+ and CD34− samples. The altered expression of ZNF465/466 in patients' samples and its absence in healthy donor haematopoietic samples indicate that ZNF465 is overexpressed in early myeloid disease and as such may represent a promising target for immunotherapy.

Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1 (ELTD1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has recently been identified as a potential cancer biomarker and a novel regulator of angiogenesis. In this mini-review, we present an overview of the current literature on ELTD1 and present bioinformatics data showing ELTD1's sequence conservation, its expression in cancer cell lines and its mutational frequency in human cancers. Additionally, we present sequence homology alignment results confirming ELTD1 to be a hybrid comprising motifs shared with individual members in both adhesion GPCR subfamilies 1 and 2. Finally, we discuss why tumour endothelial ELTD1 expression may confer a good prognosis yet still represent a therapeutic target.

Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy.

DNMT1 is a target of approved anti-cancer drugs including decitabine. However, the prognostic value of DNMT1 protein expression in R-CHOP-treated diffuse large B-cell lymphomas (DLBCLs) remains unexplored. Here we showed that DNMT1 was expressed in the majority of DLBCL cases (n = 209/230, 90.9%) with higher expression in germinal centre B-cell-like (GCB)-DLBCL subtype. Low and negative DNMT1 expression (20% cut-off, n = 33/230, 14.3%) was predictive of worse overall survival (OS; p < 0.001) and progression-free survival (PFS; p < 0.001). Nonetheless, of the 209 DNMT1 positive patients, 33% and 42% did not achieve 5-year OS and PFS, respectively, indicating that DNMT1 positive patients showed considerably heterogeneous outcomes. Moreover, DNMT1 was frequently expressed in mitotic cells and significantly correlated with Ki-67 or BCL6 expression (r = 0.60 or 0.44, respectively; p < 0.001). We demonstrate that DNMT1 is predictive of DLBCL patients' survival, and suggest that DNMT1 could be a DLBCL therapeutic target due to its significant association with Ki-67.

Aims: Transient receptor potential channel melastatin 4 (TRPM4) is an ion channel that regulates influx of calcium cations (Ca2+). Recent studies suggest that TRPM4 is an oncoprotein, and its up-regulated transcript level has been reported in diffuse large B cell lymphoma (DLBCL). We aimed to investigate TRPM4 protein expression pattern in non-malignant tissues and DLBCL cases, and its association with clinico-demographic parameters and survival in DLBCL. Methods and results: Analysis of publicly available DLBCL microarray data sets showed that TRPM4 transcripts were up-regulated in DLBCL compared to normal germinal centre B (GCB) cells, were expressed more highly in the activated B cell-like DLBCL (ABC-DLBCL) subtype and higher TRPM4 transcripts conferred worse overall survival (OS) in R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone)-treated DLBCL cases (P < 0.05). Our immunohistochemical analysis showed that TRPM4 was expressed in various human tissues but not in normal B cells within lymphoid tissues (reactive tonsil, lymph node and appendix). TRPM4 protein was present in 26% (n = 49 of 189) of our cohort of R-CHOP-treated DLBCL cases and this was associated significantly with more aggressive clinical parameters, including higher lactate dehydrogenase (LDH), Eastern Cooperative Oncology Group (ECOG) scores or stage (P < 0.01 for each of the parameters) and the ABC-DLBCL subtype (P = 0.016). TRPM4 positivity conferred significantly worse OS (P = 0.004) and progression-free survival (PFS) (P = 0.005). Worse OS remained associated significantly with TRPM4 positivity in multivariate analysis, including higher International Prognostic Index (IPI) or the non-GCB DLBCL phenotype (P < 0.05). Conclusions: TRPM4 protein expression is up-regulated in DLBCL cases compared to non-malignant B cells with preferential expression in ABC-DLBCL cases, and it confers significantly poorer DLBCL patient outcomes.

Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphomaDLBCL. These diseases can be difficult to diagnose and treat, and they lack well-characterized cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORA, IGF1, and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumor cells and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyzes active vitamin D3 production. Although Vdr and Cyp27b1 transcription in normal B cells were activated by interleukin 4 (IL-4) and CD40 signaling, respectively, unstimulated malignant plasmablastic cells lacking IL-4 expressed both VDR and CYP27B1. Positive autoregulation evidenced intact VDR function in all plasmablastic lines, and inhibition of growth by active vitamin D3 was both dependent on MYC protein inhibition and could be enhanced by cotreatment with a synthetic ROR ligand SR-1078. Furthermore, a VDR polymorphism, FOK1, was associated with greater vitamin D3–dependent growth inhibition. In summary, HLY-1 provides an important model of strongly plasmablastic lymphoma, and disruption of VDR pathway activity may be of therapeutic benefit in both plasmablastic lymphoma and myeloma.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥20% nuclear tumoral FOXP2-positivity (n=24/158) correlated with significantly inferior overall survival (OS: P=0.0017) and progression-free survival (PFS: P=0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P< 0.05 for both OS andPFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P=0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P=0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.

Objective Miller-Dieker syndrome (MDS) is a malformation of cortical development that results in lissencephaly (meaning smooth brain). This disorder is caused by heterozygous deletions on chromosome 17p13.3, including the lissencephaly 1 (LIS1) gene. Various mouse models have been used as an experimental paradigm in understanding human lissencephaly, but clear limitations exist in these studies, particularly because mice are naturally lissencephalic. Thus, the objective of this article was to establish human neural precursor cell lines from postmortem MDS tissue and to characterize the pathological cellular processes that contribute to the human lissencephalic phenotype. Methods Human neural precursors were isolated and expanded from the frontal cortices of a 33-week postmortem fetus with MDS and an age-matched control subject. Relative rates of proliferation and cell death were assessed in vitro, whereas the migration of precursors was examined after transplantation in vivo. Results Precursors showed haploinsufficiency of the LIS1 gene and a reduction in LIS1 protein. Precursors could also differentiate into both neurons and glia. MDS precursors demonstrated impairments in neuronal migration, diminished rates of cell proliferation, and increased cell death. Interpretation These results suggest that, in addition to migration, disruption in cell proliferation could play a more important role in the development of lissencephaly than previously suspected. Ann Neurol 2006

Forkhead Box P1 (FOXP1) is a member of the FOX family of transcription factors which have a broad range of functions. Foxp1 is widely expressed and has been shown to have a role in cardiac, lung and lymphocyte development. FOXP1 is targeted by recurrent chromosome translocations and its overexpression confers a poor prognosis in a number of types of lymphomas, suggesting it may function as an oncogene. In contrast, FOXP1 localises to a tumour suppressor locus at 3p14.1 and loss of FOXP1 expression in breast cancer is associated with a worse outcome, suggesting FOXP1 may function as a tumour suppressor in other tissue types. These data suggest that FOXP1 may not only be useful in prognosis but also may be used to develop FOXP1-directed therapeutic strategies.

Gains of the proto-oncogene BCL11A and nuclear accumulation of BCL11A XL protein are frequent in primary mediastinal B-cell lymphoma

Peripheral T-cell lymphomas (PTCL) are generally less common and pursue a more aggressive clinical course than B-cell lymphomas, with the T-cell phenotype itself being a poor prognostic factor in adult non-Hodgkin lymphoma (NHL). With notable exceptions such as ALK(+) anaplastic large cell lymphoma (ALCL, ALK+), the molecular abnormalities in PTCL remain poorly characterised. We had previously identified circulating antibodies to ALK in patients with ALCL, ALK(+). Thus, as a strategy to identify potential antigens associated with the pathogenesis of PTCL, not otherwise specified (PTCL, NOS), we screened a testis cDNA library with sera from four PTCL, NOS patients using the SEREX (serological analysis of recombinant cDNA expression libraries) technique. We identified nine PTCL, NOS-associated antigens whose immunological reactivity was further investigated using sera from 52 B- and T-cell lymphoma patients and 17 normal controls. The centrosomal protein CEP250 was specifically recognised by patients sera and showed increased protein expression in cell lines derived from T-cell versus B-cell malignancies. TCEB3, BECN1, and two previously uncharacterised proteins, c14orf93 and ZBTB44, were preferentially recognised by patients' sera. Transcripts for all nine genes were identified in 39 cancer cell lines and the five genes encoding preferentially lymphoma-recognised antigens were widely expressed in normal tissues and mononuclear cell subsets. In summary, this study identifies novel molecules that are immunologically recognised in vivo by patients with PTCL, NOS. Future studies are needed to determine whether these tumor antigens play a role in the pathogenesis of PTCL.

Purpose: Hans and coworkers previously developed an immunohistochemical algorithm with ∼80% concordance with the gene expression profiling (GEP) classification of diffuse large B-cell lymphoma (DLBCL) into the germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. Since then, new antibodies specific to germinal center B-cells have been developed, which might improve the performance of an immunostain algorithm.Experimental Design: We studied 84 cases of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP)–treated DLBCL (47 GCB, 37 ABC) with GCET1, CD10, BCL6, MUM1, FOXP1, BCL2, MTA3, and cyclin D2 immunostains, and compared different combinations of the immunostaining results with the GEP classification. A perturbation analysis was also applied to eliminate the possible effects of interobserver or intraobserver variations. A separate set of 63 DLBCL cases treated with rituximab plus CHOP (37 GCB, 26 ABC) was used to validate the new algorithm.Results: A new algorithm using GCET1, CD10, BCL6, MUM1, and FOXP1 was derived that closely approximated the GEP classification with 93% concordance. Perturbation analysis indicated that the algorithm was robust within the range of observer variance. The new algorithm predicted 3-year overall survival of the validation set [GCB (87%) versus ABC (44%); P < 0.001], simulating the predictive power of the GEP classification. For a group of seven primary mediastinal large B-cell lymphoma, the new algorithm is a better prognostic classifier (all “GCB”) than the Hans' algorithm (two GCB, five non-GCB).Conclusion: Our new algorithm is significantly more accurate than the Hans' algorithm and will facilitate risk stratification of DLBCL patients and future DLBCL research using archival materials. (Clin Cancer Res 2009;15(17):5494–502)

Despite advances in defining clinically relevant subtypes of diffuse large B-cell lymphoma (DLBCL), identifying high-risk patients and improved therapeutic options remains a priority. One approach is through the identification of tumor-associated antigens (TAAs), of which cancer testis antigens (CTAs) are of particular interest. Their restricted normal tissue expression, commonly widespread expression in tumors and immunogenicity makes them attractive therapeutic targets and markers for early diagnosis of cancer.1