CD229 (Ly9) a Novel Biomarker for B-Cell Malignancies and Multiple Myeloma

CD229 is a cell-surface molecule predominantly expressed on lymphocytes. Its expression in B-cell malignancies is poorly known. We tested the presence of this immunoreceptor on a large number of malignancies and normal tissue using a new monoclonal antibody and tissue microarrays. Our data show that CD229 expression is restricted to hematopoietic cells. It was strongly expressed in myeloma and marginal-zone lymphomas. Because of the high expression on multiple myeloma cells, we also analyze the presence of soluble CD229 in the sera of these patients. We showed that serum levels of soluble CD229 in myeloma patients, at the time of diagnosis, could be useful as a prognostic biomarker. Altogether, our results indicate that CD229 represents not only a useful disease biomarker but also an attractive therapeutic target. CD229 (Ly9) homophilic receptor, which belongs to the SLAM family of cell-surface molecules, is predominantly expressed on B and T cells. It acts as a signaling molecule, regulating lymphocyte homoeostasis and activation. Studies of CD229 function indicate that this receptor functions as a regulator of the development of marginal-zone B cells and other innate-like T and B lymphocytes. The expression on leukemias and lymphomas remains poorly understood due to the lack of CD229 monoclonal antibodies (mAb) for immunohistochemistry application (IHC). In this study, we used a new mAb against the cytoplasmic region of CD229 to study the expression of CD229 on normal tissues and B-cell malignancies, including multiple myeloma (MM), using tissue microarrays. We showed CD229 to be restricted to hematopoietic cells. It was strongly expressed in all cases of MM and in most marginal-zone lymphomas (MZL). Moderate CD229 expression was also found in chronic lymphocyte leukemia (CLL), follicular (FL), classic mantle-cell (MCL) and diffuse large B-cell lymphoma. Given the high expression on myeloma cells, we also analyzed for the presence of soluble CD229 in the sera of these patients. Serum levels of soluble CD229 (sCD229) at the time of diagnosis in MM patients could be useful as a prognostic biomarker. In conclusion, our results indicate that CD229 represents not only a useful biomarker but also an attractive therapeutic target

High-mobility group box (TOX) antibody a useful tool for the identification of B and T cell subpopulations.

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.S

CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.

Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.This work was supported by grants from the Fondo de Investigaciones Sanitarias - Ministerio de Ciencia e Innovacion (PI12/1832), and Spanish Cancer Research Networks-RTICC (RD12/0036/0060), cofunded by the Fondo Europeo de Desarrollo Regional (FEDER), and the Asociacion Espanola Contra el Cancer (AECC Scientific Foundation). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

CD229 (Ly9) a Novel Biomarker for B-Cell Malignancies and Multiple Myeloma

CD229 (Ly9) homophilic receptor, which belongs to the SLAM family of cell-surface molecules, is predominantly expressed on B and T cells. It acts as a signaling molecule, regulating lymphocyte homoeostasis and activation. Studies of CD229 function indicate that this receptor functions as a regulator of the development of marginal-zone B cells and other innate-like T and B lymphocytes. The expression on leukemias and lymphomas remains poorly understood due to the lack of CD229 monoclonal antibodies (mAb) for immunohistochemistry application (IHC). In this study, we used a new mAb against the cytoplasmic region of CD229 to study the expression of CD229 on normal tissues and B-cell malignancies, including multiple myeloma (MM), using tissue microarrays. We showed CD229 to be restricted to hematopoietic cells. It was strongly expressed in all cases of MM and in most marginal-zone lymphomas (MZL). Moderate CD229 expression was also found in chronic lymphocyte leukemia (CLL), follicular (FL), classic mantle-cell (MCL) and diffuse large B-cell lymphoma. Given the high expression on myeloma cells, we also analyzed for the presence of soluble CD229 in the sera of these patients. Serum levels of soluble CD229 (sCD229) at the time of diagnosis in MM patients could be useful as a prognostic biomarker. In conclusion, our results indicate that CD229 represents not only a useful biomarker but also an attractive therapeutic target

Immunohistochemical expression of CSF1R protein in normal lymphoid tissues.

<p>CSF1R+ macrophages (brown) are abundant in the paracortex of the lymph nodes (A), the red pulp of the spleen (B), and the medulla of the thymus (C). Panels D-E, double immunofluorescence experiments in tonsil: CSF1R (red) and CD68 (green) (D) and CSF1R (red) and CD163 (green) (E). It can be seen that while CSFR1+ cells also express CD68 (D), CD163 is limited to only a subpopulation of CSFR1+ cells (E).</p

Distribution of CSF1R immunohistochemical expression in lymphomas.

<p>* Manual cell counting.</p><p>** Mean of results obtained from automated scanning of protein expression.</p><p>*** CSF1R is expressed in the tumour cells in 3/7 cases.</p><p>Distribution of CSF1R immunohistochemical expression in lymphomas.</p

Immunohistochemical detection of CSF1R protein in different lymphoma types.

<p>CSF1R+ cells are less abundant in B-CLL and B- and T-LBL, and more frequent in the microenvironment of T-cell lymphomas (PTCL, AITL, and ALCL), and cHL.</p

Functional analyses of CSF1R in cHL.

<p>(A) Interaction between CSF1R+ macrophages and HRS cells in cHL; above images corresponds to double immunostaining for CSF1R (brown) and CD30 (red), below images corresponds to double immunofluorescence for CSF1R (red) and CD30 (green). While CSFR1 protein expression was observed in cells around HRS, CSFR1 protein was not detected in the CD30+ HRS cells. (B) Kaplan–Meier analyses of survival for CD68 and CSF1R. (C) Western blot analyses of CSF1R protein before and after removal of phosphatase residues with lambda protein phosphatase (AF) using protein extracts from two nodular sclerosis cHL tumour samples and two tonsils. The 150 kDa protein band present in the cHL samples was lost after lambda protein phosphatase treatment.</p

Immunohistochemistry, western blot and immunoprecipitation analyses of CSF1R.

<p>(A) IHC staining of antibody FER216 labelled the HEK-CSFR1-Fc transfectants expressing CSFR1 but not the negative control HEK-FGFR1-Fc expressing Fc transfectants. The positive anti-Fc mAb control labelled both sets of transfectants. (B) Western blot analyses of CSF1R protein using protein extracts from HEK293 cells transfected with either CSF1R or a closely related protein (FGFR1) as a negative control, normal tissues, cHL tumour samples, and cHL-derived cell lines. Antibody FER216 recognised a band of 100 kDa in tonsil extracts and two bands of 100 and 150 kDa in extract from three cHL biopsies. Comparable results were obtained using the anti-CSFR1 mAb clone 61701. The mAb to tubulin was used as the loading control. (C) IP followed by western blotting demonstrated comparable results using either the FER216 or Clone 61701. Both antibodies were able to immunoprecipitate bands of 100 kDa from tonsil and 100 and 150 kDa from a case of cHL.</p

Analysis of Telomere Maintenance Related Genes Reveals NOP10 as a New Metastatic-Risk Marker in Pheochromocytoma/Paraganglioma

One of the main problems we face with PPGL is the lack of molecular markers capable of predicting the development of metastases in patients. Telomere-related genes, such as TERT and ATRX, have been recently described in PPGL, supporting the association between the activation of immortalization mechanisms and disease progression. However, the contribution of other genes involving telomere preservation machinery has not been previously investigated. In this work, we aimed to analyze the prognostic value of a comprehensive set of genes involved in telomere maintenance. For this study, we collected 165 PPGL samples (97 non-metastatic/63 metastatic), genetically characterized, in which the expression of 29 genes of interest was studied by NGS. Three of the 29 genes studied, TERT, ATRX and NOP10, showed differential expression between metastatic and non-metastatic cases, and alterations in these genes were associated with a shorter time to progression, independent of SDHB-status. We studied telomere length by Q-FISH in patient samples and in an in vitro model. NOP10 overexpressing tumors displayed an intermediate-length telomere phenotype without ALT, and in vitro results suggest that NOP10 has a role in telomerase-dependent telomere maintenance. We also propose the implementation of NOP10 IHC to better stratify PPGL patients