Shuchun Zhao

Multiplexed ion beam imaging of human breast tumors

Immunohistochemistry (IHC) is a tool for visualizing protein expression that is employed as part of the diagnostic workup for the majority of solid tissue malignancies. Existing IHC methods use antibodies tagged with fluorophores or enzyme reporters that generate colored pigments. Because these reporters exhibit spectral and spatial overlap when used simultaneously, multiplexed IHC is not routinely used in clinical settings. We have developed a method that uses secondary ion mass spectrometry to image antibodies tagged with isotopically pure elemental metal reporters. Multiplexed ion beam imaging (MIBI) is capable of analyzing up to 100 targets simultaneously over a five-log dynamic range. Here, we used MIBI to analyze formalin-fixed, paraffin-embedded human breast tumor tissue sections stained with ten labels simultaneously. The resulting data suggest that MIBI can provide new insights into disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

LMO2 Protein Expression Predicts Survival in Patients With Diffuse Large B-Cell Lymphoma Treated With Anthracycline-Based Chemotherapy With and Without Rituximab

PURPOSE The heterogeneity of diffuse large B-cell lymphoma (DLBCL) has prompted the search for new markers that can accurately separate prognostic risk groups. We previously showed in a multivariate model that LMO2 mRNA was a strong predictor of superior outcome in DLBCL patients. Here, we tested the prognostic impact of LMO2 protein expression in DLBCL patients treated with anthracycline-based chemotherapy with or without rituximab. PATIENTS AND METHODS DLBCL patients treated with anthracycline-based chemotherapy alone (263 patients) or with the addition of rituximab (80 patients) were studied using immunohistochemistry for LMO2 on tissue microarrays of original biopsies. Staining results were correlated with outcome. RESULTS In anthracycline-treated patients, LMO2 protein expression was significantly correlated with improved overall survival (OS) and progression-free survival (PFS) in univariate analyses (OS, P = .018; PFS, P = .010) and was a significant predictor independent of the clinical International Prognostic Index (IPI) in multivariate analysis. Similarly, in patients treated with the combination of anthracycline-containing regimens and rituximab, LMO2 protein expression was also significantly correlated with improved OS and PFS (OS, P = .005; PFS, P = .009) and was a significant predictor independent of the IPI in multivariate analysis. CONCLUSION We conclude that LMO2 protein expression is a prognostic marker in DLBCL patients treated with anthracycline-based regimens alone or in combination with rituximab. After further validation, immunohistologic analysis of LMO2 protein expression may become a practical assay for newly diagnosed DLBCL patients to optimize their clinical management.

Prognostic significance of VEGF, VEGF receptors, and microvessel density in diffuse large B cell lymphoma treated with anthracycline-based chemotherapy

Vascular endothelial growth factor-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of lymphoma cell proliferation and/or survival induced by autocrine vascular endothelial growth factor receptor-mediated signaling. We have recently shown that diffuse large B cell lymphomas expressing high levels of vascular endothelial growth factor protein also express high levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether tumor vascularity, or expression of vascular endothelial growth factor protein and its receptors, contribute to patient outcomes. Our results show that increased tumor vascularity is associated with poor overall survival (P=0.047), and is independent of the international prognostic index. High expression of vascular endothelial growth factor receptor-1 by lymphoma cells by contrast is associated with improved overall survival (P=0.044). The combination of high vascular endothelial growth factor and vascular endothelial growth factor receptor-1 protein expression by lymphoma cells identifies a subgroup of patients with improved overall (P=0.003) and progression-free (P=0.026) survival; these findings are also independent of the international prognostic index. The prognostic significance of overexpression of this ligand-receptor pair suggests that autocrine signaling via vascular endothelial growth factor receptor-1 may represent a survival or proliferation pathway in diffuse large B cell lymphoma. Dependence on autocrine vascular endothelial growth factor receptor-1-mediated signaling may render a subset of diffuse large B-cell lymphomas susceptible to anthracycline-based therapy.

Immunohistochemical characterization of nasal-type extranodal NK/T-cell lymphoma using a tissue microarray: an analysis of 84 cases.

Nasal-type extranodal natural killer (NK)/T-cell lymphoma is an uncommon malignancy. By using a tissue microarray, we characterized 84 cases of extranodal NK/T-cell lymphoma with regard to expression of 18 immunohistochemical markers and the presence of Epstein-Barr virus (EBV) RNA. In our series, CD2 was positive in 69 (93%) of 74 cases, CD3 in 68 (84%) of 81, CD5 in 22 (27%) of 81, CD20 in 0 (0%) of 82, CD29 in 75 (91%) of 82, CD30 in 29 (35%) of 84, CD43 in 81 (96%) of 84, CD54 in 58 (72%) of 81, CD56 in 46 (58%) of 79, CD62L in 23 (28%) of 83, CD183 in 66 (80%) of 83, BCL2 in 33 (39%) of 84, cutaneous lymphocyte antigen in 21 (25%) of 84, granzyme B in 70 (83%) of 84, Ki-67 in 59 (71%) of 83, linker for activation of T cells in 60 (71%) of 84, perforin in 66 (86%) of 77, TIA1 in 76 (90%) of 84, and EBV in 73 (87%) of 84. Hierarchical cluster analysis separated primary cutaneous cases from cases manifesting in other sites based on lower expression of the cell adhesion molecule CD54.

Characterization of D-cyclin proteins in hematolymphoid neoplasms: lack of specificity of cyclin-D2 and D3 expression in lymphoma subtypes

D-cyclin proteins play a central role in cell-cycle regulation and are involved in the pathogenesis of lymphomas. In mantle-cell lymphoma, the t(11;14) translocation leads to overexpression of cyclin-D1, in addition to which cyclin-D1-negative mantle-cell lymphoma that overexpress cyclin-D2 or D3 have also been described. Although cyclin-D2 and D3 have been implicated in the prognosis of specific lymphoma subtypes, a thorough characterization of D-cyclin protein expression in human hematolymphoid neoplasia has not been reported. To evaluate the tissue expression patterns of D-cyclins, particularly D2 and D3, in normal and neoplastic hematolymphoid tissues, we optimized the commercially available antibodies for D-cyclins for use on paraffin-embedded tissue and stained tissue microarrays of over 700 patient samples. Our results show that cyclin-D2 and D3 proteins are expressed in many more lymphoma subtypes than cyclin-D1. Cyclin-D1, D2 and D3 were expressed in 100, 22 and 6% of mantle-cell lymphomas and 2, 49 and 20% of diffuse large B-cell lymphomas. Fluorescence in situ hybridization studies confirmed the presence of the CCND1/IGH translocation in the majority of mantle-cell lymphoma, but not in diffuse large B-cell lymphoma that expressed cyclin-D1 protein. In addition, a subset of follicular, marginal zone, lymphoplasmacytic, lymphoblastic, classical Hodgkin, mature T-cell and natural killer cell lymphomas and acute myeloid leukemias also expressed cyclin-D2 and D3. These data support the hypothesis that dysregulation of cell-cycle control by D-cyclins contribute to the pathogenesis of hematolymphoid neoplasia, and suggest a potential role for these proteins in the prognostic and therapeutic aspects of these diseases. For diagnostic purposes, however, the expression of D-cyclin proteins should be interpreted with caution in the subclassification of lymphoma types.

Germinal centre protein HGAL promotes lymphoid hyperplasia and amyloidosis via BCR-mediated Syk activation

The human germinal centre associated lymphoma (HGAL) gene is specifically expressed in germinal centre B-lymphocytes and germinal centre-derived B-cell lymphomas, but its function is largely unknown. Here we demonstrate that HGAL directly binds Syk in B-cells, increases its kinase activity upon B-cell receptor stimulation and leads to enhanced activation of Syk downstream effectors. To further investigate these findings in vivo, HGAL transgenic mice were generated. Starting from 12 months of age these mice developed polyclonal B-cell lymphoid hyperplasia, hypergammaglobulinemia and systemic reactive AA amyloidosis, leading to shortened survival. The lymphoid hyperplasia in the HGAL transgenic mice are likely attributable to enhanced B-cell receptor signalling as shown by increased Syk phosphorylation, ex vivo B-cell proliferation and increased RhoA activation. Overall, our study shows for the first time that the germinal centre protein HGAL regulates B-cell receptor signalling in B-lymphocytes which, without appropriate control, may lead to B-cell lymphoproliferation.

TdT + T-lymphoblastic Populations Are Increased in Castleman Disease, in Castleman Disease in Association With Follicular Dendritic Cell Tumors, and in Angioimmunoblastic T-cell Lymphoma

T-lymphoblastic lymphoma is an aggressive neoplasm requiring prompt clinical treatment. Conversely, indolent T-lymphoblastic proliferation mimics T-lymphoblastic lymphoma but consists of a proliferation of non-neoplastic TdT+ T cells, requiring no treatment. Recently, we identified several cases of indolent T-lymphoblastic proliferations in extrathymic lymphoid tissues: 1 in a patient suffering from Castleman disease (CD) associated with a follicular dendritic cell sarcoma/tumor, 1 in a patient with a history of angioimmunoblastic T-cell lymphoma (AITL), and 1 in association with acinic cell carcinoma. Interestingly, in the case of the patient with a history of AITL, these TdT+ T cells were seen in multiple anatomic sites over the span of 5 years. Here we review these 3 cases and extend our findings by demonstrating that TdT+ T-lymphoblastic populations are increased in lymph nodes of patients with CD (P=0.011), CD in association with follicular dendritic cell tumors, and AITL (P<0.01) compared with other T-cell or B-cell lymphomas or reactive lymph nodes. Finally, analysis of 352 nonhematolymphoid tumors including carcinomas, melanomas, and sarcomas demonstrates that TdT+ T cells are not increased in these tumors. Our studies not only present several detailed cases of indolent T-lymphoblastic proliferations, but also correlate these populations with specific hematologic diseases.

Lymphoma cell VEGFR2 expression detected by immunohistochemistry predicts poor overall survival in diffuse large B cell lymphoma treated with immunochemotherapy (R-CHOP)

Diffuse large B cell lymphoma (DLBCL) is clinically and biologically heterogeneous. In most cases of DLBCL, lymphoma cells co‐express vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, suggesting autocrine in addition to angiogenic effects. We enumerated microvessel density and scored lymphoma cell expression of VEGF, VEGFR1, VEGFR2 and phosphorylated VEGFR2 in 162 de novo DLBCL patients treated with R‐CHOP (rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone)‐like regimens. VEGFR2 expression correlated with shorter overall survival (OS) independent of International Prognostic Index (IPI) (P = 0·0028). Phosphorylated VEGFR2 (detected in 13% of cases) correlated with shorter progression‐free survival (PFS, P = 0·044) and trended toward shorter OS on univariate analysis. VEGFR1 was not predictive of survival on univariate analysis, but it did correlate with better OS on multivariate analysis with VEGF, VEGFR2 and IPI (P = 0·036); in patients with weak VEGFR2, lack of VEGFR1 coexpression was significantly correlated with poor OS independent of IPI (P = 0·01). These results are concordant with our prior finding of an association of VEGFR1 with longer OS in DLBCL treated with chemotherapy alone. We postulate that VEGFR1 may oppose autocrine VEGFR2 signalling in DLBCL by competing for VEGF binding. In contrast to our prior results with chemotherapy alone, microvessel density was not prognostic of PFS or OS with R‐CHOP‐like therapy.

Extracellular Signal-Regulated Kinase Positively Regulates the Oncogenic Activity of MCT-1 in Diffuse Large B-Cell Lymphoma

The MCT-1 oncogene was originally identified from lymphoma cell lines. Herein we establish that MCT-1 is highly expressed in 85% of human diffuse large B-cell lymphomas (DLBCL) and that knocking down MCT-1 by a specific short hairpin RNA in DLBCL cells induces apoptosis, supporting a critical role for MCT-1 in DLBCL cell survival. However, the mechanism underlying MCT-1 regulation is largely unknown. We find that MCT-1 is phosphorylated and up-regulated by extracellular signal-regulated kinase (ERK). Furthermore, by using a small inhibitory molecule targeting ERK, we interrupted MCT-1 phosphorylation and stability. Significantly, cells with distinct levels of MCT-1 protein displayed differential sensitivity to ERK inhibitor-induced apoptosis. Treatment with the ERK inhibitor showed marked in vivo antitumor activity in a human DLBCL xenograft model. Our findings establish a functional molecular interaction between MCT-1 and the MEK/ERK signaling pathway and suggest that the activation of MCT-1 function by its upstream kinase ERK plays an important role in lymphomagenesis.

CD137 Is Expressed in Follicular Dendritic Cell Tumors and in Classical Hodgkin and T-Cell Lymphomas: Diagnostic and Therapeutic Implications

CD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy.