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Megan S Lim

Publications and source records attributed to Megan S Lim.

36 records · Page 2Linked to original sources
PubMed2004

Proteomics in pathology research.

Proteomics is a multifaceted approach to study various aspects of protein expression, post-translational modification, interactions, organization and function at a global level. While DNA constitutes the 'information archive of the genome', it is the proteins that actually serve as the functional effectors of cellular processes. Thus, analysis of protein derangements on a proteome-wide scale will reveal insights into deregulated pathways and networks involved in the pathogenesis of disease. Although the field of proteomics has advanced tremendously in recent years, there are significant technical challenges that pose limitations to the routine application of mass spectrometry to clinical research. Despite these challenges, proteomic studies have yielded unparalleled information and understanding of the cellular biology of diseased states. The application of mass spectrometry to the study of diseases will ultimately lead to identification of biomarkers that are critical for the detection, diagnosis, prognosis and treatment of specific disease entities.

Biomedical Research↗
PubMed2004

Application of SELDI-TOF mass spectrometry for the identification of differentially expressed proteins in transformed follicular lymphoma.

Completion of the human genome project has focused scientific attention on the development of methods that permit rapid characterization of proteins that are encoded by the genome. Recent improvements in two-dimensional separation techniques in combination with protein identification software/databases and mass spectrometry (MS) now permit rapid comprehensive large-scale analysis of individual proteins within complex protein mixtures. We have performed pairwise comparisons of low-grade and transformed follicular lymphomas (FLs) in order to identify proteins that may be involved in FL progression using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometer (ProteinChip, Ciphergen Biosystems). This system utilizes preactivated differential binding surfaces to achieve multidimensional chromatography. The protein-bound chips were then analyzed by a SELDI-TOF mass spectrometer to generate protein profiles. In preliminary experiments, we established that the MS data obtained from SELDI-TOF MS were reproducible, and that reduction in sample complexity improved the ability to detect lower abundance proteins. With specific regard to FL transformation, we rapidly identified a number of potential candidate proteins involved in this process. These included an upregulated 32 kDa protein and a down-regulated 11.8 kDa protein. Protein database searches revealed several candidates, among them cyclin D3 (32.5 kDa) and caspase 3 (11.8 kDa) whose differential expression were confirmed by immunoblotting and/or immunohistochemical analysis on the primary tissue specimens. Our studies demonstrate the utility of SELDI-TOF-MS for the rapid discovery of differentially expressed proteins using femtomolar quantities of crude protein derived from biopsy material. The versatility of this methodology supports its application to the rapid discovery of potential biomarkers in a variety of cellular systems.

Antigens, CD20↗
PubMed2004

Ubiquitin ligases in malignant lymphoma.

The highly controlled degradation of proteins via the ubiquitin-proteasome pathway represents a key mechanism for cell regulation and homeostasis. Ubiquitin-dependent proteolysis, carried out in large part by the E3 ubiquitin ligases, is a critical mode of post-translational modification that is important in regulation of cell cycle progression, signal transduction, gene transcription, antigen receptor signaling, immune response and cell differentiation. Recent studies demonstrate that increasing numbers of proteins with ubiquitin ligase activity are being characterized. Identification and characterization of their substrates indicate that they regulate the turnover of key cell cycle proteins (p27Kip1, p21Cip1, p57Kip2, cyclin E), tumor suppressor proteins (p53, RB), signaling kinases (Src, Zap70, PI-3 kinase), apoptosis regulators (Bcl-2, Bax, Bik) and transcription factors (Myc, NF-kappaB, E1F1), all of which have been implicated in the pathogenesis of malignant lymphoma. Studies to determine the functional role of ubiquitin ligases in the pathogenesis of malignant lymphoma represent potential areas of investigation.

Animals↗
PubMed2004

T-cell posttransplant lymphoproliferative disorder occurring in a pediatric solid-organ transplant patient.

Posttransplantation lymphoproliferative disorder (PTLD) is a serious complication seen in transplant patients as a consequence of immunosuppressant therapy. Most cases are of B-cell origin and are commonly associated with Epstein-Barr virus (EBV) infection. T-cell PTLDs are rare and only 13 pediatric T-cell PTLDs with clinicopathologic correlation have been reported previously. We present the histologic, immunophenotypic, and molecular features of a monomorphic PTLD (T-cell lymphoma) identified in a pediatric patient following orthotopic liver transplantation. The lymphoma was identified in the ileum, rectum, and mesenteric lymph nodes. In situ hybridization revealed numerous EBER-1-positive tumor cells. A current review of the literature is also discussed. Of the 14 cases of pediatric T-cell PTLD reported in the literature, only 3 (21.4%) are described as being EBV positive. Most of the reported PTLDs are monoclonal, with 9 of 11 cases (82%) showing a clonal T-cell population by gene rearrangement studies. T-cell PTLD cases appear to have a poor prognosis (11 of 14 patients died of the disease), although patients with involvement of specific anatomic sites may have a better outcome.

Adolescent↗
PubMed2003

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy.

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin's lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38beta-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.

Apoptosis↗
PubMed2003

Growth regulation by p27Kip1 is abrogated by multiple mechanisms in aggressive malignant lymphomas.

The cyclin-dependent kinase inhibitor p27Kip1 is a key regulator of the G1/S transition, and an inverse relationship between p27Kip1 protein expression and proliferation index has been reported in malignant lymphomas. However, a subset of aggressive B-cell lymphomas demonstrates high p27Kip1 expression despite a high proliferation index. The aim of this study was to determine potential mechanisms by which lymphoma cells abrogate the growth inhibitory effect of high p27Kip1. The effect of transforming growth factor-beta (TGF-beta) and serum stimulation on p27Kip1 expression and cyclin E/cdk2 activity was investigated in four lymphoma cell lines, Jurkat, CEM-6, OCI-Ly1 and Nalm-6. Reactive lymphocytes responded to growth inhibitory TGF-beta by inducing p27Kip1 expression, with subsequent accumulation of cells in G0/G1. In contrast, TGF-beta did not alter the level of p27Kip1 in Jurkat, CEM-6 and OCI-Ly1 cells with no change in cyclin E/cdk2-kinase activity. Serum stimulation also did not result in a significant change in p27Kip1 expression. Western blot analysis of subcellular fractions demonstrated cytoplasmic p27Kip1, corroborated by immunocytochemistry in a subset of the lymphoma cells. Sequestration of p27Kip1 by cyclin D3 was observed in the nuclear and cytoplasmic fractions of Nalm-6, OCI-Ly-1 and NCEB cells. These results indicate that multiple mechanisms contribute to the abrogation of growth regulation by unscheduled high p27Kip1 protein expression including deficient response to TGF-beta and serum, sequestration by cyclin D3 and cytoplasmic displacement.

Blotting, Western↗
PubMed2003

BCL-2 is consistently expressed in hyperplastic marginal zones of the spleen, abdominal lymph nodes, and ileal lymphoid tissue.

BCL-2 is an antiapoptotic protein overexpressed in follicular lymphomas, principally as a result of the t(14;18)(q32;q21), and useful in distinguishing follicular lymphoma (usually BCL-2 positive) from follicular hyperplasia (BCL-2 negative). BCL-2 is also overexpressed in other lymphoma types without the t(14;18), including marginal zone B-cell lymphoma, because of other, poorly understood mechanisms. It has been suggested that BCL-2 immunoreactivity can distinguish between malignant (BCL-2 positive) and reactive (BCL-2 negative) marginal zone B cells. In this study, we evaluated 26 spleen, 10 abdominal lymph node, and 3 ileum specimens with marginal zone B-cell hyperplasia for BCL-2 expression immunohistochemically. We also analyzed these cases using polymerase chain reaction methods to evaluate for the presence of clonal rearrangements of the immunoglobulin heavy chain gene (IgH) using consensus V FRIII and J region primers, and the t(14;18) involving both the major breakpoint and the minor cluster regions of the bcl-2 gene. All (100%) cases of splenic, abdominal lymph node, and ileal marginal zone hyperplasia displayed strong BCL-2 reactivity in the marginal zone B cells. In all cases analyzed, IgH polymerase chain reaction demonstrated a polyclonal pattern, and bcl-2/JH DNA fusion sequences were not detected. Our results indicate that BCL-2 is consistently expressed by reactive marginal zone B cells of the spleen, abdominal lymph nodes, and ileal lymphoid tissue and should not be used as a criterion for discriminating between benign and malignant marginal zone B-cell proliferations involving these sites.

Abdomen↗
PubMed2003

Comparative microarray analysis of gene expression during activation of human peripheral blood T cells and leukemic Jurkat T cells.

Activation of T cells involves a complex cascade of signal transduction pathways linking T-cell receptor engagement at the cell membrane to the transcription of multiple genes within the nucleus. The T-cell leukemia-derived cell line Jurkat has generally been used as a model system for the activation of T cells. However, genome-wide comprehensive studies investigating the activation status, and thus the appropriateness, of this cell line for this purpose have not been performed. We sought to compare the transcriptional profiles of phenotypically purified human CD2(+) T cells with those of Jurkat T cells during T-cell activation, using cDNA microarrays containing 6912 genes. About 300 genes were up-regulated by more than 2-fold during activation of both peripheral blood (PB) T cells and Jurkat T cells. The number of down-regulated genes was significantly lower than that of up-regulated genes. Only 79 genes in PB T cells and 37 genes in Jurkat T cells were down-regulated by more than 2-fold during activation. Comparison of gene expression during activation of Jurkat and PB T cells revealed a common set of genes that were up-regulated, such as Rho GTPase-activating protein 1, SKP2, CDC25A, T-cell specific transcription factor 7, cytoskeletal proteins, and signaling molecules. Genes that were commonly down-regulated in both PB T cells and Jurkat T cells included CDK inhibitors (p16, p19, p27), proapoptotic caspases, and the transcription factors c-fos and jun-B. After activation, 71 genes in PB T cells and only 3 genes in Jurkat T cells were up-regulated 4-fold or more. Of these up-regulated genes and expressed sequence tags, 44 were constitutively expressed at high levels in nonactivated Jurkat cells. Quantitative real-time RT-PCR analysis confirmed our microarray data. Our findings indicate that although there is significant overlap in the activation-associated transcriptional profiles in PB T cells compared with Jurkat T cells, there is a subset of genes showing differential expression patterns during the activation of the two cell types.

Antigens, CD↗
PubMed2003

Analysis of the PI-3-Kinase-PTEN-AKT pathway in human lymphoma and leukemia using a cell line microarray.

Tissue microarray technology facilitates rapid assessment of expression of molecular markers by enabling the simultaneous analysis of hundreds of tissue specimens. We have applied this technology to establish a microarray composed of cell pellets derived from 40 human lymphoma/leukemia-derived cell lines harboring a variety of molecular abnormalities. The application of cell line microarrays to the assessment of biologic marker evaluation was validated by studying the immunohistochemical expression of PTEN and phosphorylated AKT, two mediators of the phosphatidylinositol (PI)-3-kinase pathway. In addition to the high throughout analysis of protein expression in lymphoma/leukemia cells, this methodology also enables the evaluation of subcellular localization of protein expression. Cytoplasmic PTEN expression was observed in the majority of cell lines (87%), whereas a minor subset demonstrated nuclear expression. Phosphorylated AKT was also expressed predominantly within the cytoplasm in 65% of cell lines, whereas nuclear expression was seen in a minority. An inverse relationship between PTEN and phosphorylated AKT was observed in 63% of cell lines. No cell lines showed absence of PTEN expression, whereas 50% of cell lines showed low PTEN expression. Our data support the integrity of the PI-3-kinase-PTEN-AKT pathway in a majority of cell lines derived from hematologic malignancies and clearly demonstrates the utility of microarray technology in the in situ assessment of expression of molecular markers in tumor-derived cell lines.

Biomarkers, Tumor↗
PubMed2003

Selective inhibition of cyclooxygenase 2 induces p27kip1 and skp2 in oral squamous cell carcinoma.

It has been shown that inhibition of cyclooxygenase 2 (COX-2) may cause growth arrest and reduced tumour formation in some human cancers; however, the mechanism is not fully known. In this study, we used an oral squamous cell carcinoma cell line to study growth inhibition and changes in critical cell cycle-regulating proteins induced by the selective COX-2 inhibitor celecoxib. Using cell viability assay, we established the optimal in vitro inhibitory dose of celecoxib and showed that inhibition of COX-2 markedly induces the expression of p27kip1, p21, waf1, and the F-box protein skp2. These results add new experimental data to our knowledge of the mechanism of cyclooxygenases on neoplastic cells.

Carcinoma, Squamous Cell↗
PubMed2003

High-throughput analysis of protein/peptide complexes by immunoprecipitation and automated LC-MS/MS.

BACKGROUND: The identification of interacting proteins within protein complexes is key to understanding the transduction and regulation of cell signaling pathways, and is also a useful tool for identifying novel disease markers. Immunoprecipitation of protein complexes followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been used to identify targets that bind to a protein of interest. Here, we report a high-throughput nanoflow LC-MS/MS method for analysis of protein/peptide complexes. APPROACH: To overcome the large dwell volume from connecting lines and the injector when using an autosampler in microcapillary LC-MS/MS, we employed a valve-controlled variable flow method with a peptide trap. This method enables fast trapping of peptides from samples injected by an autosampler within minutes followed by a split-controlled nanoflow of acetonitrile gradient through a microcapillary C18 column to separate and elute peptides into the ion-trap MS/MS. Over 40 protein/peptide samples at femtomole levels could be analyzed continuously using the same in-house packed microcapillary C18 column. The p38 mitogen-activated protein kinase (p38 MAP kinase) is a signaling protein that is involved in transduction of extracellular signals including oxidative stress, growth factors, and cytokines, with diverse cellular consequences such as cell proliferation, differentiation, or apoptosis. Immunocomplex of monoclonal anti-p38 beta antibodies from 2 mg total lysates from the OCI LY-1 lymphoma cell line was resolved in 1D-PAGE gel followed by silver staining of the gel. Protein bands were excised and digested with trypsin. Peptides were extracted and analyzed using the automated LC-MS/MS method. RESULTS AND CONCLUSIONS: From 37 excised protein bands in the 1D-PAGE gel, we identified more than 50 proteins, including cytoskeletal proteins, ribosomal proteins, transcription factors, and KIAA potential signaling proteins. These proteins are the potential targets that may interact directly or indirectly with the p38 MAP kinase proteins. Our studies demonstrate the utility of automated nanoflow LC-MS/MS for sensitive and high-throughput analysis of protein/peptide complexes. Utilization of methods based on this principle would greatly facilitate peptide interaction mapping and other proteomic studies requiring high-throughput pre-analytical sample preparation.

Actins↗
PubMed2002

Expression of Skp2, a p27(Kip1) ubiquitin ligase, in malignant lymphoma: correlation with p27(Kip1) and proliferation index.

Reduced levels of p27(Kip1) are frequent in human cancers and have been associated with poor prognosis. Skp2, a component of the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex, has been implicated in p27(Kip1) degradation. Increased Skp2 levels are seen in some solid tumors and are associated with reduced p27(Kip1). We examined the expression of these proteins using single and double immunolabeling in a large series of lymphomas to determine if alterations in their relative levels are associated with changes in cell proliferation and lymphoma subgroups. We studied the expression of Skp2 in low-grade and aggressive B-cell lymphomas (n = 86) and compared them with p27(Kip1) and the proliferation index (PI). Fifteen hematopoietic cell lines and peripheral blood lymphocytes were studied by Western blot analysis. In reactive tonsils, Skp2 expression was limited to proliferating germinal center and interfollicular cells. Skp2 expression in small lymphocytic lymphomas (SLLs) and follicular lymphomas (FCLs) was low (mean percentage of positive tumor cells, less than 20%) and was inversely correlated (r = -0.67; P <.0001) with p27(Kip1) and positively correlated with the PI (r = 0.82; P <.005). By contrast, whereas most mantle cell lymphomas (MCLs) demonstrated low expression of p27(Kip1) and Skp2, a subset (n = 6) expressed high Skp2 (exceeding 20%) with a high PI (exceeding 50%). Skp2 expression was highest in diffuse large B-cell lymphomas (DLBCLs) (mean, 22%) and correlated with Ki-67 (r = 0.55; P <.005) but not with p27(Kip1). Cytoplasmic Skp2 was seen in a subset of aggressive lymphomas. Our data provide evidence for p27(Kip1) degradative function of Skp2 in low-grade lymphomas. The absence of this relationship in aggressive lymphomas suggests that other factors contribute to deregulation of p27(Kip1) expression in these tumors.

Cell Cycle Proteins↗
PubMed2002

Gene expression profiling of cell lines derived from T-cell malignancies.

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated genes (greater than two-fold) in the lymphomas as a group when compared to CD4-positive T-cells. These included genes involved in cytokine signaling, cell adhesion, cytoskeletal elements, nuclear transcription factors, and known oncogenes and tumor suppressor genes. Quantitative fluorescent reverse transcription-polymerase chain reaction analysis demonstrated 70% concordance with the microarray results. While freshly isolated malignant cells may differ in their individual expression patterns relative to established cell lines from the same diagnoses, we feel that the variety of different lymphocytic cell lines that we examined provides a representative picture of the molecular pathogenesis of T-cell malignancies.

CD4-Positive T-Lymphocytes↗
PubMed2002

Fluorescence PCR quantification of cyclin D1 expression.

We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A beta-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL.

Beta-Globulins↗
PubMed2002

Microarray analysis of B-cell lymphoma cell lines with the t(14;18).

The t(14;18) is the most common genetic alteration in follicular lymphoma, and is detectable in a subset of diffuse large B-cell lymphomas (DLBCL), resulting in over-expression of the anti-apoptotic protein BCL-2. Although the t(14;18)-induced over-expression of BCL-2 is an important step in lymphomagenesis, this aberration alone is not sufficient to produce malignant lymphoma. Further analysis of these tumors is needed to identify additional genes that might be involved in the genesis of follicular lymphoma and progression to DLBCL. To address this issue, we analyzed the gene expression profiles of four t(14;18)-positive cell lines and two t(11;14)-positive mantle-cell lymphoma cell lines using cDNA microarrays containing 4364 genes, and compared them to the genetic profile of phenotypically purified B-cells obtained from hyperplastic tonsils. A total of 137 genes were differentially expressed by approximately twofold or more in the t(14;18) cell lines relative to tonsillar B-cells. 68 genes were up-regulated, 69 genes were down-regulated, and approximately 20% of the differentially regulated genes had no known function. The up-regulated genes included a number of genes involved in the promotion of cellular proliferation and survival, as well as cell metabolism. Down-regulated genes included mediators of cell adhesion and negative regulators of cell activation and growth. Hierarchical clustering analysis separated the t(14;18) and mantle-cell lines into distinct groups based on their gene expression profiles. We confirmed the differential expression of approximately 80% of selected up- and down-regulated genes identified by microarray analysis by quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) analysis and/or immunoblotting. This study demonstrates the utility of cDNA microarray analysis for the assessment of global transcriptional changes that characterize t(14;18)-positive cell lines, and also for the identification of novel genes that could potentially contribute to the genesis and progression of non-Hodgkin's lymphomas with this translocation.

Base Sequence↗
PubMed2002

T-cell analysis in identical twins reveals an impaired anti-follicular lymphoma immune response in the patient but not in the healthy twin.

In lymphomas an innate defect in the T-cell repertoire could account for the impaired tumour-specific immune response; alternatively, the tumour itself could exert an inhibitory effect on the immune system. To address this issue we analysed the T-cell responses against follicular lymphoma (FL) in identical twins as it can be postulated that their overall T-cell repertoire is identical. While differences between the T-cell response of the patient and the healthy twin would point to a tumour-induced T-cell unresponsiveness, impaired responses in both would point to a defective T-cell repertoire. We demonstrated an impaired tumour-specific proliferation (P = 0.035 and P = 0.013) and cytokine release (P = 0.004 and P = 0.0008) of both peripheral blood and tumour-derived T-cells, respectively, in the FL patient compared with the T-cell response of the healthy twin. Moreover, only syngeneic primed T cells were able to directly lyse unmodified FL cells of the patient. These data support previous findings in murine lymphomas and suggest that inhibitory mechanisms during tumour growth, rather than a defective T-cell repertoire, are responsible for the insufficient T-cell response in lymphoma.

Adult↗
PubMed2002

T-cell/histiocyte-rich large B-cell lymphoma: a heterogeneous entity with derivation from germinal center B cells.

T-cell/histiocyte-rich large B-cell non-Hodgkin's lymphoma (THRLBCL) is an unusual morphologic variant of diffuse large B-cell lymphoma. We reviewed 30 cases of THRLBCL to evaluate its heterogeneity based on morphologic, immunophenotypic, and genetic features. Cases were classified according to the appearance of the large neoplastic B cells into three morphologic variants: 1) lymphocytic and histiocytic (L&H-like) (resembling the L&H cells of nodular lymphocyte predominance Hodgkin's lymphoma (14 cases); 2) centroblast (or immunoblast)-like (10 cases), and 3) Reed-Sternberg cell-like (resembling the neoplastic cells of classic Hodgkin's lymphoma) (6 cases). We used a panel of immunohistochemical stains, including those with specificity for germinal center B cells: CD20, CD79a, CD30, CD15, epithelial membrane antigen, BCL-2, BCL-6, and CD10. The /JH polymerase chain reaction assay was further performed to investigate a relationship to follicular lymphoma. The results were correlated with Epstein-Barr virus status as determined by staining for latent membrane protein and EBER-1 in situ hybridization. All cases were of B-cell immunophenotype with strong surface CD20 reactivity in the neoplastic large lymphoid cells, although CD79a was more inconsistently and weakly expressed (10 of 17). Nuclear positivity for the BCL-6 protein was detected in the tumor cells in 26 of 29 (90%) cases. However, differences in expression of other antigens were encountered in the histologic subtypes. Epithelial membrane antigen positivity, a feature often seen in nodular lymphocyte predominance Hodgkin's lymphoma, was observed in 11 of 30 (37%) cases and was most commonly seen in cases with L&H cell morphology (8 of 14; 57%). CD30 expression was observed in 9 of 30 (30%) cases but was most frequent in cases with Reed-Sternberg-like morphology (3 of 6 [50%]). CD10 expression was infrequent overall (3 of 29; 10%), with 2 of 3 positive cases identified in the centroblastic group. The overall rarity of positivity for CD10, BCL-2 (3 of 22; 13%), and -2 JH rearrangement (1 of 28; 4%) indicates a lack of connection to follicular lymphoma for all subtypes. The three cases that were negative for BCL-6 protein were LMP-1 positive and EBER-1 positive by in situ hybridization, and 2 of 3 had neoplastic cells with Reed-Sternberg-like morphology. These results demonstrate that although a large proportion of THRLBCL represent tumors of germinal center B cell derivation, they exhibit a diversity of morphologic and immunophenotypic features. A subset of THRLBCL may be related to nodular lymphocyte predominance Hodgkin's lymphoma. A small percentage show features closely resembling classic Hodgkin's lymphoma and could be considered a variant of grey zone lymphoma.

Adult↗