Analyzing the 3D chromatin organization coordinating with gene expression regulation in B-cell lymphoma.

BACKGROUND: Eukaryotes compact chromosomes densely and non-randomly, forming three-dimensional structures. Alterations of the chromatin structures are often associated with diseases. In particular, aggressive cancer development from the disruption of the humoral immune system presents abnormal gene regulation which is accompanied by chromatin reorganizations. How the chromatin structures orchestrate the gene expression regulation is still poorly understood. Herein, we focus on chromatin dynamics in normal and abnormal B cell lymphocytes, and investigate its functional impact on the regulation of gene expression. METHODS: We conducted an integrative analysis using publicly available multi-omics data that include Hi-C, RNA-seq and ChIP-seq experiments with normal B cells, lymphoma and ES cells. We processed and re-analyzed the data exhaustively and combined different scales of genome structures with transcriptomic and epigenetic features. RESULTS: We found that the chromatin organizations are highly preserved among the cells. 5.2% of genes at the specific repressive compartment in normal pro-B cells were switched to the permissive compartment in lymphoma along with increased gene expression. The genes are involved in B-cell related biological processes. Remarkably, the boundaries of topologically associating domains were not enriched by CTCF motif, but significantly enriched with Prdm1 motif that is known to be the key factor of B-cell dysfunction in aggressive lymphoma. CONCLUSIONS: This study shows evidence of a complex relationship between chromatin reorganization and gene regulation. However, an unknown mechanism may exist to restrict the structural and functional changes of genomic regions and cognate genes in a specific manner. Our findings suggest the presence of an intricate crosstalk between the higher-order chromatin structure and cancer development.

Quantitative analysis of drug-induced complement-mediated cytotoxic effect on single tumor cells using atomic force microscopy and fluorescence microscopy.

In the antibody-based targeted therapies of B-cell lymphomas, complement-mediated cytotoxicity (CMC) is an important mechanism. CMC is activated after the binding of drugs (monoclonal antibodies) to tumor cells. The activation of CMC ultimately leads to the lysis of tumor cells. However, it remains poorly understood how CMC alters the morphology and mechanics of single tumor cells at the nanoscale. In recent years, nanoscopic observations of cellular behaviors with the use of atomic force microscopy (AFM) have contributed much to the field of cell biology. In this work, by combining AFM with fluorescence microscopy, the detailed changes in cellular ultra-microstructures and mechanical properties during the process of CMC were quantitatively investigated on single tumor cells. AFM imaging distinctly showed that the CMC effect could lead to the formation of nano holes on the tumor cells. Quantitative analysis of AFM images indicated that cell surface became lower and rougher after the CMC process. The cellular mechanics measurements showed that during the process of CMC cells firstly softened and finally stiffened, which was validated by dynamically monitoring the mechanical changes of single living cells during CMC. The experimental results provide novel insights into the antibody-dependent CMC.

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy.

The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab'effect and will facilitate the further investigation of the underlying mechanisms.

Interleukin-13 stimulation of the mediastinal B-cell lymphoma cell line Karpas-1106P induces a phenotype resembling the Hodgkin lymphoma cell line L1236.

OBJECTIVE: Primary mediastinal B-cell lymphoma (PMBCL) and Hodgkin lymphoma (HL) share many biological and clinical characteristics supporting a common pathogenetic pathway. Interleukin (IL)-13 has an important pathophysiological role in HL. In this study, we asked the question of whether IL-13 is a major contributor to the observed difference in features of inflammation between HL and PMBCL. MATERIALS AND METHODS: Expression of IL-13 and IL-4 receptors was studied by flow cytometry, expression of a functional cysteinyl leukotriene receptor type 1 (CysLT1R) was investigated by calcium flux measurement, expression and activity of 15-lipoxygenase type 1 (15-LO-1) was determined by Western blot and reversed-phase high-performance liquid chromatography, respectively, and cytokines were quantified by Bioplex detection. RESULTS: Stimulation of the PMBCL cell line Karpas-1106P with IL-13 or IL-4 induced a proinflammatory phenotype similar to that of the HL cell line L1236. Upon interleukin stimulation of the PMBCL cell line, the cellular size increased and cells became multinucleated. Cells also expressed CysLT1R and 15-LO-1, and produced the proinflammatory eoxins. The IL-13 or IL-4 treated PMBCL cell line and the HL cell line secreted a similar set of cytokines such as IL-6, tumor necrosis factor-alpha, interferon-inducible protein-10, interferon-gamma, and RANTES. CONCLUSIONS: IL-13 or IL-4 stimulation of the PMBCL cell line Karpas-1106P induced an inflammatory phenotype that resembles that of the HL cell line. Our results suggest that the autocrine release of IL-13 in HL is one critical factor that can at least partly explain the difference in phenotype between these two lymphoma entities.

Nuclear morphometry and texture analysis of B-cell non-Hodgkin lymphoma: utility in subclassification on cytosmears.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of lymphoid neoplasms and accurate subclassification is an essential prerequisite for proper management of patients. This study was aimed at evaluating the utility of nuclear morphometry and textural features on cytology smears to classify the cases of NHL on aspiration cytology. Fine needle aspiration smears of 50 cases of B-cell NHL were included. Various morphometric and texture parameters were obtained by manually tracing the nuclei on digitized images in each case and discriminant analysis performed using various features taken individually as well as all together. The percentage of cells correctly classified to a particular NHL subtype using the discriminant functions so obtained was noted.Our results show that discriminant analysis done on size parameters could correctly classify a greater number of cells than on shape parameters (36.4% vs. 21.2%, respectively). Texture parameters based on single pixel values (first order texture) were inferior (42.8%) to those based on pair of pixels (58.7%) in subtyping of cells. Discriminant analysis based on color parameters was more effective (61.9%) as compared to rest of the morphometric and textural parameters. Using all the morphometric and textural parameters together, 83.3% of cells could be correctly classified to a particular NHL subtype.The present study, perhaps the first study of detailed morphometric analysis on cytosmears, shows that satisfactory classification of NHL on aspiration cytology is possible using nuclear morphometry and textural parameters considered together. These results are promising for further studies on this subject and development of automated cytodiagnosis.

Gastric B-cell lymphoma with Mott cell differentiation in a dog.

A gastric lymphoid tumor with involvement of regional lymph nodes and spleen was diagnosed in an 8-year-old crossbreed male dog with a 6-month history of gastrointestinal disease. Despite surgical excision and palliative therapy (prednisolone and cimetidine), the dog was euthanatized due to worsening of clinical signs. At necropsy, multiple white, solid, nodular, infiltrative masses were observed in the stomach, duodenum, spleen, liver, and lungs in association with generalized lymph node enlargement. Cytology, histology, histochemistry, immunohistochemistry, and electron microscopy revealed that the neoplastic cell population was composed of B lymphocytes that contained variable amounts of round periodic acid-Schiff-positive cytoplasmic globules consistent with Russell bodies. The tumor most likely represented a variant of B-cell neoplasia with extensive Mott cell differentiation.

[Effects of infrasound therapy on proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells].

OBJECTIVE: To investigate the effect of infrasound therapy on the proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells. METHODS: Human B lymphoma Raji cells were exposed to infrasound treatment for 15, 30, 60, 90 and 120 min and cultured subsequently for 24 or 48 h. MTT assay, flow cytometry analysis, and electron microscopy were performed to examine the proliferative status, cell apoptosis and ultrastructural changes of the exposed cells, respectively. RESULTS: MTT assay revealed no significant changes in the proliferation of the cells exposed to infrasound treatment (P>0.05), nor did flow cytometry analysis identified significant variation in the cell apoptosis (P>0.05). Scanning electron microscopy, however, identified shortened or reduced cell processes and microvilli on the surface of the cells with infrasound exposure and a subsequent 24-hour culture, and the cell membrane surface became smooth. Under transmission electron microscope, the cells with infrasound treatment presented with significantly reduced microvilli, and the cell nuclei appeared homogeneous, with cytoplasmic budding and losses after a 48-hour culture. CONCLUSION: Infrasound less than 90 dB does not obviously affect the proliferation and apoptosis of Raji cells, but may directly cause cell ultrastructural changes such as reduction of the cell processes.

Precursor B-1 B cell lymphoma in a newborn calf.

A newborn Holstein female calf had neoplastic lesions in the skin and within the thoracic and abdominal cavities but not in the bone marrow, spleen, thymus, or most lymph nodes. Because the tumor cells were positive for CD79a (B cell marker), CD5 (B-1 cell marker) and terminal deoxynucleotidyl transferase (marker for immature lymphoid precursors), a diagnosis of precursor B-1 B cell lymphoma was made. The diagnosis was strongly supported by the fact that B-1 cells can develop in the fetus, unlike B-2 cells, which are produced after birth. The lymphoma was distinct from the typical calf form of lymphoma of B-2 cell origin, which does not express CD5 and is characterized by generalized lymphadenopathy and involvement of the bone marrow, blood and spleen.

The ultrastructural features of plasmablastic lymphoma.

Plasmablastic lymphoma was originally described in 1997 by Delecluse et al. and is an aggressive variant of diffuse large B-cell non-Hodgkin lymphoma seen predominantly in a setting of acquired immunodeficiency syndrome and nearly always in extranodal sites. The authors have seen 10 cases in their department between 2001 and 2005. The patients' ages ranged from 24 to 39 years and there were 7 females and 3 males. In 7 cases where human immunodeficiency virus had been tested it was positive. Eight cases were extranodal and 2 cases involved lymph nodes. Five cases were followed up and 4 were confirmed dead within 5 months of diagnosis, verifying the aggressive nature of this condition. Histology showed large, polygonal tumor cells some of which had a slightly plasmacytic appearance. Six cases had a "starry sky" background. Immunohistochemical stains were negative in 1 case, while the other 9 cases were positive for CD138. Electron microscopy showed concentrically arranged rough endoplasmic reticulum in the cytoplasm in 9 cases. In 1 case the cells were too degenerate for evaluation. This study shows that the ultrastructural features are well developed and can help in distinguishing plasmablastic lymphoma from other light microscopically undifferentiated tumors.

Expression pattern of intracellular leukocyte-associated proteins in primary mediastinal B cell lymphoma.

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

Immunophenotype and ultrastructure of B-cell lymphoproliferative disorder with cytoplasmic projection.

To identify the knowledge of rare lymphoproliferative disorder, the clinical and biological features of three kinds of lymphoproliferative disorders with cytoplasmic projections were compared. The clinical manifestations, ultrastructure and immunophenotype were analyzed. The results showed that hairy cell leukemia (HCL), splenic lymphoma with villous lymphocyte (SLVL) and hairy cell leukemia-variant (HCL-V) had some common characters including splenomegaly, peripheral blood and bone marrow infiltration by villous lymphocyte and B lymphocyte immunophenotype; but these three disorders had specific features respectively. It was concluded that overall analysis of clinical and laboratory features might be contributive to the differential diagnosis of these three disorders.

B-cell non-Hodgkin's lymphomas express heterogeneous patterns of neovascularization.

BACKGROUND AND OBJECTIVES: The role of angiogenesis in the growth and survival of hematologic malignancies was not clear until recently. We have previously demonstrated in beta-cell non Hodgkin's lymphomas (B-NHL), that neoplastic progression, as defined by its increasing malignancy grades, is clearly related to the degree of angiogenesis. DESIGN AND METHODS: In the present study we used transmission electron microscopy to examine the ultrastructural patterns of neovascularization in both low and high grade B-NHL). In low grade B-NHL the vessel lumen was formed either by endothelial cell body curving or, more frequently, by the fusion of intracellular vacuoles in poorly differentiated endothelial cells. In high grade B-NHL, on the other hand, the predominant neo-angiogenic pattern was the formation of a slit-like lumen. A remarkable ultrastructural feature in high-grade B-NHL was the intimate relationship between endothelial and tumor cells. Both low and high grade B-NHL exhibited development of transluminal bridges in larger vessels, leading to the division of the vessel. INTERPRETATION AND CONCLUSIONS: These in situ data suggest that the intrinsic process of endothelial cell proliferation and vessel formation in the stroma of B-NHL varies according to the grade of tumor malignancy and point to the important role played by the specific organ microenvironment, which determines the extent of cancer cell proliferation, angiogenesis and invasion. In fact, the microenvironment has an important influence on vascular architecture, i.e., the properties of tumor cells can also determine the outcome of the angiogenic response. Taken together our findings, which provide quantitative data on different vascular patterns in B-NHL, have potential implications for vascular targeting and cancer therapy.

Characterization of complex chromosomal abnormalities in B-cell lymphoma by a combined spectral karyotyping (SKY) analysis and fluorescence in situ hybridization (FISH) using a 14q telomere probe.

We report a case of non-Hodgkin's lymphoma of unknown origin with invasion into bone marrow and brain. This case showed complex chromosomal abnormalities, including five clonal marker chromosomes (mar) and four additional materials of unknown origin (add) that could not be identified by means of conventional G-banding. Spectral karyotyping (SKY) analysis could not only determine the origin and organization of all thus far unidentified structural chromosomal abnormalities but also detect two cryptic unbalanced translocations, which had been erroneously considered to be normal on the basis of G-banding analysis, and correct one abnormality misidentified by G banding. Among these abnormalities, we identified the new partner site of the 14q32 translocation, 22q13, and the jumping translocations involving 2p23 as a new donor chromosome. Furthermore, by using fluorescence in situ hybridization (FISH) with the probes specific for the 14q telomere, we could identify the unbalanced translocation of t(3;14)(q27;q32), which had been erroneously considered to be normal chromosome 3 on the basis of not only G-banding but also of SKY analysis. This translocation is one of the most frequent chromosomal abnormalities in B-cell lymphoma, especially diffuse large cell lymphoma. After SKY and FISH analysis, the original descriptions in the G-band karyotype were modified for a total of 13 chromosomes. The combination of SKY and FISH using the 14q telomere probe was therefore considered very useful for the characterization of complex cytogenetic cases in B-cell lymphoma.

Pituitary lymphoma: a case report and literature review.

We report the case of a B-cell type pituitary lymphoma in a 65 year-old male immunocompetent patient who presented with hypogonadotropic hypogonadism and central hypothyroidism and subsequently developed pulmonary lymphoma. Only three cases of pituitary lymphoma have been previously reported, one in a patient with acquired immunodeficiency syndrome, one case of T-cell lymphoma reported in the Japanese literature, and one case of B-cell lymphoma. The previously reported immunocompetent patients presented with signs and symptoms of optic chiasm compression as contrasted to our patient's endocrinologic presentation. B-cell lymphoma of the pituitary gland is a exceedingly rare though distinct clinical entity.

Distinguishing B and T lymphocytes by scanning electron microscopy.

This work demonstrates differences between B lymphocytes and T lymphocytes as seen in the scanning electron microscope (SEM). Slides of routinely prepared benign and malignant lymphoid tissues were viewed in the SEM. The location of prominent populations of T lymphocytes and B lymphocytes was verified by immunocytochemical staining respectively with CD3 and CD20 antisera. Benign tissues, including infant thymus and adult hyperplastic lymph node, were compared with malignant lymphomas of T-cell and B-cell types. The SEM appearance of benign B and T lymphocytes is compared utilizing the backscattered electron (BSE) mode and secondary electron (SE) mode in adult hyperplastic lymph node and infant thymus, respectively. The BSE and SE modes reveal that the sectioned T-lymphocyte nucleus has a more complex configuration than that of the B lymphocyte. T lymphocytes appear more discrete and separated one from another, while B lymphocytes exhibit close cellular association to form a syncytial array. These features are noted also in malignant lymphomas of B-lymphocyte and T-lymphocyte types, respectively. The SEM can distinguish between B and T lymphocytes by studying the differences in nuclear and chiefly the cell to cell appearances. The syncytial configuration of B lymphocytes may reflect prominent expression of cell adhesion molecules, e.g., ICAM-1, as noted in the literature.

Basement-membrane-related peri-vascular matrices not organised as a basal lamina: distribution in malignant tumours and benign lesions.

Peri-vascular matrices having a finely textured granular substructure have been identified in 27 human lesions: these were mostly malignancies but included benign tumours and reactive processes. The matrices were defined as stromal components surrounding endothelium and pericytes, and lying between vessels and adjacent lesional cells. They were identified as having a finely textured, uniform and moderately dense substructure, and differed from a conventional basal lamina expected at these sites by the absence of the typical lamina densa/lamina lucida configuration. By light microscope immunohistochemistry, vessels stained positively for laminin and collagen IV, two of the main proteins characterising a conventional basal lamina. The present observations emphasise the following. 1) The proteins laminin and collagen IV can be found in peri-vascular locations which have a finely textured granular substructure, and which have clearly defined ultrastructural differences from a conventional basal lamina. 2) While conventional light microscope immunohistochemistry demonstrates the presence and cellular location of proteins, electron microscopy is helpful for giving information on their physical organisation. 3) Peri-vascular granular matrices have a widespread distribution in malignant tumours but also exist in benign tumours and reactive lesions. This paper briefly discusses the possible functions of these matrices as modulators of cell biological processes.

Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas.

Node biopsies of 30 benign lymphadenopathies and 71 B-cell non-Hodgkin's lymphomas (B-NHLs) were investigated for microvessel and macrophage counts using immunohistochemistry and morphometric analysis. Both counts were significantly higher in B-NHL. Moreover, when these were grouped into low-grade and high-grade lymphomas, according to the Kiel classification and Working Formulation (WF), statistically significant higher counts were found in the high-grade tumours. Immunohistochemistry and electron microscopy revealed a close spatial association between microvessels and macrophages. Overall, the results suggest that, in analogy to what has already been shown in solid tumours, angiogenesis occurring in B-NHLs increases with tumour progression, and that macrophages promote the induction of angiogenesis via the release of their angiogenic factors.