R-CHOEP-14 improves overall survival in young high-risk patients with diffuse large B-cell lymphoma compared with R-CHOP-14. A population-based investigation from the Danish Lymphoma Group.

BACKGROUND: Optimal treatment of young patients with high-risk diffuse large B-cell lymphoma (DLBCL) remains a matter of debate and requires improvement. The combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) with addition of etoposide (CHOEP) has in other patient groups been shown to be effective. Further improvement has been accomplished with the use of rituximab in combination with the regimens every 2 weeks (R-CHOP-14, R-CHOEP-14). The aim of the present retrospective population-based study was to compare R-CHOP-14 with R-CHOEP-14 in a cohort of high-risk patients aged 18-60 years with two or more risk factors (stage III-IV, elevated lactate dehydrogenase levels, performance status 2-4). To our knowledge, this is the first study comparing these two regimens in this patient group. METHODS: We obtained data for the period 2004-2009 from the Danish Lymphoma Database. One hundred and fifty-nine patients were eligible to enter the study. Primary end point was overall survival (OS) and secondary end points were response to treatment, progression-free survival (PFS) and safety. RESULTS: Four-year OS was superior in the R-CHOEP-14 group: 75% compared with 62% for R-CHOP-14 (P=0.04). This superiority was also seen for PFS: 4-year PFS was 70% for the R-CHOEP-14 group compared with 58% for the R-CHOP-14 group (P=0.02). CONCLUSION: R-CHOEP-14 is a promising regimen for young patients with high-risk DLBCL with improved OS and PFS compared with R-CHOP-14.

Cytogenetic aberrations in adult acute lymphocytic leukemia: optimal technique may influence the results.

The aim of the present study was to analyse the distribution of cytogenetic aberrations in adult ALL in a population based material and compare the results with literature data. Forty-one patients were diagnosed during a 12-year period. The age varied between 14 and 82 (mean 37, median 32). Thirty-two patients were cytogenetically investigated and in all cases analysable metaphases were obtained (range 10-29, mean 24, median 25, success rate: 100%). Nine (28%) patients had a T-phenotype and 23 (72%) had a pre-B phenotype. High hyperdiploidy was found in four patients (13%). Hypodiploidy was found in 5 patients (16%), 10 (31%) had a pseudodiploid chromosome mode and four (13%) showed low hyperdiploidy (chromosome mode 47-51). Chromosomes 10 and 18 were most frequently involved in numerical aberrations. Structural aberrations most frequently involved chromosomes 6, 9 and 22. t(9;22) was seen in six cases (19%), del(6q) in five cases (16%) and der(9p) in five cases (16%). High hyperdiploid clones, which are associated with a favorable prognosis, were found with the same frequency as in other studies. The frequency of t(9;22) was 19% in our study, others have found frequencies between 11% and 30%. Compared to previously published studies our patients with t(9;22) were younger. Furthermore, those with del(6q) were older, showing a median age equivalent to the patient group as a whole. The differences between our data and previously published studies may be explained by population-based derived data and especially by an optimal technique in obtaining metaphases.

Suspected malignant lymphoma presenting with widespread granulomatous lesions in bones and lymph nodes and responding to combination chemotherapy.

A 61-year-old man presented with widespread granulomatous lesions of lymph nodes and bones, constitutional symptoms and various abnormal laboratory tests. An extensive investigation for the etiology including several biopsies, all showing granulomatous lesions, failed to reveal any specific underlying illness. A tentative diagnosis of sarcoidosis was made. However, corticosteroid therapy only temporarily improved the symptoms. Subsequently, standard chemotherapy for Hodgkin's disease induced complete clinical and biochemical remission. Rarely, the diagnosis of a lymphoma may be obscured by the presence of extensive granulomatous lesions. In these cases combination chemotherapy directed towards a malignant lymphoma may be justified in patients failing to respond to corticosteroid treatment.

Quantitation of immunogold with an interactive image analysis system. A new, practical approach to antibody-induced modulation, internalization and intracellular transport of surface antigens in viable hematopoietic cells.

This work aimed to evaluate the value of computerized quantitative analysis in ultrastructural studies on internalization and intracellular transport of leukocyte antigens traced with immunogold in viable lymphoid cells. The time of analysis and the results were compared with those obtained with a standard method based on counting gold particles on electron micrographs. A commercially available, personal computer-based, single-screen system was used for capturing and processing the ultrastructural images, counting gold particles and presenting data. The viable lymphoblasts of RAJI and NALM-6 cell lines were labeled with B4 (anti-CD19) murine monoclonal antibody followed by rabbit antimouse immunoglobulins coupled to 12.8-nm colloidal gold, cultured for six hours to allow internalization of CD19 antigen, collected after various periods of time and processed for electron microscopy. Each sample was analyzed by counting the gold particles on 30 randomly chosen, equatorial cell sections. Surface-bound particles and those located within certain intracellular compartments (that is, cytoplasmic vesicles, multivesicular bodies and lysosomes) were counted separately. No significant differences were found between the results obtained using electron micrographs and those yielded by on-screen analysis of digitized images. However, the time of analysis varied considerably--approximately eight and two hours, respectively. Also, using computerized analysis saved the cost of photographic processing. In conclusion, the system provided a reliable, rapid and inexpensive alternative for quantitation of immunogold-labeled leukocyte antigens at the ultrastructural level.

Antibody-induced modulation and intracellular transport of CD10 and CD19 antigens in human malignant B cells.

Antibody-induced antigenic modulation (AIAM) is a complex biological phenomenon closely resembling other receptor-ligand interactions. Following exposure to specific antibodies, surface antigens are usually rapidly redistributed on the cell surface and internalized. A subsequent intracellular processing results in dissociation of the antigen-antibody complexes, degradation, exocytosis and recycling. AIAM plays an important role in MoAb-targeted therapy of hematopoietic malignancies contributing to escape of tumor cells from immunodestruction. On the other hand, internalization of MoAbs used as carriers of toxins and drugs is a prerequisite of therapeutic efficacy. Even though MoAbs directed against CD10 and CD19 have been used in immunotherapy of B cell malignancies, some aspects regarding AIAM of these Ags are not yet fully understood. Both Ags are modulated by specific MoAbs and internalized through the same pathway, however, the kinetics of AIAM vary from one Ag to another and from one cell type to another. Recent studies with malignant B-cell lines show that, under certain experimental conditions, the extent and rate of surface clearing, uptake and intracellular transport are considerably higher in the case of CD19 than in CD10 and higher in less mature cells compared with more mature cells. These observations may be useful in the selection of MoAbs for immunotherapy, although they need to be confirmed with fresh malignant B cells.

Modulation and intracellular transport of CD20 and CD21 antigens induced by B1 and B2 monoclonal antibodies in RAJI and JOK-1 cells--an immunofluorescence and immunoelectron microscopy study.

By fluorescence microscopy (FM), flow cytometry (FCM) and immunoelectron microscopy (IEM) we have shown that B1 and B2 monoclonal antibodies (MoAbs) were able to induce modulation of CD20 and CD21 in RAJI and JOK-1 cell lines. Redistribution and internalization of both antigens (Ags) after binding with MoAbs was readily demonstrated by FM, and by IEM CD20 and CD21 were found to be processed by the pathway of receptor-mediated endocytosis. The rate of intracellular transport varied: CD21 > CD20 and RAJI > JOK-1. Approximately 65 and 55% of CD20 and 60 and 45% of CD21 were cleared from the surface of RAJI and JOK-1 cells, respectively (FCM and IEM). These values, however, clearly exceeded those corresponding to internalization (11, 9, 24 and 16%) indicating shedding of Ag-MoAb complexes. No evidence of recycling was found. The present data support the hypothesis that the kinetics of modulation vary from one Ag to another and probably also reflect the stage of differentiation of the malignant B-cells. The results are discussed in the context of the possible usefulness of B1 and B2 MoAbs in the therapy of B-cell malignancies.

Antibody-induced modulation and intracellular transport of CD10 and CD19 antigens in human B-cell lines: an immunofluorescence and immunoelectron microscopy study.

Antibody-induced antigenic modulation (AIAM) of CD10 and CD19 was studied on NALM-6, RAJI, and JOK-1 cell lines using fluorescence microscopy (FM), flow cytometry (FCM), and immunoelectron microscopy (IEM). Cross-linking with monoclonal antibodies (MoAbs) induced rapid redistribution of CD10 and CD19 on the cell surface (FM) followed by internalization involving uptake through plasmalemmal pits, transfer through endosomal compartment (receptor-mediated endocytosis), and, finally, delivery to lysosomes for degradation or exocytosis and recycling (IEM). Significant quantitative differences regarding modulation and intracellular processing were shown by FCM and IEM. Thus, 35%, 30%, and 25% of CD10 compared with 80%, 60%, and 40% of CD19 were internalized in NALM-6, RAJI, and JOK-1 cells, respectively. Also, the rate of intracellular transfer as well as externalization and recycling was more pronounced in the case of CD19 than of CD10 and in the NALM-6 and RAJI cells compared with the JOK-1 cells. These differences may possibly reflect the functional significance of CD10 and CD19 as well as the stage of differentiation of the malignant B cells. Although both antigens can be useful in MoAb-targeted immunotherapy, our findings suggest that anti-CD19 MoAbs would be preferable for delivery of cytotoxic agents to malignant B cells.

Immunoelectron microscopy studies on the modulation of common acute lymphoblastic leukemia antigen (CALLA) on NALM-6 cells: delineation of intracellular transport.

Even though much is known about the presence of the common acute lymphoblastic leukemia antigen (CALLA) with respect to its distribution in hematopoietic and non-hematopoietic tissues, its functional role in lymphoid cells is as yet unknown. Given the fact that CALLA is completely modulated on the surface of lymphoid cells, we have employed pre-embedding immunogold techniques at electron-microscopical level and demonstrated that J5 monoclonal antibody (MoAb)-mediated modulation of CALLA expression on the lymphoblastic cell line NALM-6 is a specific, rapid process, closely resembling receptor-mediated endocytosis. Furthermore, it was found that CALLA was internalized through plasmalemmal pits and cytoplasmic vesicles and processed intracellularly in multivesicular bodies and secondary lysosomes. In contrast, HLA-DR antigen remained at the cell surface upon contact with specific MoAb. These data suggest that CALLA might be a receptor for a hitherto unknown signal molecule.