Suppression of Burkitt's lymphoma tumorigenicity in nude mice by co-inoculation of EBV-immortalized lymphoblastoid cells

EBV-immortalized B-lymphoblastoid cell lines (LCL) inoculated s.c. into T-cell-deficient nude mice regress completely after a short initial growth period. We tested whether the putative host response underlying this phenomenon might also be directed against progressively growing Burkitt's lymphoma (BL) tumors in nude mice. Outgrowth of BL tumors was suppressed when cells of the highly tumorigenic BL cell line BL 60 were mixed with cells of the autologous LCL IARC 277 before s.c. inoculation into nude mice. Even when the cells were inoculated separately and simultaneously into contralateral flanks of the mice, regression of initially growing BL tumors could be observed, albeit with reduced frequency and dependent on the dose of LCL cells. Tumor growth of BL 60 cells could also be suppressed by co-inoculation with the non-autologous LCL IARC 174 and IARC 277 cells could suppress growth of the non-autologous BL cell line Eli. Pronounced infiltration with murine (m)CD-11b-positive mouse macrophages and mCD-8a-positive mouse lymphoid cells, most probably natural killer cells, was seen in histological tissue sections of regressing BL 60 tumors when LCL cells were inoculated contralaterally. In regressing BL tumors, these mouse cells were present not only in necrotic areas but also in vital BL tissue, indicating that infiltration of mouse cells had taken place before the development of necrosis. Since tumor-infiltrating mouse cells can be activated at least by some human cytokines, we measured cytokine production of BL 60 and IARC 277. High amounts of IL 6 and IL 10 were produced by the LCL cells, whereas IL-6 and IL-10 production by the BL 60 cells was beyond or close to the detection threshold. In addition, IL 8 was secreted up to 5-fold more by the LCL than by the BL cells. The results presented here thus suggest a host response of the nude mouse, which is triggered by cytokines released from the LCL but, once induced, is directed also against BL cells.     

Inhibition of Burkitt's lymphoma cells growth in SCID mice by a PNA specific for a regulatory sequence of the translocated c-myc

In Burkitt's lymphoma (BL) cells due to a t(8;14) chromosomal translocation c-myc is often placed in proximity to the Emu enhancer of the Ig locus and upregulated. We demonstrated that in BL cells a peptide nucleic acid (PNA), complementary to intronic Emu sequences (PNAEmuwt), specifically blocks the expression of the c-myc oncogene under the Emu enhancer control and inhibits BL cell growth in culture. Here, we investigated whether PNAEmuwt was also able to block tumor growth in SCID mice inoculated with human BL cell lines. After subcutaneous inoculum in mice BL cells reproducibly form tumors. Both pre-treatment of BL cells with PNAEmuwt before inoculum and chronic intravenous administration of PNAEmuwt to mice already inoculated with BL cells selectively caused increased latency of tumor appearance and decreased final tumor size. Tumors from PNAEmuwt-treated animals showed substantial areas of cell necrosis and of c-myc downregulation. Inhibition of tumor growth was specific and was not observed with PNAEmumut carrying sequence mutations and in BL cell lines where the translocated c-myc is not under the control of the Emu enhancer. These data confirm the potential therapeutic value of PNA targeted to regulatory non-coding regions.     

Potent anti-tumor effects of an anti-CD24 ricin A-chain immunotoxin in vitro and in a disseminated human Burkitt's lymphoma model in SCID mice

A new anti-CD24 immunotoxin (IT), SWA11.dgA, was constructed by coupling the MAb SWA11 via the bivalent linker SMPT to deglycosylated ricin A-chain (dgA). The effects of SWA11.dgA were evaluated in vitro against the B-precursor leukemia cell line REH, the non-B-non-T acute lymphoblastic leukemia cell line NALM-6 and the Burkitt's lymphoma cell lines BL-2 and BL-38. Binding of SWA11 to the CD24 antigen was assessed by flow cytometry demonstrating high affinity of the MAb for all cell lines tested. SWA11.dgA inhibited the protein synthesis of BL-38, NALM-6, REH and BL-2 cells by 50% at concentrations (IC₅₀) of 4.0 × 10⁻¹¹ M, 6.0 × 10⁻¹¹ M, 8.0 × 10⁻¹¹ M and 3.0 × 10⁻⁹ M, respectively. SWA11.dgA was subsequently used for the treatment of disseminated human BL-38 Burkitt's lymphoma in a newly developed SCID mouse model. The mean survival time (MST) of BL-38-bearing SCID mice was extended from 23 days in untreated controls to more than 230 days when 6 μg SWA11.dgA was applied intraperitoneally one day after tumor challenge. All of the animals achieved continuous complete remissions. SCID mice treated with SWA11.dgA 4 days after tumor cell challenge or a reduced dose of SWAII.dgA (67%) also had a significantly extended MST (45.0 and 51.4 days, respectively, as compared to 22.7 and 23.1 days in the controls). We conclude that SWA11.dgA might be of potential use for the treatment of lymphoma in man. © 1996 Wiley-Liss, Inc.     

Growth of human lymphoma cells in SCID mice.

Two EBV-negative human lymphoma cell lines raised in this laboratory and peripheral blood cells of a patient with large cell lymphoma in leukemic phase were injected intravenously or intraperitoneally into C.B.17 SCID mice. One line (OCI-LY18) was derived from the pleural fluid of a patient with a large cell, immunoblastic malignant lymphoma. Cells of this line are of B cell origin and characterized by multiple rearrangements of the JH locus. The second line (OCI-LY17) was grown from the peripheral blood of a patient with a large cell lymphoma of T-cell phenotype which has characteristic rearrangement of the T-cell receptor beta chain. The cells directly obtained from a patient with large cell non-cleaved malignant lymphoma were of B-cell origin. Animals carrying OCI-LY18 developed large tumor masses within 6-8 weeks of inoculation. The tumors were detected in the intestine, mesentery, retroperitoneum, lymph nodes, spleen, lung and kidney. The masses resembled the primary tumor with respect to histological appearance and immunological phenotype. It was possible to generate secondary cell lines from the tumors found in the inoculated SCID mice. Injection of one of these secondary cell lines into SCID mice resulted in the rapid development of lymphoma and as few as 10 cells were sufficient to establish the disease in the inoculated animals. In contrast cells of OCI-LY17 produced small tumor aggregates that did not appear to progress over time and did not cause death of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of lymphomatous and lymphoblastoid cell lines in the study of Burkitt's lymphoma

To better characterize the virological and cytogenetic features of Burkitt's lymphoma (BL) occurring in so-called low-incidence areas, biopsy specimens were collected from BL patients diagnosed and treated in France, and attempts were made to cultivate malignant cells in vitro in order to provide large amounts of tumour material for further laboratory investigations. Sixty new BL-derived lymphomatous cell lines have been established from 43 individuals: 24 Caucasians, 14 North Africans and five Africans. Of these, 27 lines (from 18 individuals) were established from non-Epstein-Barr virus (EBV)-associated BL tumours, indicating that the relative ease in cultivating BL malignant cells in vitro is not limited to EBV genome-containing BL tumours. Cytogenetic investigations showed that all the BL cell lines had one of the three 'BL translocations' - t(8;14), t(8;22) or t(2;8). An estimate of the frequency of each translocation, based on the analysis of 51 independent IARC BL cell lines gave the following results: t(8;14), 76%; t(8;22), 16%; t(2;8), 8%. This large panel of cell lines is now a valuable tool for analysing the various phenotypic characteristics of BL cells. Comparisons can be made between multiple lines of BL from high-, intermediate- and low-incidence areas; significant numbers of EBV genome-negative and -positive lines and of tumour cells taken at diagnosis and at various stages of the disease can also be compared. For molecular and immunological investigations, EBV-immortalized lymphoblastoid cell lines (LCL) were also established from non-malignant lymphocytes of BL patients. The 24 paired BL and LCL cell lines obtained so far will allow precise, controlled studies of the molecular consequences of chromosomal translocations and of the comparative susceptibility of BL and LCL to immune effectors.     

Expression of integrins and other adhesion molecules in Epstein-Barr virus-transformed B lymphoblastoid cells and Burkitt's lymphoma cells.

Lymphocytes adhere to cells or extracellular matrices to perform functions relating to cytotoxicity, extravasation and tissue localization, as well as modulation of lymphocyte growth and maturation. This adherence is mainly mediated by 3 families of cell-surface adhesion molecules: integrins, immunoglobulin-related molecules and selectins. Since variations in the degree of adherence may affect the pathophysiology of lymphoproliferative disorders, the expression of a large number of adhesion molecules was analysed on Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), and on EBV-positive or EBV-negative Burkitt's lymphoma (BL) lines, by immunofluorescence flow cytometry and immunoprecipitation with monoclonal antibodies. With regard to the beta 1, beta 2 and beta 3 integrin subfamilies, LCLs strongly expressed CD49d/CD29 (VLA-4), CD11a/CD18 (Leu-CAMa, LFA-1) and CD51/CD61 (vitronectin receptor). These cells also abundantly expressed CD54 (ICAM-1) and CD58 (LFA-3) as well as the "homing receptors" L-selectin (LECAM-1) and CD44. BL lines had considerably lower amounts of VLA-4 than LCLs, and ICAM-1 was expressed only by some of the tumor lines. All other adhesion molecules were absent or minimally expressed in the BL cells.     

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced,disseminated Daudi lymphoma

We describe the use of an immunotoxin (IT) cocktail (anti-CD22- and anti-CD19-ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti-tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B-cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy. © 1996 Wiley-Liss, Inc.     

Burkitt's lymphoma cell leukemia in a Turish boy.

Burkitt's lymphoma, associated with massive bone marrow and eyelid involvement that terminated with a manifested leukemic picture, was observed in a Turkish boy. Review of the world literature revealed the fact that Burkitt's lymphoma with a frankly leukemic picture is a rare condition, and usually has a very acute course with a poor prognosis.     

Tumorigenicity of EBV-carrying lymphoblastoid cell lines (LCLs): distinctive grading in SCID mice.

The in vitro and in vivo growth of lymphoblastoid cell lines (LCLs), Burkitt lymphomas (BLs) and PBL-derived immunoblastomas in SCID mice was studied in parallel. Most, but not all, of the LCLs tested were tumorigenic in SCID mice. Long-term culturing in vitro was not a necessary prerequisite for tumorigenicity. There was a good correlation between in vivo and in vitro growth. Three major classes of cells could be distinguished on the basis of their growth properties.     

Characterization of a novel human anaplastic large cell lymphoma cell line tumorigenic in SCID mice

L82, a novel anaplastic large cell lymphoma (ALCL) cell line was established from the pleural effusion of a 24-year-old patient with recurrent ALCL. L82 cells showed the typical morphologic features of ALCL cells with irregular, often indented, nuclear profiles, prominent nucleoli, and abundant cytoplasm. The immunoprofile of L82 corresponds to that seen typically in primary ALCL cells, with positivity for CD30, EMA, CD3, CD4, CD25, CD71, TIA1, and granzyme B; the cells were negative for EBV-related antigens. Cytogenetic analysis showed a complex, near triploid karyotype with 72-77 chromosomes, including the ALCL specific translocation t(2;5)(p23;q35). Chromosomal analysis revealed a number of secondary structural alterations including amplification of 7q21-31, 1q, and 6p, and gain of chromosomal material in 8q (affecting the c-myc gene). The rearrangement of the T-cell receptor-gamma locus shows that L82 is clonally derived from T-lineage lymphoid cells. mRNAs for interleukin 7 (IL-7), IL-8, IL-9, IL-10, TNF-beta, and for the IL-7 and IL-9 receptor were found. These data show that the T-helper cell (Th)1/Th2 balance was polarized to Th2. L82 were inoculated intraperitoneally into 4 week-old SCID mice and produced a disseminated tumor within 4-6 weeks. Morphological, immunohistochemical, and molecular genetic investigation confirmed that the xenograft and the original ALCL tumor were identical. SCID mice xenografted with the human ALCL cell line, L82, provide a useful model system for the investigation of the biology of ALCL and of new therapeutic approaches, such as specific immunotherapy.     

Cytogenetic study of a European Burkitt's lymphoma cell line.

The chromosomes of an Epstein-Barr virus-negative European Burkitt's lymphoma cell line were studied. All the cells carried the t(8;14) translocation. One clone had 51 chromosomes and was (+1,+7,+16,+15,+21), whereas another clone also had 51 chromosomes but was (+1q+,+7,+16,+15,+21). A third clone had 46 chromosomes (4q+/-).     

Effect of human natural killer and gammadelta T cells on the growth of human autologous melanoma xenografts in SCID mice

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, gammadelta T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and gammadelta cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vdelta1 or Vdelta2 gamma/delta T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vdelta1 gamma/delta T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vdelta1 gammadelta T lymphocytes could be detected at the s.c. tumor site. In contrast, Vdelta2 gammadelta T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vdelta1 gammadelta T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.     

The cytogenetics of human B lymphoid malignancy: studies in Burkitt's lymphoma and Epstein-Barr virus-transformed lymphoblastoid cell lines

Cells from Burkitt's lymphoma (BL) and from the majority of human B-cell neoplasms show karyotypic changes that characteristically involve chromosomal breakage and recombination in addition to some chromosome gains. These aberrations increase as the tumours progress in vivo, and a similar tendency is seen in BL-derived lymphoid lines in vitro. Epstein-Barr virus (EBV)-transformed lymphoblastoid lines of non-malignant origin also develop karyotypic abnormalities on prolonged culture, but these are predominantly nonrandom gains of whole chromosomes (i.e., non-disjunction events). They have never been observed to acquire the 8;14 translocation, which is an almost constant feature of BL. Nevertheless, there is some concordance between the pattern of chromosome gains found in long-term cultured lymphoblastoid lines and that seen in direct preparations from B-cell neoplasms. Many of the lymphoblastoid lines that have become aneuploid are tumorigenic in immunosuppressed mice, indicating that EBV-transformed human B cells can acquire a malignant phenotype in the absence of specific chromosomal translocations. It is suggested that the predominance of chromosomal breakage and recombination events in the karyotypic evolution of BL and other lymphoid neoplasms comes about because chromosomal instability (which varies within a population) is a major risk factor for lymphoid malignancy, interacting with other risk factors, including impaired T-cell function and EBV to determine the clinical and epidemiological patterns of BI and related neoplasms.     

Burkitt's lymphoma cell lines reveal different degrees of tumorigenicity in nude mice

A quantitative in vivo assay for evaluating the tumorigenicity of Burkitt's lymphoma (BL) cell lines in nude mice is described. It is based on the dose-response kinetics of BL cell lines in pre-irradiated (480 rad) nude mice following the s.c. injection of 4 different cell doses. This model system was used to estimate the xenografting potential of 26 BL cell lines derived from BL patients of different geographic and ethnic origins, as well as lymphoblastoid cell lines (LCLs) established by Epstein-Barr virus (EBV) immortalization of normal B lymphocytes. The results indicate that most BL cell lines are tumorigenic, but LCLs fail to produce progressively growing tumours in nude mice. However, BL cell lines revealed individual degrees of tumorigenicity and accordingly could be divided into 4 groups with high, moderate, low or no tumorigenicity. Preliminary attempts to correlate the xenografting phenotype of BL lines with other characteristics indicate that: (1) the aberrations of chromosome 1 are more often encountered in cell lines with high and moderate tumorigenicity; (2) EBV-positive BL lines do not reveal a higher tumorigenic phenotype in comparison with EBV-negative ones; (3) cell lines carrying translocations t(8;22) and t(2;8) might fall more frequently in the group of lines with high and moderate tumorigenicity; and (4) when LCLs grow in nu/nu mice, rejection always occurs and is associated with massive tumour necrosis. These findings suggest that the tumorigenicity of BL cell lines in immunosuppressed animals is not related with EBV, but with certain chromosomal abnormalities (BL-specific and non-specific) indicating that this in vivo model system can be instrumental for the identification of other factors or stages involved in BL development.     

Phenylbutyrate induces cell differentiation and modulates Epstein-Barr virus gene expression in Burkitt's lymphoma cells.

Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease.     

Relationship between tumorigenicity and the dosage of lymphoma- vs. normal-parent-derived chromosome 15 in somatic cell hybrids between lymphoma cells with rearranged pvt-1 gene and normal cells

Somatic cell hybrids were generated between YACUT, a doubly drug-resistant subline of YAC-1 (a Moloney-virus-induced T-cell lymphoma of strain A/Sn origin with 2 proviral insertions near the pvt-1 locus) and normal diploid fibroblasts of CBAT6T6 origin. Three independent fusions were performed. Three uncloned hybrid cultures and 9 independently-derived clones were tested for tumorigenicity by the inoculation of graded cell numbers into syngeneic hosts. One of 3 uncloned hybrid cultures and 3 of 9 clones were weakly tumorigenic (take incidence 0%), and 1 of 3 uncloned hybrid cultures and 6 clones were highly tumorigenic (take incidence greater than 80%). One weakly tumorigenic hybrid and 3 weakly tumorigenic clones carried 3 copies of the tumor-derived chromosome 15 and 2 copies of the normal fibroblast-derived t(14;15) chromosomes. In contrast, 2 highly malignant hybrid clones lost one copy of the normal-fibroblast-derived t(14;15), but contained increased numbers (3.44-4.44) of the tumor-derived chromosome 15. Four tumorigenic segregants selected from the weakly tumorigenic fibroblast hybrids by in vivo inoculation showed the same cytogenetic change as the highly tumorigenic hybrid clones, in that the ratio of the normal:tumor-derived chromosomes 15 changed from 1.18-1.55 to 4.11-5.71. Tumorigenicity was thus associated with a modified balance between the tumor vs. the normal-parent-derived 15-chromosomes. Instead of the usual 3:2 ratio, the tumor-derived 15-chromosomes increased disproportionately, whereas the relative number of the normal-parent-derived 15-chromosome decreased, as a rule. These results suggest that amplification of the lymphoma-derived chromosome 15 favors tumorigenicity, but that this effect is counteracted by some influence emanating from the normal-parent-derived homologous chromosome.     

Paired Epstein-Barr virus-carrying lymphoma and lymphoblastoid cell lines from Burkitt's lymphoma patients: comparative sensitivity to non-specific and to allo-specific cytotoxic responses in vitro

Paired Epstein-Barr (EB) virus-carrying cell lines have been established from Burkitt's lymphoma (BL) patients; one the BL-cell line derived from the malignant cells of the tumour and bearing the relevant chromosomal translocation, the other the diploid lymphoblastoid cell line (LCL) derived from the patient's normal B cells by experimental infection with the virus. Comparative studies have shown that both BL and LCL cells are relatively resistant to fresh NK cells but are more susceptible to in vitro-activated NK cells; individual pairs differ as to whether the BL or LCL cells are more sensitive to such effectors. With most cell pairs studied, the two cell types were equally efficient at inducing NK-cell activation in vitro. When the in vitro stimulation protocol was changed to favour the induction of allo-specific responses, most BL cells were noticeably poorer stimulators than the corresponding LCL although both cell types induced a similar pattern of cytotoxicity; this appeared to be directed against HLA class I allo-antigens and could be inhibited by class I antigen-specific monoclonal antibodies. Most BL target lines, though susceptible to this allo-specific lysis, were significantly less so than the corresponding LCL However, only 1/7 BL-cell lines tested vis-à-vis the LCL gave evidence of a gross reduction in HLA antigen expression and this was immediately apparent from both serological typing and cellular analysis.