Guidelines for the characterization and publication of human malignant hematopoietic cell lines.

Continuous human malignant hematopoietic (MH) cell lines have become invaluable tools for hematological diagnosis and research. Over the last 35 years several hundred cell lines spanning almost the whole spectrum of hematopoietic cell lineages have been described. The cardinal features of MH cell lines are their monoclonal origin, arrest of differentiation, genetic alterations, and unlimited proliferation; the major advantages of cell lines are the unlimited supply of cell material and the infinite storability and recoverability at will of the cells. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed and comparative characterization of both primary and cultured cells is absolutely necessary. New cell lines, in particular, must be adequately characterized. While clinical and cell culture data and immunological and cytogenetic features are the most important data, cell lines should be described in as much additional detail as possible allowing any singular features to be pointed out. In addition to detailed characterization, immortality of the culture, proof of neoplasticity, authentication of the true origin of the cells, scientific significance and availability of the cell line for other investigators are of paramount importance. In summary, MH cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described.     

Establishment and characterization of a human histiocytic lymphoma cell line (U-937).

A human hematopoietic cell line (U-937) with exceptional characteristics was derived from a patient with generalized histiocytic lymphoma. The morphology of the cell line was identical to that of the tumor cells in the pleural effusion from which the line was derived. Since Epstein-Barr virus (EBV) carrying diploid lymphoblastoid cell lines unrelated to the tumor population often become established in vitro from non-Burkitt lymphoma explants, several parameters were studied to discriminate the U-937 from such lines: morphology in vitro, growth characteristics, cytochemistry, surface receptor pattern, Ig production, lysozyme production, beta2-microglobulin production, presence of EBV genome and karyotype. In all these respects U-937 differed from prototype lymphoblastoid cell lines. The histiocytic origin of the cell line was shown by its capacity for lysozyme production and the strong esterase activity (naphtol AS-D acetate esterase inhibited by NaF) of the cells. It is therefore concluded that the U-937 is a neoplastic, histiocytic cell line.     

Combined modality therapy for advanced, diffuse lymphocytic and histiocytic lymphomas.

Forty-six previously untreated patients with advanced aggressive non-Hodgkin's (34 poorly differentiated and mixed diffuse, 8 histiocytic and 4 undifferentiated) were treated with a 3 phase combined modality program employing cyclophosphamide (C), hydroxyl-daunomycin (H), vincristine (O), prednisone (P), procarbazine (P) [CHOP(P)] combination chemotherapy in an initial induction phase, radiotherapy and nonmarrow toxic chemotherapy as a second consolidation phase, followed by a third phase of CHOP(P) chemotherapy for four more cycles. Long-term maintenance therapy was not given. High dose involved field radiation in phase II was limited to volumes encompasing less than 50% of the marrow bearing skeleton. The large majority of patients (82%) had such widespread involvement that this limitation precluded the use of local radiation and were treated instead with a mean of 132 rad of fractionated total body irradiation (TBI). Thirty-eight patients (83%) achieved complete remission. Twenty-nine (66%) of the 44 patients evaluable for follow-up, and 22 (61%) of the 36 patients receiving TBI, remain alive in complete remission for observation periods of up to 26 months.     

Somatic cell hybrids between human lymphoma lines. IV. Establishment and characterization of a P3HR-1/Daudi hybrid.

A new human B-lymphoma hybrid line designated DIP-1 was derived from the fusion of the Burkitt lines Daudi and P3HR-1. Cytogenetic analyses proved hybridity and showed that the hybrid was nearly complete from the chromosomal point of view. Hybridity was also confirmed by isozyme marker tests. The Daudi-derived IgM-kappa ring was fully exposed on DIP-1, together with the P3HR-1-derived human beta-2-microglobulin. Expression of C3 receptors was intermediate between the two parents and EBV receptors were some what reduced in comparison with both. The spontaneous low-level EBV antigen (EA and VCA) production of the parents was maintained and amplified in the hybrid. The hybrid, like its Daudi parent, was highly inducible with IdUrd. In P3HR-1 virus super-infection experiments, the Daudi parent was more permissive than the BU-P3HR-1 parent. The hybrid was intermediate, resembling the previously studied Raji/Daudi hybrid and contrasting with Raji/Namalwa and Raji/BJAB hybrids. Virus DNA replication patterns after P3HR-1 virus superinfection resembled the antigen pattern. The implication of these findings for the understanding of virus-host cell relationships and the regulation of the viral cycle is discussed The findings are meant to imply that genetically determined isozyme markers are autonomously expressed, as in other systems. Differentiation-related markers and EBV-cycle-related characteristics are autonomously expressed in some cases (surface immunoglobulin, beta-2-microglobulin, spontaneous EBV-production, IdUrd inducibility) and appear to be under a restrictive control in others (EA inducibility by P3HR-1 virus superinfection, C3 and EBV receptors, viral DNA replication).     

Nodular lymphocytic lymphoma eventuating into diffuse histiocytic lymphoma: immunoperoxidase demonstration of monoclonality.

The patient described here had a nodular, poorly differentiated lymphocytic lymphoma associated with a serum monoclonal protein, IgG lambda. Following a three year period of radiation-induced clinical remission she developed generalized diffuse histiocytic lymphoma. Direct immunoperoxidase staining of the tissue sections demonstrated that the neoplastic cells of each biopsy only contained IgG lambda immunoglobulin, identical to the serum monoclonal protein. This is presumptive evidence that these two histopathologically distinctive malignant lymphomas, occurring consecutively in the same patient, were responsible for the synthesis and secretion of the same serum M component. This strongly suggests that both lymphoid neoplasms arose from the same malignant clone. The results 1) confirm the light microscopic observation that nodular lymphocytic lymphoma may progress to diffuse histiocytic lymphoma and 2) offer further evidence that histiocytic lymphomas arising in patients with previous B cell malignancies are most probably related to the original B cell proliferation and do not represent the emergence of a second, separate malignant clone.     

Long-term survival of patients with localized diffuse histiocytic lymphoma

From January 1970 to March 1981, localized diffuse histiocytic lymphoma (DHL) was identified in 31 patients by exploratory laparotomy and splenectomy (pathologic stage I, 17 patients; pathologic stage II, 14 patients) at the University of Chicago. The median follow-up time was 72 months. All patients were previously untreated and received radiation therapy as their primary treatment modality. Chemotherapy was administered only at the time of relapse. All but two patients achieved a complete remission (CR) with radiation therapy. The actuarial disease-free survival for patients with stage I disease is 94% at 5 years and 72% at 10 years. For stage II disease, the disease-free survival is 56% at 5 years and 31% at 10 years. The difference in the disease-free survival between stage I and II is statistically significant (P = .02). The survival at 10 years is 70% for stage I disease and 46% for stage II disease. Five patients had documented relapses (four had stage II disease). Only two of those who relapsed achieved a second CR with salvage chemotherapy. Our data show an excellent outcome in patients with pathologic stage I disease, indicating that a high percentage of these cases can be cured with radiotherapy alone. Patients with clinical stage II disease might be served better with chemotherapy.     

Feeder layer and nutritional requirements for the establishment and cloning of human malignant lymphoma cell lines.

Cell lines were successfully established in continuous suspension culture from 10 patients with a histopathological diagnosis of diffuse histiocytic lymphoma (SU-DHL-1 to SU-DHL-10), two with North American Burkitt's lymphoma (SU-AmB-1 and SU-AmB-2), and one with acute lymphoblastic leukemia (SU-ALL-1). By screening a variety of parameters, including media, sera, effusion fluids, feeder layers, and chemical supplements, the nutritive growth requirements of lymphoma cells obtained from malignant effusions and lymph node biopsies were determined for each tumor. Most of these cell lines initially required human skin fibroblast or epithelial cell feeder layers from which they could be weaned after one to six weeks in culture and maintained in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing 20% fetal calf serum and 10% pooled human serum. Several of these cell lines were successfully cloned on 0.5% Noble agar substrates. In the presence of human serum and selected feeder monolayers, cloning efficiencies increased significantly from less than 1% to 15 to 25%. In addition, the cloning efficiencies of certain cell lines showed a concentration-dependent increase with specific chemical supplements including L-cysteine and dithiothreitol. Placental colony-stimulating factor, nerve growth factor, epithelial growth factor, and fibroblastic growth factor were ineffective in augmenting the cloning efficiencies of the human lymphoma cell lines. After a single passage on agar, cells subpassaged from visible colonies showed markedly increased cloning efficiencies to levels as high as 50%. Such cloning efficiencies, coupled with the use of replica plating, make this technique applicable to genetic and quantitative radiobiological, immunological, and chemotherapeutic studies. Although these methods have thus far been used only with lymphoreticular tumors, they may also be applicable to the cell culture of other human neoplasms and normal tissues.     

Biology of the human malignant lymphomas. III. Intracranial heterotransplantation in the nude, athymic mouse.

Intracranial heterotransplantation in the nude, athymic mouse has been found to be an effective method for the experimental growth of human malignant lymphomas. Transplants of 11 primary lymphomas and six derived cell lines yielded a high take rate (90%) and a low mean latent period (36 days). Relatively small inocula produced extensive intracerebral infiltrates which could be identified as human in origin by immunofluorescence. Although confined to the central nervous system and meninges, the tumors were highly invasive and displayed morphologic features strikingly similar to those of the original primary tumors. Heterotransplantation of the lymphomas to extracranial sites was only rarely successful. Nude mice previously grafted with isologous neonatal thymuses failed to develop intracerebral tumors. Secondary cell cultures successfully established from several of the intracranial heterotransplants were found to be infected with NIH type-C xenotropic virus. The distinctive growth patterns and other neuropathologic features of the heterotransplants are described, and the relevance of these observations to the development of intracerebral lymphomas in immunosuppressed organ transplant recipients is noted. This method of studying human malignant lymphomas in vivo may permit better histopathologic characterization of the tumors, and may serve as a basis for further experimental lines of investigation, including viral, immunologic, and therapeutic studies.     

Immunological characterization of cell lines established from malignant and normal human urothelium

Cell lines from normal or malignant human urothelium, described elsewhere by morphological, cell kinetic and genotypical criteria, have been characterized further by their response to antibody or lymphocyte mediated cytotoxicity in vitro. Four groups of cell lines were included: (1) two fast proliferating cell lines from normal urothelium, (2) three fast proliferating cell lines from transitional cell carcinoma (TCC) together with two transformed sublines of normal derived cell lines, (3) six slowly proliferating TCC lines, and (4) a line from squamous cell carcinoma. HLA antigen expression was demonstrated in the cell lines of groups 1 and 3, but not in lines from group 2 or 4. The sensitivity to spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells in group 1 and 2 exceeded that of cells from group 3 by a factor of 8. The TCC line, HU 456, was more susceptible to SLMC than T 24. Specifically increased cytotoxicity of lymphocytes from patients suffering from interstitial cystitis (IC) against HU 609 from normal urothelium indicated that this line expresses tissue specific antigens, and at the same time that an immune reaction may be part of the pathogenesis of IC. By a sensitive test for antibody-dependent, cell-mediated cytotoxicity (ADCC) antibody activity against HU 456 was found in only one of nine TCC patients and none of five clinical controls. A pronounced non-disease-related blocking serum activity, frequently found, may account for some of the negative findings. The SLMC against HU 456 could be inhibited but not abolished by Fab fragments of rabbit anti-human immunoglobulin, indicating an ADCC mechanism as part of the SLMC.     

Epstein-Barr virus integration in human lymphomas and lymphoid cell lines.

BACKGROUND. Epstein-Barr virus (EBV) is maintained as an episome in most infected cells. The presence of fused terminal restriction enzyme fragments distinguishes the circular DNA form from the linear virion form. METHODS. EBV genomic structure was analyzed in 8 lymphoid cell lines and 21 human lymphoma specimens by the Southern blot technique. RESULTS. Evidence of viral integration into host chromosomal DNA was identified in four cell lines. In the Namalwa and BL30-B95.8 cell lines, integration occurred through the terminal repeat (TR) sequences. In the BL41-P3HR1 and BL41-B95.8 cell lines, there was loss of left-end viral genomic sequences, including ori-P sequences required for episome maintenance, implying that integration was required for viral genome persistence. Integration was not detected in four other cell lines (Raji, Daudi, B95.8, and BL30-P3HR1). In 21 EBV-containing human lymphomas, including 18 immunodeficiency-related lymphomas, fused TR sequences were identified without evidence of viral genomic integration. CONCLUSIONS. These findings suggest that, although viral integration is common in Burkitt lymphoma cell lines infected in vitro, integration is not common in human lymphomas that develop in vivo in normal or immunodeficient people.     

Establishment and characterization of five human malignant mesothelioma cell lines derived from pleural effusions.

Malignant mesothelioma (MM) is an aggressive tumour of the serosal cavities which is associated with exposure to asbestos. Studies of this tumour have been limited by a paucity of well-characterized human MM cell lines. In this study, 5 human MM cell lines were established from pleural effusions of patients with this malignancy. All 5 patients were males with known crocidolite asbestos exposure, who had received no treatment for their disease and in whom the diagnosis was confirmed by cytology, histology and electron microscopy (EM). These lines have been in culture from 11 to 25 months, and all of them for more than 18 passages. The appearance of the cells in culture was extremely varied; in 3 of the lines they were spindle-shaped with few vacuoles (JU77, LO68 and ONE58); in 1 line they had a thick, stellate shape with vacuoles (NO36) and in 1 they were very pleomorphic in both shape and size with irregular membranes and numerous vacuoles [DeH128 (M)]. Upon reaching confluence, cells in 3 of the 5 lines assumed the cobblestone-like pattern characteristic of epithelial-type cells, whereas in the other 2 (LO68 and ONE58) they remained spindle-shaped. All 5 lines demonstrated a loss of contact inhibition (i.e., piling) at confluence. Minimum doubling times varied significantly from 18 hr (JU77) to more than 30 hr [DeH128 (M)]. Cytological examination showed characteristic mesothelial/mesothelioma morphology, and epithelial membrane antigen (EMA) and cytokeratin were demonstrated in cells from all 5 lines. These cells lacked CEA and epithelial mucin. The presence of cell junctions, glycogen and numerous long, thin, branching microvilli was readily demonstrable by EM. All lines had abnormal karyotypes, with the modal chromosome number varying from 40 to 80. Variable chromosome numbers, numerous structural rearrangements and unrecognizable marker chromosomes were readily observed; however, the only consistent change seen was del 6q21 in 4 of the 5 lines. The establishment of these 5 cultured human MM cell lines now provides an opportunity for comparative study of several aspects of the biology of MM in vitro as well as screening new treatment modalities.     

耐糖皮质激素人类弥漫大B细胞淋巴瘤细胞系的建立及其生物学特性观察

目的 采用长期间歇小剂量糖皮质激素（GC）递增诱导法建立耐GC的人类弥漫大B细胞淋巴瘤细胞系Toledo／地塞米松（DEX）,并观察耐药和亲本细胞株两者之间的生物学特性差异,探讨其耐药机制.方法 以DEX为诱导剂,将对数期亲本Toledo细胞接种在含DEX起始浓度为1×10-s mol／L的培养液中培养96 h后,改为不含药物的培养液培养,待细胞增殖再次进入对数生长期,提高DEX浓度1倍,直至在含DEX 1.024×10-5 mol／L的浓度中稳定生长.并通过细胞学、遗传学、免疫学、分子生物学和裸鼠致瘤实验等多种方法观察耐药和亲本细胞系两者之间的生物学特性差异,探讨GC耐药机制.结果 Toledo／DEX细胞系可以在含DEX 1.024×10-5 mol／L的浓度中稳定生长.耐药及亲本细胞系在形态及免疫表型上完全一致.对超微结构观察可见Toledo／DEX细胞表面局部可见片层状突起,有细长微绒毛.耐药细胞致瘤性强,对多种化疗药有耐药性且耐药指数高,提示耐药细胞更具侵袭性.耐药细胞增殖慢,较多被阻滞在G1期,染色体变异多,但未形成两个相关克隆.此外,GC受体（GR）α、GR β表达异常参与GC耐药的形成.结论 Toledo／DEX是一株生物学特性稳定的高耐药细胞系,为研究GC耐药机制及探索逆转GC耐药性的药物提供了生物学基础.     

Adjuvant radiotherapy in stage IV diffuse large cell lymphoma improves outcome

The role of adjuvant radiotherapy to sites of nodal bulky disease in patients with aggressive diffuse large cell lymphoma (DLCL), and stage IV remain undefined. We began a prospective controlled clinical trial to evaluate impact in event free survival (EFS) and overall survival (OS) in a large cohort of patients with a longer follow-up. Between 1989 and 1995; 341 patients with aggressive DLCL and presence of nodal bulky disease (tumor mass > 10 cm) in pathological proven complete response after intensive chemotherapy were randomized to received either radiotherapy (involved fields, 40 Gy) or not. The 5-year EFS and OS in radiated patients were respectively: 82% (95% Confidence interval (CI): 70-89%) and 87% (95% 80-99%), that were statistically significant to control group: 55% (41-64%) (P < 0.001) and 66% (95% CI: 51-73%) (P < 0.01) respectively. Radiotherapy was well tolerated, acute toxicity was mild and until now late toxicity did not appear. The use of adjuvant radiotherapy improve EFS and OS and probably the possibility of cure in patients diffuse large cell lymphoma with worse prognostic factors. Thus, we felt that adjuvant radiotherapy will be considered as part of the initial treatment in this setting of patients.     

Chromosomal characterization and isoenzyme pattern of non-malignant and malignant human urothelial cell lines

The karyotypes and selected isoenzymes of five cell lines, HU 961b, HU 1922, HU 609, HCV-29 and HU 1703 He, were characterized. These cell lines were derived either from histologically normal human urothelial cells or from transitional cell carcinoma (TCC) of the urinary bladder. They displayed different life span in vitro and transplantability in nude mice, as well as a unique isoenzyme and chromosomal pattern. In addition, various chromosomes were involved in the formation of marker chromosomes in these cell lines. Since there was a lack of consistency in chromosomal changes in the cell lines with TCC origin, the cause of malignancy could not be attributed to the abnormality of specific chromosomes.     

Establishment of human malignant mesothelioma cell lines

Seventeen human malignant mesothelioma cell lines were isolated from 61 samples (46 effusions, 9 biopsies and 6 tumors obtained at autopsy) collected from patients with a confirmed malignant mesothelioma. The method used is given in detail. Cytogenetic analysis of growing cultures is the best indicator to determine whether the observed proliferation concerns malignant or normal mesothelial cells. The addition of epidermal growth factor (EGF) and hydrocortisone (HC), or EGF alone, to the culture medium increases the chances of successful isolation of a malignant mesothelioma cell line.     

Primary histiocytic lymphoma of the epididymis.

The first documented case of primary histiocytic lymphoma of the epididymis is presented. The tumor showed distinct nodularity, which is unusual for extranodal lymphomas, and marked sclerosis. Extensive staging work-up showed no evidence of extraepididymal spread. Unusual features included the youth of the patient at presentation and the severe diffuse atrophy of the adjacent testicular parenchyma. Following orchiectomy and radiotherapy, there has been no subsequent clinical evidence of systemic disease.