Clinical and flow cytometry characteristics of malignant pleural effusions in patients after intracavitary administration of methylprednisolone acetate.

Ten patients with recurrent pleural effusions due to advanced cancer were treated by intracavitary methylprednisolone acetate (Depo-Medrol [DM], Upjohn, Kalamazoo, MI). They received one to six courses of DM (median, three courses per patient) with doses ranging from 80 to 160 mg per course. Effusion cells were cryopreserved before and during DM installation for subsequent determination of ploidy by flow cytometry. Pleural effusion in all three patients with advanced breast cancer resolved and did not reaccumulate throughout follow-up for 11+, 10+, and 8+ months. Pleural effusion in a patient with metastatic gastric cancer and in two of four patients with adenocarcinoma of unknown origin partially resolved. Altogether six of ten patients (60%) subjectively and objectively benefited from this therapy. All patients tolerated the treatment well with no local or systemic side effects. Flow cytometry showed a reduction in ploidy of effusion cells in all three patients with breast cancer, from a peak mean channel of 6C to nearly 2C after therapy. Transient reduction of ploidy was seen also in the effusion of a patient with unknown primary tumor associated with clinical improvement. The clinical and laboratory data reported offers initial evidence that DM when instilled into the pleural cavity after incomplete thoracentesis may act as effective palliative therapy either alone or in combination with other anticancer agents.

Competitive ELISA for detection of native ras gene-related products in sera of cancer patients.

A solid phase, enzyme-linked immunosorbent assay (ELISA) competition kit was developed to detect circulating native ras gene-related products in sera of 151 healthy volunteers and cancer patients. This assay uses monoclonal antibody (mAb) BST-6A generated against a yeast-derived, native ras-related polypeptide Yp20. Only 2% (1 of 58) of normal control sera showed strong competition, as compared to 15% (5 of 34) of patients with early stage or no evidence of disease, and 44% (26 of 59) of patients with advanced disease. These differences were statistically significant (x2, P less than 0.05-0.001). Eleven sera samples of cancer patients found to be strong competitors in the ELISA competition kit were tested for the presence of anti-ras antibodies by ELISA. None showed higher ELISA values as compared with pooled normal human serum and control sera. It is thus suggested that our procedure detected circulating ras-related onco-proteins in sera of cancer patients mainly with advanced disease.

In vivo detection of retroviral particles in mice with hybridoma induced ascites.

Ascites induced by hybridoma cells following their injection into pristane-primed peritoneal cavities of BALB/c mice is often associated with the formation of intra-abdominal tumors related to the transplanted cells. These tumors and adjacent abdominal and extra-abdominal organs were excised and examined by electron microscopy. Clusters of viral particles were seen in nearly all cells composing the hybridoma tumors. The viral particles were usually present in rough ER cisternae of the transplanted tumor cells and at times were observed budding from the cisternal membranes. Individual viral units were doughnut shaped, 80-85 nm in diameter, with two concentric shells of which the outer one was more electron dense. No exogenous viral particles were detected in the peritoneally growing hybridoma cells. All non-tumor tissues examined such as liver, spleen, lung, brain and circulating WBC were free of virus. These findings provide further evidence of viral contamination of hybridomas and demonstrate the mode of persistence of such contamination when cells are placed in-vivo.

A yeast-derived ras-gene-related protein expressed in human tumor cells. I. Detection by polyclonal antibodies.

Rabbit polyclonal antibodies (pAb) were raised against a yeast ras-related protein YP20 and shown to be immunoreactive with human normal as well as altered Ha-ras and Ki-ras p21 gene products using immunoblotting and immunoprecipitation techniques. The p21 protein revealed by anti-YP20 antibodies comigrates with p21 protein detected by anti-p21 monoclonal antibody (Cetus Diagnostics). These pAbs were tested against a panel of human acetone-fixed tumor cell lines and malignant effusions and nonfixed fresh-frozen tissue sections obtained from cancer patients by the indirect immunofluorescence assay (IFA). Twelve of sixteen (75%) sarcoma and carcinoma cells lines and one fibroblast cell line were stained by the anti-YP20 pAb. The binding occurred most commonly in the cytoplasm. Six of eight fresh-frozen colon and breast cancer tissue sections were immunostained and normal sections from these organs or skin showed only low level of binding to the pAbs. Four of five malignant effusions were distinctively immunostained. These antibodies are suggested to serve as additional probes for assessing the expression of ras gene-related proteins in human malignancy.

Dupuytren's contracture studied with monoclonal antibodies to connective tissue differentiation antigens.

Seventeen patients with Dupuytren's contracture underwent partial fasciectomy, and frozen tissue sections from the involved palmar fascia were prepared for binding studies with hybridoma-derived murine monoclonal antibodies (MoAb) recognizing connective tissue differentiation antigens. The two MoAb used were both generated using human sarcomas as immunizing agents, 23H7 known to bind to an antigen shared by selected sarcomas and carcinomas but not normal adult tissues except a subset of granulocytes, and 12C9 shown to recognize a common fibroblastic marker. MoAb 23H7 was discovered to bind to a subset of fibroblasts within the lesions of six of 17 patients with Dupuytren's disease. Occasionally it immunostained a single cell population associated with tissue granulocytes dispersed in the surroundings of the lesions. MoAb 12C9 was found to be expressed in only 12 of 17 specimens prepared from involved lesions from Dupuytren's disease. It is suggested that fibroblasts from selected patients with Dupuytren's contracture express a novel antigen, defined by MoAb 23H7, previously shown to be associated with human sarcomas and other neoplasia. The other fibroblast marker which is defined by MoAb 12C9 and known to be a common connective tissue antigen, is only occasionally expressed in lesions involved with this disease. Though additional markers associated with Dupuytren's contracture remain to be defined, the MoAb, capable of defining connective tissue differentiation markers, reported in this study may serve as new immunological probes for immunodissecting this syndrome into subsets of diseases which may better define the variety of clinical patterns presented by patients.

Labeling of sarcoma associated monoclonal antibody with 111In, 67Ga and 125I.

Labeling of human sarcoma-associated murine monoclonal antibody (MAb) 23H7 with 67Ga and 111In by the bifunctional ligand method is reported. 67Ga was chelated to the MAb via desferrioxamine B and 111In via the cyclic anhydride of DTPA. Higher specific activity was obtained with 67Ga (4-5 microCi/micrograms) as compared with 111In (2 microCi/micrograms). The binding capacity of the MAb was confirmed by repeated indirect immuno-fluorescence assays performed before and after labeling. A fast blood clearance was observed: 33% recovered dose (R.D.) blood level 3 h post-injection as compared with 56% after injection of control polyclonal IgG. Preliminary results on chemically induced sarcoma bearing mice showed a relatively high tumor uptake of the labeled antibody.

Human sarcoma-associated murine monoclonal antibody labeled with indium-111, gallium-67, and iodine-125.

Monoclonal antibody (MAb) 23H7 is a hybridoma-derived IgG that is generated following fusion of mouse myeloma cell line P3U1 and spleen cells from BALB/c mice hyperimmunized with a human fibrosarcoma. It detects a mesenchymal antigen of 23KD expressed on human sarcoma tissues and other neoplasms, including myeloid leukemias, but it rarely binds to normal tissues. The MAb 23H7 was labeled with 67Ga and 111In using the bifunctional ligand method. The 67Ga was chelated to the MAb via desferrioxamine B, while 111In was chelated via the cyclic anhydride of diethylenetriamine pentaacetic acid. Higher specific activity was obtained with 67Ga than with 111In (4.5 and 2 muCi/microgram, respectively); both gave stable complexes. When 23H7 was labeled with 125I, considerable breakdown was observed. This, together with the physical shortcomings of this isotope, emphasizes the advantages of labeling with 111In and 67Ga. The rapid blood clearance of the labeled sarcoma-associated MAb may be beneficial for early tumor uptake and for imaging shortly after injection.

Novel approach to MER/BCG administration in cancer patients.

Forty-four patients with previously untreated advanced lung cancer were randomized to receive radiochemotherapy (RC) or radiochemotherapy plus MER/BCG (RCM). Prior to immunotherapy administration cutaneous reactivity to 5 log dilutions of MER/BCG was determined starting at 100 micrograms per injected site to 10 sites. Reactive patients were injected with 10, 1.0, or 0.1 micrograms while anergic patients were given 200 or 100 micrograms to each of 10 cutaneous sites, the dose being inversely related to the strength of the pretreatment reaction. Injected doses were subsequently further increased or decreased to achieve tolerable local erythema and induration. This modification resulted in a marked reduction in cutaneous toxicity previously observed, and made it possible to nearly double the mean number of MER courses per patient. It is suggested that intradermal MER/BCG may prove to be a considerably more useful therapeutic agent if doses are adjusted to patients' individual cutaneous responsiveness.

The effect of insulin on human-mouse hybridoma formation.

To evaluate the effect of insulin on the formation of human-mouse hybridoma clones, P3U1 mouse plasmacytoma cells were fused with human lymph-node lymphocytes in the presence of polyethylene glycol. After fusion, cells were grown for 2 weeks in HAT medium supplemented with insulin (H1AT, 10(-1)-10(-5) units/ml) or in HAT medium alone. The addition of 10(-3) units/ml of insulin to HAT medium resulted in an over two fold increase in the number of clones formed and in the average colony size compared to growing the fused cells in HAT medium alone. In view of the recent increasing interest in human-mouse hybridoma fusions it is suggested that selective medium HAT should be supplemented by insulin (H1AT) to enhance the number of colonies formed and provide a more efficient way for stabilizing the newly formed hybrids.

Monoclonal antibody defining fibroblasts appearing in fetal and neoplastic tissues.

VIF3 is a hybridoma-derived mouse IgG monoclonal antibody (MoAb) generated in a fusion with the use of a malignant fibrous histiocytoma as the immunizing agent and shown to recognize a 70-kilodalton antigen expressed within connective tissues. Of 55 human tissue culture cell lines tested by indirect immunofluorescence, VIF3 was shown to bind to 20 of 35 (57%) sarcomas, 4 of 9 (44%) normal fibroblasts, and none of 11 carcinomas and other neoplasm-derived cell lines. A panel of over 259 human frozen tissue sections obtained from surgical pathology specimens, postmortem studies, and elective abortions was used to further determine the histopathologic specificity of VIF3. MoAb VIF3 was found to bind to 15 of 19 (79%) human sarcoma tissue sections tested. It was also shown to recognize an antigen expressed on a subset of fibroblasts dispersed within the stroma of carcinomas obtained from all 46 patients tested, as well as a subset of cells within 3 of 10 benign hyperplastic tissues (30%). VIF3-positive cells were detected in all 60 fetal tissues tested including amniotic membranes, placentas, and umbilical cords. In contrast, fibroblasts of normal adult tissues tested stained infrequently (22/97 or 23%) with this reagent. The results confirm that this MoAb is directed against a human connective tissue-specific marker. VIF3 detects a marker appearing on fetal fibroblasts that is typically not present in normal adult tissues, but reappears in association with neoplastic diseases. MoAb VIF3 therefore defines a fibroblast-oncofetal antigen and as such may serve as a probe for immunopathologic studies.

Detection of retroviral particles in hybridomas secreting monoclonal antibodies.

Electronmicroscopy of hybridoma clones derived by fusing BALB/c mouse spleen cells with P3U1 mouse plasmacytoma cells to generate monoclonal antibodies against human sarcoma antigens, revealed the presence of large number of viral particles. These particles were also seen budding from the cell surfaces. The intracytoplasmic particles were intracisternal and resembled type-A oncornavirus, while the budding and extracellular forms, with a centrally located nucleoid, resembled mature type-C oncornaviruses. Cells of the parental P3U1 palsmacytoma cell line and of the NS-1 myeloma cell line contained morphologically identical viral structures. The scientific and medical communities engaged in hybridoma research should be alert to the possible presence of viruses in hybridomas and their products. The question is raised as to whether it is safe to use mouse monoclonal antibodies for clinical purposes, both diagnostic and therapeutic.

Modulation of hybridoma formation by dexamethasone.

In an effort to determine the effect of dexamethasone on hybridoma formation, spleen cells from BALB/c mice hyperimmunized with sheep red blood cells (SRBC) were fused with mouse plasmacytoma cells (P3U1) in the presence of polyethylene glycol (PEG). Dexamethasone was added in decreasing doses (10(-3) to 10(-9) mM) to the hypoxanthine-aminopterin-thymide (HAT) medium immediately after the PEG-mediated cell fusion. 10(-3) mM of this steroid was found to inhibit markedly the number and size of hybridoma clones generated, while 10(-5) mM dexamethasone was shown to enhance hybridoma formation. The effect of 10(-3) mM dexamethasone was most pronounced when added immediately after fusion. When this dose was given 48 or 120 h after cell fusion, the extent of the inhibitory effect was less pronounced. High concentration of dexamethasone may also inhibit monoclonal antibody production by hybridomas once generated. An increase in the number of clones formed was observed when 10(-5) mM dexamethasone was added to HAT medium as well as an increase in the average colony size. Large clones were also observed with lower dexamethasone doses ranging from 10(-7) to 10(-9) mM. Possible mechanisms on the effect of dexamethasone on hybridoma formation are discussed.

The use of insulin-enriched culture medium and human-human hybridoma formation: a preliminary study.

Human lymphoblastoid cell line WI-L2-729-HFZ was fused with human lymph-node lymphocytes in one fusion and with human spleen cells in another fusion to generate human-human hybridomas. In both, increasing doses of insulin were added to the HAT medium immediately after the PEG-mediated cell fusion (10(-1)-10(-5) IU/ml) and the number of clones formed was determined 3 weeks later. 10(-3) IU/ml of insulin resulted in a 2- to 5-fold increase in the number of clones generated compared to the control plates. In view of the known difficulties in generating high-yield human-human hybridoma fusions, it is suggested that the use of insulin-supplemented HAT medium may provide a more efficient way in obtaining such clones.

A high-affinity monoclonal antibody (GIF-1) to human gamma-interferon: neutralization of interferon mediated inhibition of retrovirus production and 2'-5' (A) synthetase induction.

An hybridoma clone secreting an IgG1 monoclonal antibody (GIF-1) specific for human gamma-interferon (HuIFN-gamma) has been generated using HAT medium supplemented with insulin (HIAT) at the initial stage of cell fusion. This antibody is capable of neutralizing the antiviral activity of HuIFN-gamma, the ability of HuIFN-gamma to inhibit retroviral replication in RD-114 cells, and the ability of HuIFN-gamma to induce the 2'-5' oligoadenylate (A) synthetase in RD-114 and HeLa cells. Eluate from an immunoaffinity column containing GIF-1 yielded two protein bands of molecular weight of 20 and 25 kd when subjected to SDS-PAGE.

The interaction of Kaposi's sarcoma with monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

Four monoclonal antibodies (McAbs) previously generated against human soft tissue sarcomas and reacting with connective tissue differentiation antigens were evaluated for their interaction with tissues obtained from patients with classic Kaposi's sarcoma. Biopsy was performed on active neoplastic lesions from the skin of 26 patients, frozen sections were prepared, and the binding of the McAbs was tested using the indirect immunofluorescence assay. Clinically uninvolved skin from the same patients as well as skin and muscle from eight non-cancer patients were treated similarly and served as controls. McAbs IXG11, 23H7, IIIE5, and 15G5 interacted strongly with the Kaposi's sarcoma lesions and weakly with the uninvolved skin in 22 of 26 (84%), 23 of 26 (88%), 12 of 14 (85%), and 1 of 6 (16%) of the patients, respectively. IXG11, 23H7, and IIIE5 interacted weakly with the skin of seven of eight non-cancer patients. McAb 15G5 was found to bind strongly to tumor lesions, to the respective uninvolved skin in four of five Kaposi's sarcoma patients, and also to skin and connective tissues of muscle from non-cancer patients. The mode of interaction was morphologically different for each McAb. It is suggested that McAbs IXG11, 23H7 and IIIE5 identify markers whose expression is markedly increased in Kaposi's sarcoma lesions as compared with uninvolved skin of the same patients. These markers may serve as immunologic probes for the investigation of this neoplastic process.

Malignant melanoma appearing in a post-mastectomy lymphedematous arm: a novel association of double primary tumors.

The association of a malignant melanoma appearing as an additional primary tumor in the swollen arm adjacent to a mastectomy for breast cancer is reported. A review of the literature revealed only one similar patient previously reported. In both patients, the melanoma and its metastasis were restricted to the lymphedematous arm, appeared 10 years post-mastectomy, and responded to therapy. The similarity to Stewart-Treves syndrome is emphasized. It is suggested that nevi developing in the lymphedematous arm post-mastectomy should be carefully monitored and excised early whenever indicated.

Monoclonal antibodies to human sarcoma and connective tissue differentiation antigens.

The use of monoclonal antibodies to distinguish human sarcoma from carcinoma cells has been explored. Spleen cells from a BALB/c mouse immunized with a human malignant fibrohistiocytoma were fused with cells of the mouse P3U1 plasmacytoma cell line. Antibodies were then screened for reactivity against human sarcoma and carcinoma cells growing in culture. This work has yielded 2 immunoglobulin G monoclonal antibodies VIE4 and VIF3 which, respectively, reacted with 85% (17 of 20) and 90% (18 of 20) of sarcoma lines tested but with none of eight carcinoma cell line preparations. Reactivity against normal fibroblasts was also demonstrated. By immunofluorescence, the antigens detected by the two antibodies appear to have distinctive intracellular distributions. Immunoprecipitation with VIF3 has shown that it is detecting a protein with a molecular weight of 70,000. When tested against pathological frozen tissue sections, VIF3 reacted with four of 11 and VIE4 with three of 11 human sarcomas but with none of ten carcinomas tested. VIF3 occasionally bound to normal adult connective tissues, whereas no such reactivity was seen with VIE4. These antibodies appear to be directed to fibroblastic markers associated with sarcomas and connective tissue differentiation antigens.

The addition of insulin to HAT medium (HIAT) enhances hybridoma formation.

To assess the effects of insulin on the formation of hybridomas, sheep red blood cell (SRBC) immunized spleen cells from BALB/c mice were fused with P3U1 mouse myeloma cells. After fusion, cells were grown for 2 weeks in HAT medium containing insulin (HIAT) (doses ranging between 10(-1) to 10(-9) units/ml) or HAT medium alone. The number of hybridoma colonies was found to be significantly increased in the presence of HIAT medium compared to HAT alone. In addition, the average size of the hybridoma clones was at least doubled and the cumulative colony size index per plate increased several folds. A significant rise in the number of wells containing clones secreting anti-SRBC monoclonal antibodies was again shown in the presence of HIAT compared to HAT medium alone. The maximal effect of insulin on the above biological parameters ranged between 10(-1) and 10(-4) units/ml. It is concluded that the addition of insulin to HAT medium (HIAT) enhances hybridoma formation and thus, its more frequent use may considerably expediate ongoing research efforts on the production of monoclonal antibodies.

Enhancement of hybridoma formation by addition of insulin to HAT medium (HIAT).

The effects of insulin on the formation of hybridoma clones following fusion experiments with SRBC immunized BALB/c mouse spleen cells and P3U1 mouse plasmacytoma cells were evaluated. The addition of insulin to HAT medium (HIAT) resulted in significant increases in the number and size of hybridoma colonies generated. The total number of anti-SRBC antibody-secreting clones also increased as much as sevenfold using insulin-supplemented medium compared to HAT alone. In view of the increasing interest in hybridoma technology for monoclonal antibody production, the use of insulin-supplemented medium (HIAT) may significantly expedite ongoing work by providing a more efficient method for the establishment of stable clones.