In vivo localization of anti-osteogenic sarcoma 791T monoclonal antibody in osteogenic sarcoma xenografts

Monoclonal antibody 791T/36, (mouse IgG_2b) to the human osteogenic sarcoma 791T was isolated from hybridoma supernatants or ascites fluid and labelled with radioiodine. Following injection into immuno-deprived CBA mice with subcutaneous xenografts of human osteogenic sarcomas 791T, 788T and 20s, reactive antibody was detectable in the circulation and there was a preferential uptake into tumour tissue compared with muscle, bone or visceral organs. The tumour uptake of radioactivity correlated with the size of the xenograft, up to 20% of the total body counts being within the tumour. Autoradiography of tissue sections showed activity mainly within the subcapsular regions of the xenografts. There was no tumour localization of radio-labelled normal IgG. Furthermore, with a colon carcinoma and a bladder carcinoma, both unreactive with the 791T/36 antibody, there was no localization of radiolabelled antibody in xenograft growths. With 791T/36 monoclonal antibody labelled with ¹³¹I localization into 791T xenografts could be visualized by whole-body gamma scintigraphy with computerized ^99mTc-pertechnetate or ^113mIn-indium chloride-labelled blood pool subtraction. These studies demonstrate the potential of monoclonal antibody to a human tumour for in vivo localization both for diagnostic purposes and as a carrier for therapeutic agents.     

Patterns of reactivity of the monoclonal antibody 791T/36 with different tumour metastases in the liver

Reactivity of the monoclonal antibody 791T/36 with secondary malignant deposits has been investigated in frozen sections of 74 human liver biopsy specimens. There was no reactivity with hepatocytes but in some instances binding to fibrous tissues and in one case to portal tract lymphocytes was observed. Sections from 9 biopsy specimens contained malignant deposits. In seven of these 791T/36 bound either to malignant cells or to pseudoacinar contents (3 colorectal adenocarcinomas; 1 probable pancreatic adenocarcinoma; 1 medullary cell carcinoma of thyroid; 1 oat cell carcinoma of bronchus and 1 deposit of nodular sclerosing Hodgkins Disease). Two undifferentiated tumours (1 gastric adenocarcinoma and 1 oat cell bronchial carcinoma) showed no antibody binding. The histological pattern of reactivity previously reported with primary tumours appears to be similar in secondary deposits. A wider range of tumours than recognised hitherto binds 791T/36. Whether the binding to fibrous tissue seen in some instances is sufficient to cause diagnostic uncertainty when 791T/36 is used for scanning requires further investigation.     

Complement-dependent cytotoxicity of anti-human osteogenic sarcoma monoclonal antibodies.

Two mouse monoclonal antibodies against the human osteogenic sarcoma 791T were examined for their capacity to exert complement-dependent cytotoxicity against a panel of human tumour cell lines. Cytotoxicity was most evident against the immunizing tumour 791T although significant reactivity was directed against other osteogenic sarcomas. In admixture, the 2 antibodies displayed synergism in their cytotoxicity although this was only demonstrable with defined ranges of antibody concentration. The cytotoxicity of these antibodies was dependent upon the use of rabbit serum as complement and no tumour-cell lysis was produced using human, guinea-pig or mouse serum complement. The more potent cytotoxic antibody failed to modify the outgrowth of 791T tumour xenografts in immunodeprived mice even though localization of antibody at the tumour site has been demonstrated (Pimm et al., 1982).     

Induction of delayed hypersensitivity to human tumor cells with a human monoclonal anti-idiotypic antibody

There is considerable interest in the development of anti-idiotypic antibodies as vaccines in a number of diseases, including cancer. We have developed a human anti-idiotypic monoclonal antibody (105AD7) which binds at or very near to the binding site of mouse antitumor monoclonal antibody 791T/36. The 791T/36 antibody binds to a tumor-associated antigen (gp72) expressed on a number of human tumors, including colorectal and ovarian carcinomas and osteogenic sarcoma. This study shows that, in rats and mice, 105AD7 induces delayed-type hypersensitivity to human tumor cells bearing the gp72 antigen. Local transfer of delayed hypersensitivity was also demonstrated using lymphocytes from mice primed with 105AD7. These findings show that the human monoclonal anti-idiotypic antibody 105AD7 is likely to induce cellular immune responses to tumors in cancer patients.     

Association of antigen expression and DNA ploidy in human colorectal tumors

Fifty colorectal tumors were screened by indirect immunofluorescence and flow cytometry for antigen expression using a panel of monoclonal antibodies that recognize determinants preferentially expressed on tumor cells (carcinoembryonic antigen, Y haptenic blood group, 791T/36 defined antigen 791T-P72). Fifty % of the tumors expressed all three antigens, 41%, two, and 9%, one. Over a third reacted strongly with at least one monoclonal antibody, although the majority of tumors stained with a moderate intensity. Extranuclear membranes from tumors showed similar antigen expression to disaggregated tumor cells and were particularly useful for providing the relative tumor:normal tissue binding ratios. The carcinoembryonic antigen specific monoclonal antibody showed the strongest tumor selectivity with a tumor:normal tissue ratio of 24 +/- 7:1. Lack of correlation between expression of the three antigens suggested that the monoclonal antibodies recognizing them may have potential as a "cocktail." One-third of the tumors contained cells with an aneuploid DNA content and expressed elevated levels of carcinoembryonic antigen and Y haptenic blood group antigen when compared to tumors with diploid DNA content. Aneuploid cells within a tumor were also preferentially stained with all of the monoclonal antibodies.     

Prognostic significance of HLA class I expression in osteosarcoma defined by anti-pan HLA class I monoclonal antibody, EMR8-5

With the goal of establishing efficacious peptide-based immunotherapy for patients with bone and soft tissue sarcomas, we previously identified the cytotoxic T lymphocyte-defined osteosarcoma antigenic gene Papillomavirus binding factor. The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas. Seventy-four formalin-fixed paraffin-embedded specimens of various bone and soft tissue sarcomas, including 33 osteosarcomas, were stained with the anti-HLA class I monoclonal antibody EMR8-5, which we recently generated. The expression of HLA class I was lost or downregulated in 46 of these specimens (62%). With respect to osteosarcoma, loss or downregulation of HLA class I expression was seen in 13 (52%) of 25 primary tumors and seven (88%) of eight metastatic tumors. In six of 11 HLA class I-negative osteosarcoma specimens, the expression of beta-2 microglobulin was also lost. Subsequently the prognostic significance of HLA class I expression was analyzed in 21 patients with osteosarcoma who had completed multidrug neoadjuvant chemotherapy and undergone adequate surgery. Patients with osteosarcoma highly expressing HLA class I showed significantly better overall and event-free survival than those with HLA class I-negative osteosarcoma. In contrast, such prognostic significance of HLA class I expression was not found in 15 patients with malignant fibrous histiocytoma of soft tissue. These findings suggest that the class I-restricted cytotoxic T lymphocyte pathway plays a major role in immune surveillance of patients with osteosarcoma.     

Endocytosis of immunotoxin-791T/36-RTA by tumor cells in relation to its cytotoxic action

Ricin A chain immunotoxin constructed with monoclonal antibody 791T/36, which recognizes a tumor associated glycoprotein Mr 72,000 antigen present on sarcomas and colon and ovarian cancer cells, is cytotoxic for cell lines from tumors expressing this antigen. Incubation of sarcoma 791T cells with immunotoxin for only 5 min is sufficient to produce greater than 95% inhibition of tumor cell growth. Papain treatment of these cells to remove immunotoxin from the cell surface indicated that the cell surface acts as a reservoir for continued internalization of immunotoxin over several hours, but even so, 50% inhibition of cell survival was produced over a 2- to 3-h period. Analysis of the rate of endocytosis demonstrated that 30-50% of cell bound immunotoxin was internalized over a 180-min period. This was primarily dictated by the antibody moiety, regardless of the degree of conjugation to ricin A chain. This rate is much slower than that of other cell surface ligands such as transferrin. Cell cytosol acidification experiments were performed to determine whether this immunotoxin was internalized by clathrin coated pits, which is relatively rapid, or by smooth pits, which is slower, and the results indicated the latter mechanism is almost exclusively used. Intracellular trafficking of antibody 791T/36, conjugated to human serum albumin-tetramethylrhodamine was investigated by flow cytometry. The movement of the conjugate into the lysosomal compartment was delayed so that degradation products were only detected after a lag phase of 30-60 min. The lack of potentiator dependence of 791T/36 immunotoxin is in keeping with these findings.     

Immunohistochemical localization of ganglioside GD3 in human malignant melanoma, epithelial tumors, and normal tissues

The specific tissue distribution of melanoma-associated ganglioside II3-alpha-N-acetylneuraminosyl-alpha 2----8-N-acetylneuraminosyllactosylceramide (GD3) was studied on 175 cryopreserved, unfixed human tissue sections with R-24 mouse monoclonal antibody by indirect immunoperoxidase staining. A striking specificity of monoclonal antibody R-24 for malignant melanoma tissues was established. Ganglioside GD3 was detected in all 21 tissue sections of 21 patients with primary melanoma and in all 37 probes of 24 patients with metastatic malignant melanoma. The majority of tumor cells in the samples of primary malignant melanoma expressed GD3; however, GD3 expression was more heterogeneous in samples of metastatic lesions even in different metastases of the same patient. Of 11 nevi, 9 reacted with monoclonal antibody R-24, while melanocytes in the basal layer of normal skin stained only weakly and irregularly. None of the 32 normal and 12 fetal human tissue types were R-24 positive, but a strong cytoplasmic staining was observed with single cells in the dermis and in the interstitial tissue of the gastrointestinal tract, in the interlobular septa of the thymus, and in other distinct locations. Only two malignant carcinoid tumors of 38 nonmelanomatous tumors tested reacted with monoclonal antibody R-24.     

Absence of telomerase activity in malignant bone tumors and soft-tissue sarcomas

Purpose: Telomerase activity appears to play a crucial role in the development of many tumors. More than 80% of all malignant human tumors show an increased telomerase activity. However, conflicting results have been reported about telomerase activity in sarcomas. The aim of the study was to obtain more information about telomerase activity in sarcomas based on a large number of cases.Methods: Telomerase activity was measured in 69 different tumor samples (33 malignant bone tumors and 36 soft tissue sarcomas). Tumor samples were obtained intraoperatively and frozen immediately in liquid nitrogen. Telomerase activity was detected by the telomeric repeat amplification assay (TRAP-assay).Results: Only 7% of the samples showed telomerase activity. No correlation between staging and telomerase activity could be observed.Discussion: The fact that only five out of 69 examined tumor samples showed a telomerase activity provides experimental evidence that in sarcomas the reactivation of telomerase may play a subordinate role. Our results suggest that alternative mechanisms for cell immortalization, yet to be determined, seem to be involved in the development and/or maintenance of soft-tissue sarcomas and malignant bone tumors.     

Leiomyosarcoma of bone. A clinicopathologic, immunohistochemical, and ultrastructural study of five cases.

The authors identified five leiomyosarcomas (LMS) in a review of 13 nonmatrix-producing spindle cell sarcomas of bone. Only two were initially recognized as LMS; the others had been diagnosed as malignant fibrous histiocytoma (two) and fibrosarcoma (one). The patients, four of whom were women, ranged in age from 32 to 70 years. Sites included proximal humerus (two), distal femur (two), and rib (one). All tumors presented with clinical and radiographic features consistent with a diagnosis of primary bone neoplasms, although one probably represented a solitary metastasis from a primary uterine LMS. Radiographs showed lytic bone destruction with a moth-eaten appearance, and three cases had soft tissue extension. Histologically, all tumors showed broad, interlacing fascicles of spindle cells with pleomorphic nuclei, frequent mitoses, and necrosis. Two cases had a focal storiform pattern and bizarre multinucleated cells, and two other cases had focally prominent osteoclast-like giant cells. Extensive immunoreactivity for muscle actin was seen in all cases and for desmin in three. In each case, electron microscopy showed definite smooth muscle differentiation including cytoplasmic filaments with densities. At this writing, two patients are free of disease (including the patient with a presumed metastasis), one is alive with locally recurrent disease, and two are dead of disease. Experience suggests that LMS of bone is a distinct clinicopathologic entity that may be more common than previously recognized. Application of immunohistochemistry and electron microscopy to nonmatrix-producing bone sarcomas should facilitate diagnosis of additional cases.     

Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody

The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.     

Sensitivity and selectivity of ricin toxin A chain-monoclonal antibody 791T/36 conjugates against human tumor cell lines

Ricin toxin A chain (RTA) was conjugated to monoclonal antibody 791T/36, which was raised originally against human osteogenic sarcoma cell line 791T. The resultant conjugates were characterized and tested for cytotoxicity against a panel of human tumor cell lines representing a defined range of antigenicity with regard to 791T/36. Conjugates were highly cytotoxic for cells expressing high antigen density, inhibiting cell survival at RTA concentrations three to four orders of magnitude lower than that possible with RTA alone. Cytotoxicity of conjugates diminished with decreasing 791T/36 antigen concentration on target cells, but significant effects were seen against cells of low or intermediate antigenicity. Cytotoxicity could be blocked specifically by excess 791T/36 antibody, clearly indicating that antigen binding was a necessary part of the mechanism of action. Comparison with drug-antibody conjugates indicated that RTA immunotoxins are much more active, but discriminate less readily than drug-antibody conjugates between cells of different antigenicity. It is suggested that these properties be taken into account with regard to practical application and future development.     

Primary mediastinal germ cell tumors. Histologic patterns of treatment failures at autopsy

Twenty-five patients presented with primary mediastinal germ cell tumors at Roswell Park Memorial Institute between 1959 and 1984. All patients were treated by surgery and chemotherapy with or without radiotherapy. Four patients are still alive, and 21 patients died of mediastinal germ cell tumor and its sequelae. Two patients were found to have testicular scars and were dropped from the study. Nongerm cell malignant transformation of a teratoma occurred in five of the remaining 17 patients (29%), resulting in three adenocarcinomas and two sarcomas. Another patient developed leukemia. Metastatic disease most commonly involved the lungs, mediastinal lymph nodes, liver, bone, retroperitoneum, and heart. Respiratory failure was the cause of death in 12 patients. Of the possible mechanisms of germ cell transformation into malignant nongerm cell tumors discussed, this study suggests that chemotherapy alone is unlikely to induce stem cell differentiation. The presence of mature, differentiated teratoma within the primary lesion may be indicative of a poorer prognosis.     

Antigenicity of newly established colorectal carcinoma cell lines

Cells from two adenocarcinomas, an adenoma and a metastatic node were isolated in soft agar. Expression of antigens, CEA, Y haptenic blood group and 791T-p72, defined by a range of candidate antibodies for tumour targeting was assessed. All of the cells expressed low levels of CEA but high levels of the Y haptenic blood group antigen although there was enormous inter and intraclonal variation. Of particular interest was the membrane expression of 791T-p72 antigen on all of the dividing tumour cells as previous studies had shown that 791T/36 antibody reacted with tumour stromal elements rather than malignant cell surfaces. The DNA content was abnormal in all of the cells whether they were derived from diploid or aneuploid primary tumours. They all grew readily in athymic mice and at least one monoclonal antibody, 791T/36, localised efficiently within these xenografts. Clonogenic cells therefore expressed the three tumour-associated antigens, several at higher levels than observed in the primary tumour. Monoclonal antibody 'cocktails' should therefore allow antibody mediated drug cytotoxicity to be effective at eradicating rapidly dividing tumour cells.     

Evaluation of immunolocalization in gastrointestinal cancer

Tumor localization by a 131I-labeled monoclonal antibody to CEA has been evaluated in a series of 50 patients with clinically suspected primary or recurrent gastrointestinal cancer. Eighty-five percent of the primary tumors were correctly detected, as were 43% of associated nodal metastases. Localization was compared with computerized tomography in the detection of recurrent disease. Each technique correctly identified 61% of the sites but missed 39%. In addition, labeled antibody localization produced a significant number of false-positive images. Radioactivity accumulated by tumors, both primary and secondary, was significantly higher than that in surrounding normal tissue (P less than .01). However, less than 0.8% of the injected radioactivity and 0.01% of the injected antibody were detectable in the tumors. Radiolabeled antibody was rapidly cleared from the circulation, and this may reflect a recipient reaction to the foreign protein.     

Immunohistochemical localization of prostate carcinoma-associated antigens

The immunoperoxidase technique was used to study the localization and distribution of antigens reactive with two monoclonal antibodies, D83.21 and P6.2, produced against cultured prostate tumor cells, in formalin-fixed, paraffin-embedded histological sections of human tissues. Monoclonal D83.21 reacted with 11 of 19 (58%) primary prostate carcinomas and 1 of 6 (17%) metastatic tumors, whereas monoclonal P6.2 reacted with 14 of 19 (68%) primary and 4 of 6 (67%) metastatic prostate tumors. Neither antibody reacted with five primary prostate tumors and one metastatic prostate tumor. In some tumor cells, the antigens recognized by these monoclonals were localized in either the cytoplasm or cell membrane, while in other tumor cells, both diffuse cytoplasmic and membrane or focal staining patterns were observed. In addition to the variable staining patterns, antigenic heterogeneity was also noted within most prostate tumors examined. Two types of staining variability were observed: (a) tumor cells in one area of the tissue section stained positive, but in another area they did not react with the antibody; and (b) both stained and unstained tumor cells were adjacent to each other. These results would suggest that a panel of monoclonals will be required to detect the different subpopulations of prostate tumor cells. Neither antibody reacted with 6 normal or 12 benign prostate tissues, nor any of a variety of other normal human tissues except for staining of the proximal tubules of normal kidneys. The antigen detected by P6.2 demonstrated a wider tissue distribution being found on bladder, breast, lung, and pancreatic tumors, whereas the antigen recognized by D83.21 was restricted to prostate and bladder carcinomas. These antibodies may have clinical applicability for the identification of prostate tumor cells in biopsy specimens and for immunohistopathological classification of prostate carcinomas.     

Radioimmunolocalization of colorectal carcinoma. A correlation among RIL results, surgical findings, serum tumor marker levels and the presence of CEA and CA 19.9 in tumor tissue: the experience of the Hospital de la Santa Creu i Sant Pau.

We report a prospective study in two groups of colorectal cancer patients carried out by radio-immunolocalization (RIL) with F(ab') fragments of monoclonal antibodies against CEA and CA 19.9 labeled with 131-I. Twenty-two patients were studied before radical surgery and 12 patients after initial surgery, when progressive increase in CEA was registered. Scintigraphic images obtained in vivo in RIL studies were compared with scintigraphic images of the corresponding surgical specimens. Results were compared with known serum marker levels and with the presence and localization of markers in the excised specimens. RIL images correctly identified 13 of 23 (52%) primary tumors, with only one false positive image. Scintigraphy of surgical specimens correlated with RIL findings in 14 of 19 cases (74%). Four specimens which showed antibody uptake had not been visualized preoperatively in the RIL study. Two of them were retrovesical and were obscured by residual activity in the bladder. Nine of 13 (64%) patients with at least one elevated tumor marker were imaged. Staining pattern or intensity of antigen staining in the specimens did not correlate with RIL findings. Recurrent disease was confirmed by laparotomy or other exploration in 10 of the 12 patients with progressive CEA elevation during follow-up. Spontaneous normalization of CEA levels was observed in the remaining 2 patients. RIL studies were positive in 7 of the 10 patients with confirmed recurrent disease. Of the 3 false negative patients 2 had liver metastases and one developed clinical lung, bone and adrenal metastases 11 months later. No false positive studies were observed in this group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soft tissue sarcoma as a second malignant neoplasm in the pediatric age group

BACKGROUND: Survivors of childhood malignancies have an increased risk of developing second malignant neoplasms (SMN) due to their prior treatment and/or genetic susceptibility. A small proportion of SMNs are soft tissue sarcomas (STS), whose prognosis is generally thought to be poor, though publications on such patients' treatment and outcome is limited. METHODS: The authors analyzed 25 patients who were registered for the Italian Cooperative Group protocols for pediatric STS from 1979 to 2000. The primary tumor was STS in five patients; Hodgkin disease in five patients; leukemia in four patients; retinoblastoma, neuroblastoma, and Wilms tumor in two patients each; and other tumor types in five patients. SMNs occurred after a median of 8 years (range, 1.9-15.0 years) and included rhabdomyosarcoma (RMS) in 4 patients, malignant peripheral nerve sheath tumor in 4 patients, extraosseous Ewing family tumor (EFT) in 4 patients, leiomyosarcoma in 3 patients, fibrosarcoma in 2 patients, synovial sarcoma in 2 patients, and other tumor types in 6 patients. Treatment generally was administered according to the guidelines for primary STS. RESULTS: Seven non-RMS patients with STS underwent surgery alone, whereas 18 patients received chemotherapy and 8 patients received radiotherapy. Retreatment was feasible with acceptable toxicity. Fifteen patients were alive in complete remission of their SMN at the time of last follow-up. Responses to chemotherapy and survival were satisfactory for patients with tumors such as RMS and EFT. Complete tumor resection was correlated with a favorable prognosis in patients with other types of STS and in patients with postirradiation sarcoma. Two patients developed a third malignancy. CONCLUSIONS: Although prior treatment may hinder the management of these patients, pediatric STS second malignancies can be cured using the same strategies used for de novo pediatric sarcomas. Long-term follow-up is mandatory given the risks of further malignancies and more severe, treatment-related side effects.     

Phase I study of monoclonal antibody-ricin A chain immunotoxin XomaZyme-791 in patients with metastatic colon cancer

Monoclonal antibody 791T/36, recognizing a Mr 72,000 antigen on the surface of colon carcinoma cells, has been used to construct an immunotoxin by conjugating to it the ribosomal inhibitor protein, ricin toxin A chain. The antibody 791T/36 has been shown to bind to membranes of freshly disaggregated tumor cells from human colon tumors, and to localize in tumors in vivo. Subacute toxicology testing in rats receiving immunotoxin i.v. showed, at highest doses, weight loss, decreased serum albumin, and hepatocyte vacuolization without elevation in liver function tests. A Phase I dose escalation study was carried out in which 17 patients with metastatic colorectal cancer were treated with doses of immunotoxin ranging from 0.02 to 0.2 mg/kg/day in 1-h i.v. infusions for a 5-day course. Side-effects included a composite of signs and symptoms thought to be generic to ricin A chain immunotoxins, including decreased serum albumin, mild fever, and flu-like symptoms, all being reversible. Two additional findings, reversible proteinuria and mental status changes, were also noted which may be characteristic of this immunotoxin. By 10-20 days after therapy, most patients developed IgM and IgG antibodies against both the ricin toxin A chain and the immunoglobulin portion of the immunotoxin, which were asymptomatic. A strong anticombining site antibody response was seen. Biological activity manifest as mixed tumor regression was seen in five patients.     

Biological Study of R24 Mouse Monoclonal Antibody in Patients Undergoing Thoracotomy for Pulmonary Metastases from Soft Tissue Sarcoma

The disialoganglioside GD3 is expressed on the surface of soft tissue sarcoma, malignant melanoma, and other malignant cells and is, therefore, a potential target for therapeutic monoclonal antibodies. Intravenously administered R24, a murine IgG₃ monoclonal antibody to GD3, induces inflammation and tumor regression at sites of metastatic malignant melanoma. R24 5 mg/m² was given intravenously every other day for six doses to 10 patients with pulmonary metastases from a primary soft tissue sarcoma of the extremity for whom thoracotomy was planned. Resected tissue was available from 7 patients. All metastases expressed GD3; however, expression was heterogeneous within tumors, and in no tumor were more than 80% of the cells GD3 positive. A mild to moderate infiltrate consisting of mononuclear cells with T-cell markers was identified around or within pulmonary metastases in 6 patients. Tolerable acute allergic reactions occurred in all patients, but 3 patients had severe chest tightness and bronchospasm that limited the planned therapy. The setting of thoracotomy for metastatic disease provides an ideal system for studies on the pharmacology and biological effects of monoclonal antibodies that target soft tissue sarcoma antigens.