Specific antigen in serum of patients with colon carcinoma.

The binding of monoclonal antibody specific for colon carcinoma was inhibited by serum from patients with adenocarcinoma of the colon but not by serum from patients with other bowel diseases or from healthy volunteers. Of other malignancies studied, serum from two patients with gastric carcinoma and two patients with pancreatic carcinoma also inhibited the specific binding of monoclonal antibody. The levels of carcinoembryonic antigen in these serum samples were not correlated with their levels of binding inhibition. Such monoclonal antibodies may prove useful for the detection of colorectal carcinoma.

Advantages of cryoperserved lymphocytes for sequential evaluation of human immune competence. I. Mitogen stimulation.

Human lymphocytes were harvested from normal volunteer donors and cryopreserved. Various concentrations of dimethyl sulfoxide and human serum in the cryoprotective media were evaluated to optimize the recovery and function of viable cells. Multiple samples were then drawn from volunteers over a number of days, processed individually, and used in mitogen stimulation assays. These cells were also cryopreserved, immediately thawed, and cultured simultaneously with the fresh cells. In all instances fresh and cryopreserved lymphocytes exhibited similar levels of stimulation by mitogens. Cryopreserved cells from these sequential bleedings were then recovered and cultured simultaneously in mitogen stimulation assays. The results obtained in these assays with cryopreserved cell had less day-to-day variagion than those with cells cultured individually. Coefficients of variance of individual cultures of mitogen stimulation assays were reduced from 26-59% to 5-17% by use of cryopreserved cells. The findings suggested that use of cryopreservation techniques decreases the variability of cellular immune assays and thus alows more accurate longitudinal study of the immune competence of patients.

Advantages of cryopreserved lymphocytes for sequential evaluation of human immune competence. II. Mixed lymphocyte cultures and mononuclear cell subpopulations.

Peripheral blood lymphocytes were obtained from normal volunteer donors over a 2-week period. One-way or two-way mixed lymphocyte reactions, percentages of T-cells and B-cells, and percentages of monocytes were quantitated on both fresh and cryopreserved lymphocytes. These comparisons revealed that the measurement of mixed leukocyte reactions, fractions of E-rosettes and EA rosettes, and the fraction of esterase-staining cells were not altered by cryopreservation and storage at -196 degrees C up to 1 year. Cryopreserved reference lymphocytes provided a stable standard for use as stimulating cells in mixed lymphocyte cultures and could be irradiated or treated with mitomycin C prior to being frozen, without alteration of their function as stimulator cells. Cryopreserved cells thus appeared to be utilizable in the sequential measurement of the immune competence of humans.

Multiparameter evaluation of the expression in situ of normal and tumor-associated antigens in human colorectal carcinoma.

The immunoreactivity of a panel of monoclonal antibodies (MoAb) was studied in a series of patients with colorectal carcinomas to test the association of antigen expression with other parameters such as histopathologic stage, differentiation, and clinical outcome. Low-level binding to normal tissue and high-level binding to malignant tissue were observed with MoAb defining, respectively, a gastrointestinal cancer antigen (GICA), Leb (distal colon only), A, H type 2 antigen, X-like antigen, and the 200-kilodalton (Kd) protein of carcinoembryonic antigen (CEA). The degree of histologic differentiation correlated with the expression of Lea antigen, A, and Y haptens, whereas a progressive loss of these antigens coincided with loss of differentiation. Two undifferentiated carcinomas expressed only two, H type 2 antigen and a highly glycosylated protein of 20-50 Kd, of the 14 antigens investigated. An interesting, but not significant, association between Leb antigen expression and more extensive disease was found: Whereas 71% of Dukes C tumors were positive for Leb, only 48% of patients with Dukes A and B2 tumors showed the presence of Leb antigen. On the other hand, the presence of B72.3-defined antigen is significantly associated with an earlier stage of disease. Chi-square tests to assess the association of antigen positivity with disease recurrence indicated a significant binding association with tumor recurrence over a broad range of percent positive cells for two MoAb defining different determinants of GICA. Similar associations, but over a narrow range of positive cells, were found for H type 2 antigen and the 200-Kd protein of CEA.

Detection of the tumor-associated glycoprotein antigen (TAG-72) in premalignant lesions of the colon.

We used monoclonal antibody B72.3 to study the expression of the colorectal carcinoma-associated antigen TAG-72 in premalignant colonic lesions with the immunoperoxidase technique. This antigen, which is rarely detectable in the normal colonic epithelium, was expressed in 13 of 19 adenomas with moderate to severe dysplasia and nine of nine cases of inflammatory bowel disease. The antibody reacted with the normal-appearing mucosa adjacent to a carcinoma in 10 of 12 cases, although only eight of the tumors expressed the antigen. The expression of the TAG-72 antigen in the colonic epithelium may be an early marker of malignant transformation.

Phase II clinical trial of a murine monoclonal antibody cytotoxic for gastrointestinal adenocarcinoma.

A murine monoclonal antibody (MAb) which binds to human metastatic gastrointestinal adenocarcinomas can be administered safely and has tumor effects in some patients. Its therapeutic effect was assessed in 20 patients with measurable advanced colorectal carcinoma that was refractory to prior surgical resection, chemotherapy, and/or radiotherapy. All patients had agreed to receive no other therapy at the time of MAb administration and follow-up evaluation. In one patient, tumor at all known sites responded after a single i.v. injection of antibody. One other patient had a marked reduction in a hepatic metastasis where binding of 131I-labeled F(ab')2 MAb fragments was demonstrated but not in his abdominal wall metastases where no MAb binding could be demonstrated. In a third patient, stabilization persisting for 12 mo of an aggressively growing tumor was observed. The antibody was well tolerated in all patients, although 10 patients mounted an anti-murine immunoglobulin antibody response.

Gastrointestinal cancer-associated antigen in immunoperoxidase assay.

A monosialoganglioside antigen of gastrointestinal adenocarcinomas defined by murine monoclonal antibody was demonstrated by immunoperoxidase (IP) assay in fixed paraffin-embedded tumors in 59% of colonic adenocarcinomas, 86% of pancreatic adenocarcinomas, and 89% of all gastric adenocarcinomas. In all patients with detectable levels of antigen in circulation, the resected tumors also expressed the antigen in IP assay. Six of eight individuals with no detectable levels of antigen in their serum samples expressed the antigen in the tumor tissue. Removal of the sialic acid residue of the antigen abolished the IP reaction. The successful use of the IP assay on fixed tissue to demonstrate the specific sites of gastrointestinal cancer antigen localization in human tumors and normal tissues provides an important tool for the study of developing neoplasia.

Phenotypic characteristics of cells derived from precursors of human melanoma.

Of 66 specimens from benign melanocytic nevi, including common acquired and congenital nevi, Spitz tumors (epithelioid cell nevi), and melanocytic nevi with dysplasia, 57 could be grown in tissue culture. The cultured cells were identified as melanocytes by the presence of premelanosomes and melanosomes. Cells from 28 of 32 nevus cultures grew in an anchorage-independent way in soft agar with a colony-forming efficiency between 0.001 and 1%. Clones derived from single cells and soft agar-selected colonies showed marked phenotypic heterogeneity, but all had a limited life span and did not undergo transformation in culture. These cells were nontumorigenic in nude mice. Cultured nevus cells expressed antigens present on melanoma but absent on normal fibroblasts and/or melanocytes as tested with monoclonal anti-melanoma antibodies. The anti-melanoma antibodies bound equally well to dysplastic, congenital, and common acquired nevi. Antigens are released by nevus cells similar to melanoma cells.

A cell surface glycoprotein expressed by colorectal carcinomas including poorly differentiated, noncarcinoembryonic antigen-producing colorectal tumors.

A monoclonal antibody to a cell surface glycoprotein on human colorectal carcinomas was raised using the undifferentiated colon carcinoma cell line MIP 101 as the immunogen. This antibody, ND4, is an IgG2a which does not cross-react with carcinoembryonic antigen (CEA), non-specific cross-reacting antigen, or blood group substances A, B, and H. Immunoprecipitation using lysates of cells grown in [35S]methionine or [3H]glucosamine and lysates of cells surface labeled with 125I showed binding to a cell surface glycoprotein with a molecular weight of approximately 160,000. Indirect immunofluorescence showed binding to the cell surface of 14 of 15 human colorectal carcinoma cell lines including six of six that do not secrete CEA. Two of seven human noncolorectal carcinoma lines and one of six nonhuman cell lines also bound antibody. Immunoperoxidase staining of formalin-fixed tissues showed prominent antibody binding with 19 of 33 (58%) human colorectal carcinomas, including five of six poorly differentiated tumors, five of 43 (12%) normal colonic mucosal biopsies, and one of 17 (6%) normal noncolonic tissues. One of 11 (9%) noncolonic tumors, a gastric adenocarcinoma, stained with ND4. Preliminary data obtained by a nonquantitative nitrocellulose dot-immunoassay have tentatively identified this glycoprotein in the serum of 15 of 37 (41%) patients with colorectal cancer. Three of the 15 patients had early stage disease and normal CEA levels (less than 2.5 ng/ml). Three patients had circulating antigen detectable preoperatively but not after tumor resection. Only one of 11 (9%) sera samples from normal subjects was positive. The characteristics of ND4 suggest that it may be of value in monitoring patients with colorectal carcinomas who do not have plasma CEA elevations. It may also be of value in the differential diagnosis of metastatic, poorly differentiated adenocarcinomas of unknown primary origin.

Increased sensitivity in detecting tumor-associated antigens in sera of patients with colorectal carcinoma.

A monoclonal antibody defining the Lewis blood group determinant was used to immobilize antigen in sera of patients with adenocarcinoma of the gastrointestinal tract, and a second radiolabeled antibody, which defines a gastrointestinal cancer-associated antigen (GICA), was used to detect the immobilized antigen. With this approach, elevated antigen levels were found in 34 of 49 (69%) of sera from patients with advanced colorectal carcinoma as compared with 9 of 292 (3%) of sera from patients with non-malignant gastrointestinal diseases and of healthy donors. For early primary colorectal carcinoma, the combination of anti-Lewis and anti-GICA monoclonal antibodies was more sensitive in detecting GICA than using anti-GICA antibody alone. Double determinant radioimmunoassay revealed the glycolipid determinant lacto-N-fucopentaose (LNF) III circulating in colorectal carcinoma patients' sera. 53% of patients older than 65 years had elevated levels of the LNF III determinant compared to none of age-matched, apparently healthy donors or patients with benign gastrointestinal tract lesions, and 18% of patients with inflammatory gastrointestinal tract diseases. In younger patients, the differences were less marked. Our results suggest the potential usefulness of Lewis and LNF III determinants as markers for the detection of gastrointestinal tract malignancies.

Selection of monoclonal antibodies detecting serodiagnostic human tumor markers.

Monoclonal antibodies (MAbs) were selected for specific binding to spent media of cultured tumor cells. Out of more than 12,000 hybridomas screened, 19 were selected in preliminary inhibition assays for secretion of MAbs which detected antigens in cancer patients' sera. Antibody CO 29.11, which was studied in detail, bound to an antigen shed and expressed by adenocarcinoma cells of colon, stomach, pancreas and urinary bladder. CO 29.11 bound to purified sialylated Lewis a (Lea) antigen but to a different epitope and with a higher binding affinity than the MAb CA 19-9. CO 29.11 but not CA 19-9 bound weakly to unsialylated Lea antigen. In double-determinant radioimmunoassay with sera of patients with colorectal carcinoma, CO 29.11 was found to be a more sensitive marker than CA 19-9 for the detection in serum of sialylated Lea antigen.

Extraction of circulating gastrointestinal cancer antigen using solid-phase immunoadsorption system of monoclonal antibody-coupled membrane.

An immunoadsorption system of monoclonal antibody immobilized on a polyolefin alloy fiber is described for extraction of serum gastrointestinal cancer antigen (GICA). Continuous circulation or single passage of plasma from gastrointestinal cancer patients through this antibody-fiber matrix resulted in 90% depletion of circulating GICA in 2 h using 0.6 mg immobilized antibody, and 90% depletion in 5 min using 8 mg antibody. Continual circulation resulted in total GICA removal in both cases. Desorption of antibody or of antibody-containing complexes was minimal. This methodology provides a selective and convenient means of removing any targeted substance by monoclonal antibody from the serum, and thus overcomes many of the shortcomings associated with conventional plasmapheresis.

Hepatic adenoma associated with oral contraceptive use: an unusual clinical presentation.

A young woman who had taken contraceptive steroids for many years had the acute onset of abdominal pain because of central necrosis and hemorrhage into a hepatic adenoma. She had multiple lesions confined to one lobe of the liver. Persistent pyrexia and leukocytosis were also prominent clinical findings. She has had no evidence of recurrence of this problem during the seven years following right hepatic lobectomy. A review of the anabolic and contraceptive steroid-associated hepatic neoplasms is presented with comments directed toward the recognition of the critical clinical sequelae that can befall the patient with hepatic adenoma. Although all the patients in the steroid-treated group have tumors with benign and striking histologic similarity, microscopic evidence of malignant invasion of surrounding tissue is occassionally noted.

Human immune response to monoclonal antibody administration is dose-dependent.

When mouse monoclonal antibodies (MAbs) are injected into patients they usually induce an immune response. The resultant human anti-mouse-immunoglobulin antibody (Hu-aMAb) limits multiple injections of these reagents. A strategy to decrease the production of Hu-aMAb was tested in 20 patients with advanced gastrointestinal carcinoma. Ten patients received 700 mg of MAb as their initial exposure to mouse immunoglobulin, while the other ten patients received 100-mg of immunoglobulin initially. Each group received the same maintenance regimen until Hu-aMAb or disease progression was detected. Six patients in the high-dose group did not produce detectable Hu-aMAb for up to five months after initial exposure. All ten of the patients who received the low initial dose developed Hu-aMAb. Allergic reaction did not occur in the absence of Hu-aMAb. This larger initial dose in vivo injection strategy may allow repetitive exposure to MAb reagents without Hu-aMAb limiting further diagnostic or therapeutic use of murine immunoglobulin.

Complication with mechanical stapling device in creation of ileoconduit.

Of 296 patients with pelvic malignancy and ileal urinary conduits, urinary tract calculi developed in 14. Calculi which ordinarily require surgical intervention because of their size may pass spontaneously in patients with ileoconduits because of the presence of a chronically dilated colllecting system and the surgical elimination of three of the four sites of stone impaction (pelvic brim, ureterovesical junction, and ureteral orifice). In 1 patient multiple calculi developed around the surgical staples used to create the proximal end of the ileal conduit. We recommend that autosuture with stapling devices not be used to create the proximal end of an ileal urinary conduit.

Citrate anticoagulation and cell washing for intraoperative autotransfusion in the baboon.

Extracorporeal citrate was used for anticoagulation during autotransfusion of baboons. A cell-washing plasmaphoresis procedure was added in one group of animals in order to remove activated clotting materials. Both groups became hypocoagulable, but the cell-washed group had less evidence of disseminated intravascular coagulation as well as lower plasma hemoglobin levels. Citrate anticoagulation plus cell washing is a potential alternative to heparinization for autotransfusion.

Circulating tumor markers and assessment of response to intrahepatic chemotherapy of colon carcinoma.

To assess the change in concentrations of circulating gastrointestinal cancer-associated antigens in response to therapy, we analyzed the sera of patients with hepatic metastasis from colorectal carcinoma who were treated with intrahepatic arterial chemotherapy. Serial serum samples were assessed for the tumor-associated antigens, carcinoembryonic antigen (CEA) and the gastrointestinal cancer antigen CA 19-9. Computed axial tomographic (CAT) scans were made to assess the size of the hepatic metastasis. In 9/10 of these patients the CEA predicted tumor response within 2-6 weeks after initiation of treatment, and in 7/10 the information was supported or more dramatically demonstrated by the CA 19-9. Combining data from both tumor markers may provide a more accurate assessment of the clinical response than one antigen alone. Recurrence of hepatic metastatic growth or extrahepatic tumor also was identified by elevation of one or both circulating tumor-associated antigens prior to other laboratory or clinical evidence of tumor growth.

Initial clinical evaluation of two murine IgG2a monoclonal antibodies for immunotherapy of gastrointestinal carcinoma.

Eleven patients with advanced gastrointestinal (GI) carcinoma were entered in Phase I initial clinical trials with IgG2a antiGI carcinoma monoclonal antibodies (MAbs) GA733 (five patients) or CO19-9 (six patients). Infusion of MAb GA733 in doses greater than 30 mg was accompanied by mild and short-lasting GI toxicity. Infused MAb GA733 was bound to each patient's tumor tissue in vivo. MAb circulated in the blood for 10-25 days. All patients developed anti-mouse antibodies between 15 and 60 days post infusion. Furthermore, all but one patient raised anti-idiotypic antibodies against MAb GA733. Following administration of 10-600 mg of MAb CO19-9, no immediate or delayed toxicity symptoms were noted. Binding of infused MAb CO19-9 to tumor cells in vivo could not be detected in any of the six patients studied. The MAb circulated in the bloodstream between 5 and 12 days. Human anti-mouse antibody was detected in sera of three patients. None of the eleven patients treated with either MAb had anti-tumor responses in this Phase I clinical trial. The strong binding reactivity of MAb GA733 to tumors in vivo suggests the use of this MAb in cancer patients with less tumor burden to determine the tumoricidal efficacy of this antibody.

Detection of monoclonal antibody-defined colorectal carcinoma antigen by solid-phase binding inhibition radioimmunoassay.

We have established a solid-phase binding inhibition radioimmunoassay for the detection of colorectal carcinoma-specific antigens in tissue culture supernatants of human colorectal carcinoma cell lines and in serum and urine of colorectal carcinoma patients. Using the [3H]glucosamine-labeled cell membrane glycolipid antigen and colorectal carcinoma-specific monoclonal antibodies in this assay, we have been able to detect several human colorectal carcinoma membrane-specific antigens that are released from the cell membrane into tissue culture supernatants, and an antigen detected by antibodies 1116-NS-19-9 and 1116-NS-52a that is found only in the serum and urine of cancer patients.

Biologic effects of gamma interferon pre-treatment followed by monoclonal antibody 17-1A administration in patients with gastrointestinal carcinoma.

Twenty-seven patients with metastatic adenocarcinoma of the colon or pancreas were treated with 400mg of monoclonal antibody 17-1A. This antibody, which binds to a cell surface glycoprotein moiety preferentially expressed by adenocarcinomas of the rectum, colon, pancreas, and stomach, is postulated to induce antibody-dependent monocyte cytotoxicity (ADMC) as a mechanism of tumor lysis. Therapy was preceded by four days of gamma interferon infusions, with the intent of activating peripheral blood monocytes, enhancing monocyte Fc receptor expression and increasing the likelihood of tumor lysis as reflected by enhanced ADMC directed against a colon carcinoma cell line (SW1116) which expresses 17-1A's target antigen. In this Phase I study patients were treated daily at one of the following gamma interferon dose levels (X 10(6) U/M2/day): 0.001, 0.01, 0.1, 1.0, 10.0, 40.0, 60.0, 80.0. Addition of 100 U/ml of rIFN-gamma in vitro to monocytes isolated from normal controls or from patients prior to treatment significantly enhanced monocyte Fc receptor expression and ADMC. in vitro tumor cell killing by monocytes and monoclonal antibody was enhanced by treatment with low doses of rIFN-gamma, while treatment with high doses of rIFN-gamma did not enhance ADMC. No objective clinical responses were noted, although serum tumor markers dropped transiently in 36% of the treated patients. Seven of 11 assayed patients developed human anti-idiotype antibodies. With better scheduling of rIFN- and 17-1A we hope to duplicate optimal in vitro conditions for antibody-mediated cytotoxicity, hopefully enhancing in vivo antibody mediated tumor lysis.