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Choriocarcinoma: Blocking factor and monoclonal antibody iodine 131 imaging
Choriocarcinoma: Blocking factor and monoclonal antibody iodine 131 imaging R. A. Pattillo, M. B. Khazaeli, A. C. F. Ruckert, R. O. Hussa, B. D. Collier, W. Beierwaltes, and R. F. Mattingly Milwaukee, Wisconsin, and Ann Arbor, Michigan Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with 131 1 and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease. (AM. J. OesTET. GVNECOL. 148:1040, 1984.)
Monoclonal antibodies have recently escalated to the central position of application to virtually every remaining biologic challenge. 1 The production of monoclonal antibody has been made possible through the development of cellular hybrids (Fig. 1). Antigen injected into a mouse causes the generation of antibodyproducing cells in the spleen of the animal. The spleen is removed; when its cells are fused with tumor cells, cellular hybrids are produced. The hybrids produce monoclonal antibodies, and cloning of these antibodies results in hybridomas of unlimited growth potential. The clones are separated, isolated, and grown up, and the desired monoclonal antibody is harvested in large quantities. The purposes of this study were to describe the application of this new technology to tumor localization,2-6 to explore conditions, namely, "blocking factors," that may impose limitations on its application, to relate blocking factor to resolution of tumor growth, and to test the hypothesis that monoclonal antibody specifically localizes at the site of tumor growth. Trophoblastic neoplasms have the unique endocrine property of production of human chorionic gonado-
From the Departments of Gynecology and Obstetrics and Nuclear Medicine at the Medical College of Wisconsin, and the Department of Nuclear Medicine at the University of Michigan. This work was supported in part by the American Cancer Society, Milwaukee Division, and the Patrick and Anna Cudahy Fund. Presented at the Second Annual Meeting of the American Gynecological and Obstetrical Society, Phoenix, Arizona, September 7-10, 1983. Reprint requests: Dr. R. A. Pattillo, Department of Gynecology and Obstetrics, The Medical College of Wisconsin, 8700 West Wisconsin Ave., Milwaukee, Wisconsin 53226. 1040
tropin (heG), the ideal tumor marker, which permits awareness of the presence of tumor although the number of cells or location of the lesion may not be detectable. These are the circumstances under which localization and imaging of such a tumor may permit its surgical removal. On the other hand, certain factors may interfere with antibody localization and concentration in tumor cells, and some of these factors will be dealt with in this article. Material and methods
Monoclonal antibody to choriocarcinoma cells was prepared by Dr. M. B. Khazaeli of the University of Michigan at Ann Arbor from nonseminomatous male choriocarcinoma. Approximately one billion whole viable cells were harvested from the tumor. Six BALB/c mice were injected with 20 million cells each, three times at 3-week intervals. Antibody titers were identified in the subject animals, and the antibody-producing spleen cells were isolated and fused with myeloma cell line 653. Approximately 100 hybridomas were identified. Three of these did not react with the normal cells of the patient or other tumor lines. These strongest reactors were taken and the antibody was expanded in mice by peritoneal injection. The ascites antibody was further purified by affinity chromatography on Staphylococcus protein A columns. The harvested antibody reacted only with choriocarcinoma cell line BeWo, which was previously derived from a patient with gestational choriocarcinoma. 7 The BeWo choriocarcinoma cell line was transplanted to nude mice and the above monoclonal antibody was conjugated wid] iodine 131, which successfully imaged this tumor in the nude mice (Fig. 2). It was presumed that
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Cf.Ll CUt fUHt ~YI:.LOMA LINE
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Fig. 2. Cone imaging of transplanted choriocarcinoma in the nude mouse with monoclonal 13lI-labeled antibody. FOR ANTl800Y
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Fig. 1. Monoclonal antibody production procedure. (Reprinted with permission from Milstein, C.: Monoclonal antibodies, Sci. Am. 243:66, 1981.)
131
1 would also image the tumor of the patient since lymphocytes from all patients with choriocarcinoma studied recognized BeWo choriocarcinoma cells in the blocking factor assay. Blocking factor assays at this center have recently been added to hCG monitoring of patients with trophoblastic disease because of an approximate 10% incidence of recurrent disease after chemotherapy. Determination of blocking factor is a new test for agents in the serum of the patient that prevent the host from reacting against the tumor. These agents may be tumor antigen, tumor antibody, or antigen-antibody complex. During the course of treatment of the patient who was selected to undergo imaging, the hCG became undetectable at several points; however, testing for blocking
factor remained positive. This suggests that final tumor elimination may depend upon an immunobiologic reaction of the host to the tumor. Blocking of this reaction could prevent complete cure and lead to recurrent disease. The blocking factor assay consists of incubation of choriocarcinoma target cells with lymphocytes of the patient and control lymphocytes (Fig. 3). The choriocarcinoma cells are grown without lymphocytes, with normal control lymphocytes (which do not antigenically recognize the tumor cells and, therefore, do not adhere), and with the lymphocytes of the patient which are specifically sensitized to the tumor, recognize it, and cytotoxically destroy it unless the serum of the patient, which contains the blocking factor and prevents the lymphocytes from adhering and destroying the tumor, is added. The end point of the assay is the terminal tritiated thymidine uptake of target tumor cells not destroyed by the cytotoxic lymphocytes. The blocking index is calculated by the difference of cytotoxicity in the presence and absence of the serum of the patient containing the blocking factor. The choriocarcinoma cells were derived from five different patients, including the patient whose tumor was transplanted to nude mice and was imaged by the l3lI-labeled monoclonal antibody against the male choriocarcinoma, and provided five different assays. Lymphocytes from the patient who was to undergo imaging and the monoclonal antibody specifically recognized the BeWo choriocarcinoma tumor, one of the target systems in the blocking factor assay. The blocking factor calculation was performed as previously described8 , 9 (Table I). The choriocarcinoma
1042 Pattillo et al.
April I, 1984 Am. J. Obstet. Gynecol.
Fig. 3. Cellular observations of blocking factor assay in Patient LiWi with choriocarcinoma studied in 131I_labeled monoclonal antibody localization: A: Choriocarcinoma target cells grown in Lab Tek assay chamber without lymphocytes. Note mitotic figure of uninhibited growth. (x 1,000.) B: Choriocarcinoma target cells with normal control lymphocytes. No lymphocyte attachment or cytotoxicity. (X 1,000.) C: Lymphocytes of Patient LiWi in control serum. A single choriocarcinoma target cell is recognized by patient lymphocytes, and the choriocarcinoma cell is being cytotoxically destroyed (nuclear karyorrhexis and karylysis) by the patient lymphocytes. (X 1,000.) D: Choriocarcinoma target cells incubated with patient lymphocytes but in the presence of patient serum. Note lymphocytes are "blocked" from antigenic recognition and cytotoxicity toward the target cell. The lymphocytes do not adhere to the target cells. (x450.)
cells were grown in tissue culture flasks and transferred to Lab Tek chambers for the assay. One thousand target choriocarcinoma cells were incubated with 100,000 lymphocytes from the patient in the absence and in the presence of serum of the patient. If blocking was present, cytotoxic destruction of the choriocarcinoma cells would be blocked when serum of the patient was added to the incubation. The blocking index could be calculated as the difference between cytotoxicity in the absence and in the presence of serum of the patient. A pulse procedure with tritiated thymidine was used to detect remaining undestroyed tumor cells after the incubation. Target cells (103 cells per well) were incubated in eight-well Lab Tek chambers at 37° C in an atmosphere of 5% carbon dioxide in air. After 24 hours' incubation, the medium was removed and re-
placed with fresh 3520 medium* containing 105 lymphocytes or 3520 medium with 20% patient serum containing 105 lymphocytes. After incubation for 24 hours, the chambers were washed four times with balanced salt solution to remove the lymphocytes. The remaining target cells were incubated for 2 hours in 3520 medium with (methyPH) thymidine (10 /LCi/ml, 5 to 30 Ci/mmol). The pulse medium was removed, the cells were washed twice with balanced salt solution containing excess unlabeled thymidine, and frozen. After thawing, the amount of radioactivity incorporated into acid-precipitable material was determined. Determina*3520 medium, 30% Gey's balanced salt solution, 50% Waymouth medium, 20% fetal calf serum; all tissue culture media were obtained from Grand Island Biological Co., Grand Island, New York.
Choriocarcinoma 1043
Volume 148 Number 7
100
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100
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Fig. 4. Blocking factor assay in Patient CoCI with OZ cells. This patient had resistant choriocarcinoma and underwent operation after multiple chemotherapy with rising hCG levels and positive assay for blocking factor.
tions were made in triplicate. Values were expressed as the percentage of radioactivity incorporated by target cells incubated in the absence of lymphocytes. Cytotoxicity of lymphocytes of the patient was assessed by comparing values obtained from incubations of patient lymphocytes with those of normal donor lymphocytes incubated with the target cells in the same assay. Blocking of cytotoxicity was assessed by comparing the values obtained from incubation of patient lymphocytes in fetal calf serum-containing medium with values obtained with the use of autologous serum-containing medium with target cells in the same assay. Significance was assessed by Student's t test with p $ 0.05 considered significant. Results
In the concurrent continuing study, patients with metastatic trophoblastic disease evaluated by blocking factor assessment showed a pattern of resolution of blocking factor activity with resolution of trophoblastic disease. Representative of patients studied during the period of time in which the monoclonal antibody was being produced are the following: Patient CoC! (Fig. 4) developed resistant postgestational choriocarcinoma after three courses of methotrexate. Blocking factor and hCG became undetectable after total abdominal hysterectomy with removal of all tumor that was still confined to the uterus. Patient LaDe (Fig. 5) who experienced recurrent pulmonary metastatic trophoblastic disease after undetectable hCG levels for 1 year was treated with multiagent chemotherapy, and blocking factor resolved after treatment. After 2 years of follow-up and undetectable blocking factor, the patient was allowed to become pregnant and was delivered of a healthy infant at 33 weeks' gestation.
0
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-----
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.
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JAN FEB FEB MAR FEB 1983 1981
Fig. 5. Blocking factor assay in Patient LaDe with JAr cells. Metastatic trophoblast disease.
Table I. Blocking factor assay Control versus 10' Target cells
Control versus 10' Target cells
10' Patient lymphocytes
10' Patient lymphocytes
with patient serum 24-hour incubation 2-hour thymidine pulse
without patient serum 24-hour incubation 2·hour thymidine pulse
Blocking index = lymphocyte killing with patient se· rum - lymphocyte killing without patient serum.
Patient LiLi (Fig. 6) experienced pulmonary metastasis of postgestational choriocarcinoma and demonstrated the presence of blocking factor until complete resolution of the tumor by chemotherapy. The patient subsequently had a healthy postchoriocarcinoma pregnancy and delivery. Lymphocytes of patients ElFa and JAr were tested against their choriocarcinomas as well as other target tumors and showed uniform recognition of these tumors of the same histologic type. Complete resolution of disease and resolution of blocking factor were observed in both patients. Patient Bar N was delivered of a term infant and within 3 weeks experienced massive pulmonary metastasis from choriocarcinoma. Blocking factor resolution occurred promptly after chemotherapy, and the patient now has undetectable hCG and blocking factor levels. Patient DoSmi was an elderly gravida who had a hydatidiform mole evacuated; however, because of persistent bleeding, she underwent hysterectomy and malignant trophoblastic disease was found. Blocking factor assays and hCG testing have remained negative. Patient MaBr experienced pulmonary metastasis after evacuation of a
1044 Pattillo et al.
April I, 1984 Am. J. Obstet. Gynecol.
100 90 80 )(
70
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30 20 10 JULY AUG OCT FEB 1980 1981
Fig. 6. Blocking factor assay in Patient LiLi with JAr cells. Blocking factor and hCG were undetectable after chemotherapy for postgestational choriocarcinoma with lung metastasis and pleural effusion.
hydatid mole; after successful chemotherpay, hCG and blocking became undetectable. The patient subsequently conceived and gave birth to a normal child. Patients DiBI and CaPI experienced metastatic trophoblastic disease (the latter with brain metastasis). Each developed undetectable blocking factor and hCG and are free of disease. Patients LuLu and DeWe currently have blocking factor and hCG present and have resistant disease. Patients SEb (Table II) and DoMi both experienced early metastatic trophoblastic disease which responded to methotrexate, and blocking factor and hCG tests became negative after therapy.* In review, the lymphocytes of all 15 patients studied (Table III) showed recognition of choriocarcinoma and blocking factor activity. The survival in our 15 high-risk patients studied with blocking factor and hCG monitoring is 88%. Thirteen patients are in remission and two have persistent disease. All 15 patients have survived for more than 2 years. With these studies completed and the monoclonal antibody cleared for human use, the study patient was admitted for imaging.
*Individual details on the findings in each patient will be furnished to any reader upon request.
Fig. 7. Gamma camera scan of resected choriocarcinoma lung lesion. Lesion/lung specific activity: 2.65: 1 (8,914 cpm/gm of tumor versus 3,368 cpm/gm of normal lung).
Case presentation The 29-year-old patient had resistant choriocarcinoma. She had initially been evacuated of a hydatidiform mole in December, 1979. After several negative titers the hCG level began to rise; 11 months after evacuation of the mole, repeat curettage revealed choriocarcinoma. There was resistance to multiple forms of chemotherapy (methotrexate, actinomycin D, bleomycin, cis-platinum, vinblastine, and Adriamycin), and although low levels of hCG persisted, no lesions could be identified by roentgenography, computerized axial tomography, or ultrasound. For this reason, imaging with the monoclonal antibody was planned; however, just before imaging, a small lesion appeared. The lymphocytes of this patient showed positive blocking factor activity with BeWo gestational choriocarcinoma. Because the male choriocarcinoma monoclonal antibody also recognized BeWo gestational choriocarcinoma, it was anticipated that it would recognize and image the tumor of this patient. During the course of the treatment, the hCG became undetectable at several points; however, assays for blocking factor remained positive. The monoclonal antibody study was carried out, after pyrogen testing, by intravenous injection of 5 /Lg of antibody with 5 mCi of 131 1 in 30 ml of saline over a lO-minute period. The thyroid was blocked by oral administration of a saturated solution of potassium iodide given three times per day, beginning 24 hours prior to injection. One tenth milliliter of the antibody was skin tested 30 minutes prior to injection. The imaging was done with the Technicare Gamma Camera, Columbus, Ohio. The l31I-labeled monoclonal antichoriocarcinoma
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120 110 100
90 ~
80
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70
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60
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30 20 10 FEB. FEB MAR. DEC. JAN. FEB FEB APR. APR. APR. APR. MAY MAY MAY JUNE JUNE FEB MAR. APR. MAY
1981
1982
1983
Fig. 8. Blocking factor assay in Patient LiWi with JAr cells. Levels of blocking factor dropped but remained detectable after resection of the lung lesion. heG dropped to a low of 22 mIU/ml after excision of the lung lesion.
Table II. Blocking factor assay in Patient SEb with BeWo choriocarcinoma target cells Control lymphocytes (mean ± SE)
Patient lvmphocytes (mean ± SE)
Patient lymphocytes with autologous serum (mean ± SE)
Date of assay
('lo)
('lo)
('lo)
Recognition
Blocking
October, 1977 November, ]977
]07.6' ± 20.6 B 93.8 ± 10.7
3.9 ± 2.1 39.6 ± 6.2
57.3 ± ]9.3 53.4 ± 9.0
+*
+t
*Differs significantly (p tDiffers significantly (p
~ ~
+
0.05) from value of target cells incubated with control lymphocytes. 0.05) from value of target cells incubated with patient lymphocytes.
antibody was injected without complication; however, subsequent daily body scanning for 5 days failed to reveal isotope differences over the 2.5 cm area of the left upper lung field wherein the lesion had been identified on the computerized axial tomographic scan. Fourteen days later, at the time of thoracotomy, the lesion was resected and found to be clearly isolated in an anomalous lower segment of the left upper lobe. Antibody-isotope localization was verified in the lesion (Fig. 7), which consisted of malignant cytotrophoblast and synctiotrophoblast, hemorrhage, calcification, and fibrosis. On the basis of counts from the resected lung lesion and the adjacent normal lung, the lesion/lung specific activity ratio was 2.65: 1 (8,914 cpm/gm of tumor versus 3,368 cpm/gm of normal lung). Although the 131I-labeled monoclonal antibody had localized in the tumor, the preoperative isotope antibody concentration was not sufficient to differentiate it from the blood pool and background activity. The cellular observations and blocking factor activity of the monoclonal antibody study patient are seen in Figs. 3 and 8, respectively. Just as a serum factor blocks the lymphocytes from contact with the choriocarcinoma cells of the patient, the monoclonal antibody may have been partially blocked in the diagnostic imaging study. This may have prevented the dense localization of 131I-labeled
antibody in the patient compared to that seen in the transplanted choriocarcinoma. Dense imaging of the latter was seen preoperatively in the nude mouse. During the course of the chemotherapy treatment, assays for blocking factor remained positive, while tests for heG became negative on several occasions. Efforts to dilute or remove the blocking factor by plasmapheresis may improve imaging in future human diagnostic studies. Comment
The presence of a "blocking factor," which blocks the contact of lymphocytes with the choriocarcinoma cell, may be antibody, antigen-antibody complex, or antigen. Under either circumstance, it could prevent adequate concentration of the monoclonal 131I-labeled antibody and thus prevent satisfactory preoperative imaging. Further studies to examine these possibilities are in progress. Monoclonal antibody technology has provided a means to address a leading problem in the use of antibodies in medical science. The problem has been that no matter how pure an antigen one can produce, the body's discernment of molecular differences still leads to the production of a numbet of antibodies, and these
1046
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April I, 1984 Am.
Table III. Blocking factor assay results in 15 patients Patient
Recognition and cytotoxicity
CoC! LaDe LiLi ElFa JAr BarN DoSmi MaBr DiBI CaPI LuLu DeWe LiWi DoMi SEb
+ + + + + + + + + + + + + + +
cannot be readily separated for use. The present advance in monoclonal antibody capability has provided the means to reach into the "alphabet soup" of spleen cells producing antibodies to multiple antigens and grow one kind by fusing it with a tumor cell that can grow indefinitely and provide unlimited antibody. Conjugation of monoclonal antibodies with radioisotopes offers the next step for scanning to image the location of the tumor identified by the specific antibody. However, the body produces its own antibodies to tumors, the tumors shed antigens, and antigen-antibody complexes are generated; all of these may serve to block sufficient localization of even a monoclonal antibody in the subject tumor. Further, because monoclonal antibodies are produced in rats and mice, the potential for anaphylactic reactions in human subjects limits the numbers of investigation one can perform. The study case being reported is an example of an Institutional Research Committee's permission of a single compassionate study case when all other alternatives had been explored. Now that this study has been completed without complication, an Investigational New Drug permit has been issued by the Food and Drug Administration and additional cases can be examined in various stages of blocking factor activity. Such studies of trophoblastic tumor patients in the absence of significant blocking factor will permit further evaluation of the role of blocking factor in immune localization of specific antibody in the choriocarcinoma model. The need of preoperative imaging has not yet been met. The computerized axial tomographic scan and, indeed, radiographic localization permitted identification of this lesion. Only after the tumor was removed was it possible to verify the presence of 2.65: 1 specific activity of the labeled antibody in tumor versus normal lung. These current studies in the monoclonal antibody
J.
Obstet. Gynecol.
patient and the series of trophoblastic disease patients have revealed the presence of a serum blocking factor that blocked the immune localization and cytotoxicity by lymphocytes of these patients against their choriocarcinomas. Data from these studies suggest that attenuation of specific antibody or receptor concentration may interefere with immune localization. This blocking factor may have prevented the preoperative concentration of the monoclonal antibody and may serve as an obstruction to sufficient tumor localization in patients with high levels of blocking activity in general. Alternatively, serum halogenases may have cleared radioactive iodine from the antibody. The blocking factor assays proved to be useful as they remained positive when the tests for hCG became negative. This second modality for trophoblastic disease monitoring may have unique application because of its immunobiologic nature, a parameter of critical importance in tumor-host response. We conclude from this work: (1) All 15 patients with metastatic trophoblastic disease have been shown to exhibit blocking factor which correlated with the presence of tumor; (2) 1311-labeled monoclonal antichoriocarcinoma antibody was postoperatively localized in a metastatic lung lesion; (3) blocking factor may interfere with adequate antibody isotope concentration for preoperative imaging; (4) blocking factor assays proved to be useful as they remained positive when hCG tests became negative in the presence of small tumor volume. We would like to thank Mrs. Martha Rinke and Mr. James Kurtz for technical assistance in this work.
REFERENCES 1. Milstein, C.: Monoclonal antibodies, Sci. Am. 243:66, 1981.
2. Goldenberg, D. M., Kim, E. E., DeLand, F. H., et al.: Clinical radioimmunodetection of cancer with radioactive antibodies to human chorionic gonadotropin, Science 208: 1284, 1980. 3. Ballou, B., Levine, G., Hakala, T. R., et al.: Tumor location detected with radioactively labeled monoclonal antibody and external scintigraphy, Science 206:844, 1979. 4. DeLand, F. H., Kim, E. E., Primus, F. ]., et al.: In vivo radioimmunodetection of occult recurrent colonic carcinoma, AJR 138:145, January 1982. 5. Bergmann, S. R., Lerch, R. A., Mathias, C.]., et al.: Noninvasive detection of coronary thrombi with In-Ill platelets: concise communication,]. Nucl. Med. 24:130,1983. 6. Larson, S. M., Brown,]. P., Wright, P. W., et al.: Imaging of melanoma with 1-131 labeled monoclonal antibodies,]. Nucl. Med. 24:123, 1983. 7. Pattillo, R. A., and Gey, G.: Establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro, Cancer Res. 28:1231, 1968. 8. Pattillo, R. A., Story, M. T., and Ruckert, A. C. F.: Expression of cell-mediated immunity and blocking factor using a new line of ovarian cancer cells in vitro, Cancer Res. 39: 1185, 1979. 9. Pattillo, R. A., Ruckert, A. C. F., Story, M. T., et al.: Im-
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munodiagnosis in ovarian cancer: Blocking factor activity. J. OBSTET. GYNECOL. 133:791, 1979.
AM.
Discussion
DR. HUGH R. K. BARBER, New York, New York. In the discussion I shall raise more questions than may be considered proper for a discussant. In defense I must say that the article has many complex features. Although the article contains a great deal of good material, there are statements that are difficult to correlate with the conclusions. As an introduction it must be stated that the immune system is complex and changes from second to second. Therefore, it is difficult to study radioactive tagged monoclonal antibodies as to localization and blocking factor as an isolated event. However, Dr. Pattillo and his colleagues have achieved this and reported it to their satisfaction. Having said this, I must raise challenges to statements in the manuscript. The procedures for selecting hybrid cells to produce monoclonal antibodies that exhibit tumor specificity are not described nor are the results of tests used to screen for reactivity with similar or shared antigenic determinants in normal serum. Therefore, the reviewer must take for granted that the antibody used for tumor localization has no cross-reactivity for other normal tissue components which may be present. This is open to question. If there was, in fact, such cross-reactivity with a serum component, results identical to those described would be obtained for reasons quite different from those proposed. The statement that imaging was possible with tumor grown in nude mice does not guard against this possibility, since the interfering component might well be species-specific. For an article depending so heavily on the characteristics of a given monoclonal antibody, a more detailed description or definition of that antibody should be included. The monoclonal antibody to choriocarcinoma cells was prepared from nonseminomatous male choriocarcinoma (a teratoma). Gestational trophoblastic disease is folic acid-dependent whereas choriocarcinoma arising in a teratoma is folic acid-dependent. This accounts for some of the differences in the response to chemotherapy among these two seemingly different cancers. It is conceivable that a subtle change in the antibody may be identified in a larger series. By taking advantage of the extraordinary power of discrimination of the immune defense mechanism, the antibody produced against teratomatous choriocarcinoma may not always react against gestational trophoblastic choriocarcinoma cells as described in this article. There are discrepancies in the manuscript: The authors stated that no lesion could be identified by roentgenography, computerized axial tomography, or ultrasound. However, they later state that they scanned the area of the upper left lung field where a lesion had
Choriocarcinoma
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been identified on the computerized axial tomographic scan. In addition, the authors claimed that blocking factor correlates with the presence of tumor, yet they described Patient BarN who developed massive pulmonary metastases but say that "Blocking factor resolution occurred promptly ... , and the patient now has undetectable hCG and blocking factor levels." This is scientifically impossible to explain from the premise advanced in their paper. The figures and tables, although complicated, are good. The lack of more detailed characterization of the antibody and speculation, on the part of the authors, about a specific blocking factor that is circulating and blocks localization of the monoclonal 1311-labeled antibody raises more questions than have been answered. Since one case of monoclonal antibody 131 I imaging and blocking factor to choriocarcinoma is reported in detail and blocking factor activity is reported in 13 additional patients, there is "a paper within a paper." In conclusion, at best, I can say only that the results from the material presented are suggestive but not conclusive. DR. JAMES C. WARREN, St. Louis, Missouri. I wonder whether you could have studied the tumor in vitro and then obtained serum from the patient to determine if it was related to the uptake or binding of the antibody. Could you have done that test? DR. JOHN L. LEWIS,JR., New York, New York. I have a very brief comment, and it is a sideline to the paper. It concerns the woman who had an increase in blocking factor activity; the authors were concerned about a recurrence, but she was pregnant. Her situation indirectly raises the question of what the antibody is working against. More than 20 years ago Kaliss proposed that, in mice, antibodies generated to trophoblastic tissue had the effect of blocking antibodies, thus enabling pregnancy to proceed without immunologic rejection. This would suggest that the antibody is not directed against trophoblastic tissue. I think you will agree that it is not a specific antibody if it has been raised to one tumor of one sex and it is reacting with another tumor in the other sex, so I am interested in what you think the antibody has been responding to. What is the antigen or antigens? DR. PATTILLO (Closing). In response to Dr. Barber's questions, the procedures to identify the nature of the monoclonal antibody and its production are being published in the Journal of Nuclear Medicine. Controls for the monoclonal antibody are dealt with in this publication and could not be presented in the short time allotted here. The computerized axial tomographic scan identification of a chest lesion just prior to the imaging study is included in the paper and was noted in the presentation. This proved to be an asset because there was not sufficient isotope in the lesion to distinguish it from the blood pool and background. The fact that we knew there was a gross lesion permitted us to excise that lesion at thoracotomy and show that localization to
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the tumor by the monoclonal antibody had occurred. With regard to Dr. Barber's final question concerning the BaRn patient, there was a typographical omission in the manuscript. It should have been stated that: The blocking factor and hCG became undetectable after appropriate chemotherapy. Dr. Warren's approach for evaluating the antibody by immunoprecipitation is a good suggestion. The in vitro system that we used, however, was to identify the monoclonal antibody's specific recognition of the patient's choriocarcinoma in an indirect fashion. The lymphocytes of the patient recognized the choriocarcinoma that was established in culture and the monoclonal antibody also showed cytotoxicity to it. We felt
April I, 1984 Am. J. Obstet. Gynecol.
that this was appropriate documentation for the specificity of the antibody. In answer to Dr. Lewis' comments with regard to pregnancy systems, the blocking factor assay was negative in normal pregnancy, which suggests that there is no cross-reactivity of the choriocarcinoma-specific antibody with normal pregnancy trophoblast, that is, patients with normal pregnancies do not show recognition of choriocarcinoma cells. Their lymphocytes do not show recognition of choriocarcinoma cells in culture; therefore, we presume that the antigen that we are dealing with in choriocarcinoma is a trophoblastic tumor-associated antigen.
Erratum John W. C. Johnson, M.D., was inadvertently omitted from the list of Consultants for the AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY, published in the December 15, 1983, issue, on page 857.
Monoclonal antibodies have recently escalated to the central position of application to virtually every remaining biologic challenge. 1 The production of monoclonal antibody has been made possible through the development of cellular hybrids (Fig. 1). Antigen injected into a mouse causes the generation of antibodyproducing cells in the spleen of the animal. The spleen is removed; when its cells are fused with tumor cells, cellular hybrids are produced. The hybrids produce monoclonal antibodies, and cloning of these antibodies results in hybridomas of unlimited growth potential. The clones are separated, isolated, and grown up, and the desired monoclonal antibody is harvested in large quantities. The purposes of this study were to describe the application of this new technology to tumor localization,2-6 to explore conditions, namely, "blocking factors," that may impose limitations on its application, to relate blocking factor to resolution of tumor growth, and to test the hypothesis that monoclonal antibody specifically localizes at the site of tumor growth. Trophoblastic neoplasms have the unique endocrine property of production of human chorionic gonado-
From the Departments of Gynecology and Obstetrics and Nuclear Medicine at the Medical College of Wisconsin, and the Department of Nuclear Medicine at the University of Michigan. This work was supported in part by the American Cancer Society, Milwaukee Division, and the Patrick and Anna Cudahy Fund. Presented at the Second Annual Meeting of the American Gynecological and Obstetrical Society, Phoenix, Arizona, September 7-10, 1983. Reprint requests: Dr. R. A. Pattillo, Department of Gynecology and Obstetrics, The Medical College of Wisconsin, 8700 West Wisconsin Ave., Milwaukee, Wisconsin 53226. 1040
tropin (heG), the ideal tumor marker, which permits awareness of the presence of tumor although the number of cells or location of the lesion may not be detectable. These are the circumstances under which localization and imaging of such a tumor may permit its surgical removal. On the other hand, certain factors may interfere with antibody localization and concentration in tumor cells, and some of these factors will be dealt with in this article. Material and methods
Monoclonal antibody to choriocarcinoma cells was prepared by Dr. M. B. Khazaeli of the University of Michigan at Ann Arbor from nonseminomatous male choriocarcinoma. Approximately one billion whole viable cells were harvested from the tumor. Six BALB/c mice were injected with 20 million cells each, three times at 3-week intervals. Antibody titers were identified in the subject animals, and the antibody-producing spleen cells were isolated and fused with myeloma cell line 653. Approximately 100 hybridomas were identified. Three of these did not react with the normal cells of the patient or other tumor lines. These strongest reactors were taken and the antibody was expanded in mice by peritoneal injection. The ascites antibody was further purified by affinity chromatography on Staphylococcus protein A columns. The harvested antibody reacted only with choriocarcinoma cell line BeWo, which was previously derived from a patient with gestational choriocarcinoma. 7 The BeWo choriocarcinoma cell line was transplanted to nude mice and the above monoclonal antibody was conjugated wid] iodine 131, which successfully imaged this tumor in the nude mice (Fig. 2). It was presumed that
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Cf.Ll CUt fUHt ~YI:.LOMA LINE
; ... f
11~I'
HAT MEDIUM
~~~:
.1 ASSAy
SELECT HYSRIO CEllS
FOft AffflBOOV )0 FREeZE
1-v
Fig. 2. Cone imaging of transplanted choriocarcinoma in the nude mouse with monoclonal 13lI-labeled antibody. FOR ANTl800Y
.. FREEZE
RECLONE
I
t ANTISODY
Fig. 1. Monoclonal antibody production procedure. (Reprinted with permission from Milstein, C.: Monoclonal antibodies, Sci. Am. 243:66, 1981.)
131
1 would also image the tumor of the patient since lymphocytes from all patients with choriocarcinoma studied recognized BeWo choriocarcinoma cells in the blocking factor assay. Blocking factor assays at this center have recently been added to hCG monitoring of patients with trophoblastic disease because of an approximate 10% incidence of recurrent disease after chemotherapy. Determination of blocking factor is a new test for agents in the serum of the patient that prevent the host from reacting against the tumor. These agents may be tumor antigen, tumor antibody, or antigen-antibody complex. During the course of treatment of the patient who was selected to undergo imaging, the hCG became undetectable at several points; however, testing for blocking
factor remained positive. This suggests that final tumor elimination may depend upon an immunobiologic reaction of the host to the tumor. Blocking of this reaction could prevent complete cure and lead to recurrent disease. The blocking factor assay consists of incubation of choriocarcinoma target cells with lymphocytes of the patient and control lymphocytes (Fig. 3). The choriocarcinoma cells are grown without lymphocytes, with normal control lymphocytes (which do not antigenically recognize the tumor cells and, therefore, do not adhere), and with the lymphocytes of the patient which are specifically sensitized to the tumor, recognize it, and cytotoxically destroy it unless the serum of the patient, which contains the blocking factor and prevents the lymphocytes from adhering and destroying the tumor, is added. The end point of the assay is the terminal tritiated thymidine uptake of target tumor cells not destroyed by the cytotoxic lymphocytes. The blocking index is calculated by the difference of cytotoxicity in the presence and absence of the serum of the patient containing the blocking factor. The choriocarcinoma cells were derived from five different patients, including the patient whose tumor was transplanted to nude mice and was imaged by the l3lI-labeled monoclonal antibody against the male choriocarcinoma, and provided five different assays. Lymphocytes from the patient who was to undergo imaging and the monoclonal antibody specifically recognized the BeWo choriocarcinoma tumor, one of the target systems in the blocking factor assay. The blocking factor calculation was performed as previously described8 , 9 (Table I). The choriocarcinoma
1042 Pattillo et al.
April I, 1984 Am. J. Obstet. Gynecol.
Fig. 3. Cellular observations of blocking factor assay in Patient LiWi with choriocarcinoma studied in 131I_labeled monoclonal antibody localization: A: Choriocarcinoma target cells grown in Lab Tek assay chamber without lymphocytes. Note mitotic figure of uninhibited growth. (x 1,000.) B: Choriocarcinoma target cells with normal control lymphocytes. No lymphocyte attachment or cytotoxicity. (X 1,000.) C: Lymphocytes of Patient LiWi in control serum. A single choriocarcinoma target cell is recognized by patient lymphocytes, and the choriocarcinoma cell is being cytotoxically destroyed (nuclear karyorrhexis and karylysis) by the patient lymphocytes. (X 1,000.) D: Choriocarcinoma target cells incubated with patient lymphocytes but in the presence of patient serum. Note lymphocytes are "blocked" from antigenic recognition and cytotoxicity toward the target cell. The lymphocytes do not adhere to the target cells. (x450.)
cells were grown in tissue culture flasks and transferred to Lab Tek chambers for the assay. One thousand target choriocarcinoma cells were incubated with 100,000 lymphocytes from the patient in the absence and in the presence of serum of the patient. If blocking was present, cytotoxic destruction of the choriocarcinoma cells would be blocked when serum of the patient was added to the incubation. The blocking index could be calculated as the difference between cytotoxicity in the absence and in the presence of serum of the patient. A pulse procedure with tritiated thymidine was used to detect remaining undestroyed tumor cells after the incubation. Target cells (103 cells per well) were incubated in eight-well Lab Tek chambers at 37° C in an atmosphere of 5% carbon dioxide in air. After 24 hours' incubation, the medium was removed and re-
placed with fresh 3520 medium* containing 105 lymphocytes or 3520 medium with 20% patient serum containing 105 lymphocytes. After incubation for 24 hours, the chambers were washed four times with balanced salt solution to remove the lymphocytes. The remaining target cells were incubated for 2 hours in 3520 medium with (methyPH) thymidine (10 /LCi/ml, 5 to 30 Ci/mmol). The pulse medium was removed, the cells were washed twice with balanced salt solution containing excess unlabeled thymidine, and frozen. After thawing, the amount of radioactivity incorporated into acid-precipitable material was determined. Determina*3520 medium, 30% Gey's balanced salt solution, 50% Waymouth medium, 20% fetal calf serum; all tissue culture media were obtained from Grand Island Biological Co., Grand Island, New York.
Choriocarcinoma 1043
Volume 148 Number 7
100
Surgery (TAH)
100
><
Q)
"0
E
C1'
c
:>< u 0
iii
\
90
90 80 70
80 ><
ChemoRx(MTX)
t
Q)
60 50 40 30 20 10
'0
E
60
C1'
50
.S ..::t:.
(,)
----l-----------
J
ISD
Jan Feb Feb Mar Apr
1981
Fig. 4. Blocking factor assay in Patient CoCI with OZ cells. This patient had resistant choriocarcinoma and underwent operation after multiple chemotherapy with rising hCG levels and positive assay for blocking factor.
tions were made in triplicate. Values were expressed as the percentage of radioactivity incorporated by target cells incubated in the absence of lymphocytes. Cytotoxicity of lymphocytes of the patient was assessed by comparing values obtained from incubations of patient lymphocytes with those of normal donor lymphocytes incubated with the target cells in the same assay. Blocking of cytotoxicity was assessed by comparing the values obtained from incubation of patient lymphocytes in fetal calf serum-containing medium with values obtained with the use of autologous serum-containing medium with target cells in the same assay. Significance was assessed by Student's t test with p $ 0.05 considered significant. Results
In the concurrent continuing study, patients with metastatic trophoblastic disease evaluated by blocking factor assessment showed a pattern of resolution of blocking factor activity with resolution of trophoblastic disease. Representative of patients studied during the period of time in which the monoclonal antibody was being produced are the following: Patient CoC! (Fig. 4) developed resistant postgestational choriocarcinoma after three courses of methotrexate. Blocking factor and hCG became undetectable after total abdominal hysterectomy with removal of all tumor that was still confined to the uterus. Patient LaDe (Fig. 5) who experienced recurrent pulmonary metastatic trophoblastic disease after undetectable hCG levels for 1 year was treated with multiagent chemotherapy, and blocking factor resolved after treatment. After 2 years of follow-up and undetectable blocking factor, the patient was allowed to become pregnant and was delivered of a healthy infant at 33 weeks' gestation.
0
CD
Act. 0
\
70
40 30 20 10
-----
---~-r---(
.
I SD
, Iii
JAN FEB FEB MAR FEB 1983 1981
Fig. 5. Blocking factor assay in Patient LaDe with JAr cells. Metastatic trophoblast disease.
Table I. Blocking factor assay Control versus 10' Target cells
Control versus 10' Target cells
10' Patient lymphocytes
10' Patient lymphocytes
with patient serum 24-hour incubation 2-hour thymidine pulse
without patient serum 24-hour incubation 2·hour thymidine pulse
Blocking index = lymphocyte killing with patient se· rum - lymphocyte killing without patient serum.
Patient LiLi (Fig. 6) experienced pulmonary metastasis of postgestational choriocarcinoma and demonstrated the presence of blocking factor until complete resolution of the tumor by chemotherapy. The patient subsequently had a healthy postchoriocarcinoma pregnancy and delivery. Lymphocytes of patients ElFa and JAr were tested against their choriocarcinomas as well as other target tumors and showed uniform recognition of these tumors of the same histologic type. Complete resolution of disease and resolution of blocking factor were observed in both patients. Patient Bar N was delivered of a term infant and within 3 weeks experienced massive pulmonary metastasis from choriocarcinoma. Blocking factor resolution occurred promptly after chemotherapy, and the patient now has undetectable hCG and blocking factor levels. Patient DoSmi was an elderly gravida who had a hydatidiform mole evacuated; however, because of persistent bleeding, she underwent hysterectomy and malignant trophoblastic disease was found. Blocking factor assays and hCG testing have remained negative. Patient MaBr experienced pulmonary metastasis after evacuation of a
1044 Pattillo et al.
April I, 1984 Am. J. Obstet. Gynecol.
100 90 80 )(
70
(])
"0
c
60
Q'\ c 50 :i:
u
0
CI)
ChemoRx (MTX)
t
40
30 20 10 JULY AUG OCT FEB 1980 1981
Fig. 6. Blocking factor assay in Patient LiLi with JAr cells. Blocking factor and hCG were undetectable after chemotherapy for postgestational choriocarcinoma with lung metastasis and pleural effusion.
hydatid mole; after successful chemotherpay, hCG and blocking became undetectable. The patient subsequently conceived and gave birth to a normal child. Patients DiBI and CaPI experienced metastatic trophoblastic disease (the latter with brain metastasis). Each developed undetectable blocking factor and hCG and are free of disease. Patients LuLu and DeWe currently have blocking factor and hCG present and have resistant disease. Patients SEb (Table II) and DoMi both experienced early metastatic trophoblastic disease which responded to methotrexate, and blocking factor and hCG tests became negative after therapy.* In review, the lymphocytes of all 15 patients studied (Table III) showed recognition of choriocarcinoma and blocking factor activity. The survival in our 15 high-risk patients studied with blocking factor and hCG monitoring is 88%. Thirteen patients are in remission and two have persistent disease. All 15 patients have survived for more than 2 years. With these studies completed and the monoclonal antibody cleared for human use, the study patient was admitted for imaging.
*Individual details on the findings in each patient will be furnished to any reader upon request.
Fig. 7. Gamma camera scan of resected choriocarcinoma lung lesion. Lesion/lung specific activity: 2.65: 1 (8,914 cpm/gm of tumor versus 3,368 cpm/gm of normal lung).
Case presentation The 29-year-old patient had resistant choriocarcinoma. She had initially been evacuated of a hydatidiform mole in December, 1979. After several negative titers the hCG level began to rise; 11 months after evacuation of the mole, repeat curettage revealed choriocarcinoma. There was resistance to multiple forms of chemotherapy (methotrexate, actinomycin D, bleomycin, cis-platinum, vinblastine, and Adriamycin), and although low levels of hCG persisted, no lesions could be identified by roentgenography, computerized axial tomography, or ultrasound. For this reason, imaging with the monoclonal antibody was planned; however, just before imaging, a small lesion appeared. The lymphocytes of this patient showed positive blocking factor activity with BeWo gestational choriocarcinoma. Because the male choriocarcinoma monoclonal antibody also recognized BeWo gestational choriocarcinoma, it was anticipated that it would recognize and image the tumor of this patient. During the course of the treatment, the hCG became undetectable at several points; however, assays for blocking factor remained positive. The monoclonal antibody study was carried out, after pyrogen testing, by intravenous injection of 5 /Lg of antibody with 5 mCi of 131 1 in 30 ml of saline over a lO-minute period. The thyroid was blocked by oral administration of a saturated solution of potassium iodide given three times per day, beginning 24 hours prior to injection. One tenth milliliter of the antibody was skin tested 30 minutes prior to injection. The imaging was done with the Technicare Gamma Camera, Columbus, Ohio. The l31I-labeled monoclonal antichoriocarcinoma
Choriocarcinoma
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120 110 100
90 ~
80
]
70
c>
60
c: ~ 50
.2 40 CD
Vv/
30 20 10 FEB. FEB MAR. DEC. JAN. FEB FEB APR. APR. APR. APR. MAY MAY MAY JUNE JUNE FEB MAR. APR. MAY
1981
1982
1983
Fig. 8. Blocking factor assay in Patient LiWi with JAr cells. Levels of blocking factor dropped but remained detectable after resection of the lung lesion. heG dropped to a low of 22 mIU/ml after excision of the lung lesion.
Table II. Blocking factor assay in Patient SEb with BeWo choriocarcinoma target cells Control lymphocytes (mean ± SE)
Patient lvmphocytes (mean ± SE)
Patient lymphocytes with autologous serum (mean ± SE)
Date of assay
('lo)
('lo)
('lo)
Recognition
Blocking
October, 1977 November, ]977
]07.6' ± 20.6 B 93.8 ± 10.7
3.9 ± 2.1 39.6 ± 6.2
57.3 ± ]9.3 53.4 ± 9.0
+*
+t
*Differs significantly (p tDiffers significantly (p
~ ~
+
0.05) from value of target cells incubated with control lymphocytes. 0.05) from value of target cells incubated with patient lymphocytes.
antibody was injected without complication; however, subsequent daily body scanning for 5 days failed to reveal isotope differences over the 2.5 cm area of the left upper lung field wherein the lesion had been identified on the computerized axial tomographic scan. Fourteen days later, at the time of thoracotomy, the lesion was resected and found to be clearly isolated in an anomalous lower segment of the left upper lobe. Antibody-isotope localization was verified in the lesion (Fig. 7), which consisted of malignant cytotrophoblast and synctiotrophoblast, hemorrhage, calcification, and fibrosis. On the basis of counts from the resected lung lesion and the adjacent normal lung, the lesion/lung specific activity ratio was 2.65: 1 (8,914 cpm/gm of tumor versus 3,368 cpm/gm of normal lung). Although the 131I-labeled monoclonal antibody had localized in the tumor, the preoperative isotope antibody concentration was not sufficient to differentiate it from the blood pool and background activity. The cellular observations and blocking factor activity of the monoclonal antibody study patient are seen in Figs. 3 and 8, respectively. Just as a serum factor blocks the lymphocytes from contact with the choriocarcinoma cells of the patient, the monoclonal antibody may have been partially blocked in the diagnostic imaging study. This may have prevented the dense localization of 131I-labeled
antibody in the patient compared to that seen in the transplanted choriocarcinoma. Dense imaging of the latter was seen preoperatively in the nude mouse. During the course of the chemotherapy treatment, assays for blocking factor remained positive, while tests for heG became negative on several occasions. Efforts to dilute or remove the blocking factor by plasmapheresis may improve imaging in future human diagnostic studies. Comment
The presence of a "blocking factor," which blocks the contact of lymphocytes with the choriocarcinoma cell, may be antibody, antigen-antibody complex, or antigen. Under either circumstance, it could prevent adequate concentration of the monoclonal 131I-labeled antibody and thus prevent satisfactory preoperative imaging. Further studies to examine these possibilities are in progress. Monoclonal antibody technology has provided a means to address a leading problem in the use of antibodies in medical science. The problem has been that no matter how pure an antigen one can produce, the body's discernment of molecular differences still leads to the production of a numbet of antibodies, and these
1046
Pattillo et al.
April I, 1984 Am.
Table III. Blocking factor assay results in 15 patients Patient
Recognition and cytotoxicity
CoC! LaDe LiLi ElFa JAr BarN DoSmi MaBr DiBI CaPI LuLu DeWe LiWi DoMi SEb
+ + + + + + + + + + + + + + +
cannot be readily separated for use. The present advance in monoclonal antibody capability has provided the means to reach into the "alphabet soup" of spleen cells producing antibodies to multiple antigens and grow one kind by fusing it with a tumor cell that can grow indefinitely and provide unlimited antibody. Conjugation of monoclonal antibodies with radioisotopes offers the next step for scanning to image the location of the tumor identified by the specific antibody. However, the body produces its own antibodies to tumors, the tumors shed antigens, and antigen-antibody complexes are generated; all of these may serve to block sufficient localization of even a monoclonal antibody in the subject tumor. Further, because monoclonal antibodies are produced in rats and mice, the potential for anaphylactic reactions in human subjects limits the numbers of investigation one can perform. The study case being reported is an example of an Institutional Research Committee's permission of a single compassionate study case when all other alternatives had been explored. Now that this study has been completed without complication, an Investigational New Drug permit has been issued by the Food and Drug Administration and additional cases can be examined in various stages of blocking factor activity. Such studies of trophoblastic tumor patients in the absence of significant blocking factor will permit further evaluation of the role of blocking factor in immune localization of specific antibody in the choriocarcinoma model. The need of preoperative imaging has not yet been met. The computerized axial tomographic scan and, indeed, radiographic localization permitted identification of this lesion. Only after the tumor was removed was it possible to verify the presence of 2.65: 1 specific activity of the labeled antibody in tumor versus normal lung. These current studies in the monoclonal antibody
J.
Obstet. Gynecol.
patient and the series of trophoblastic disease patients have revealed the presence of a serum blocking factor that blocked the immune localization and cytotoxicity by lymphocytes of these patients against their choriocarcinomas. Data from these studies suggest that attenuation of specific antibody or receptor concentration may interefere with immune localization. This blocking factor may have prevented the preoperative concentration of the monoclonal antibody and may serve as an obstruction to sufficient tumor localization in patients with high levels of blocking activity in general. Alternatively, serum halogenases may have cleared radioactive iodine from the antibody. The blocking factor assays proved to be useful as they remained positive when the tests for hCG became negative. This second modality for trophoblastic disease monitoring may have unique application because of its immunobiologic nature, a parameter of critical importance in tumor-host response. We conclude from this work: (1) All 15 patients with metastatic trophoblastic disease have been shown to exhibit blocking factor which correlated with the presence of tumor; (2) 1311-labeled monoclonal antichoriocarcinoma antibody was postoperatively localized in a metastatic lung lesion; (3) blocking factor may interfere with adequate antibody isotope concentration for preoperative imaging; (4) blocking factor assays proved to be useful as they remained positive when hCG tests became negative in the presence of small tumor volume. We would like to thank Mrs. Martha Rinke and Mr. James Kurtz for technical assistance in this work.
REFERENCES 1. Milstein, C.: Monoclonal antibodies, Sci. Am. 243:66, 1981.
2. Goldenberg, D. M., Kim, E. E., DeLand, F. H., et al.: Clinical radioimmunodetection of cancer with radioactive antibodies to human chorionic gonadotropin, Science 208: 1284, 1980. 3. Ballou, B., Levine, G., Hakala, T. R., et al.: Tumor location detected with radioactively labeled monoclonal antibody and external scintigraphy, Science 206:844, 1979. 4. DeLand, F. H., Kim, E. E., Primus, F. ]., et al.: In vivo radioimmunodetection of occult recurrent colonic carcinoma, AJR 138:145, January 1982. 5. Bergmann, S. R., Lerch, R. A., Mathias, C.]., et al.: Noninvasive detection of coronary thrombi with In-Ill platelets: concise communication,]. Nucl. Med. 24:130,1983. 6. Larson, S. M., Brown,]. P., Wright, P. W., et al.: Imaging of melanoma with 1-131 labeled monoclonal antibodies,]. Nucl. Med. 24:123, 1983. 7. Pattillo, R. A., and Gey, G.: Establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro, Cancer Res. 28:1231, 1968. 8. Pattillo, R. A., Story, M. T., and Ruckert, A. C. F.: Expression of cell-mediated immunity and blocking factor using a new line of ovarian cancer cells in vitro, Cancer Res. 39: 1185, 1979. 9. Pattillo, R. A., Ruckert, A. C. F., Story, M. T., et al.: Im-
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munodiagnosis in ovarian cancer: Blocking factor activity. J. OBSTET. GYNECOL. 133:791, 1979.
AM.
Discussion
DR. HUGH R. K. BARBER, New York, New York. In the discussion I shall raise more questions than may be considered proper for a discussant. In defense I must say that the article has many complex features. Although the article contains a great deal of good material, there are statements that are difficult to correlate with the conclusions. As an introduction it must be stated that the immune system is complex and changes from second to second. Therefore, it is difficult to study radioactive tagged monoclonal antibodies as to localization and blocking factor as an isolated event. However, Dr. Pattillo and his colleagues have achieved this and reported it to their satisfaction. Having said this, I must raise challenges to statements in the manuscript. The procedures for selecting hybrid cells to produce monoclonal antibodies that exhibit tumor specificity are not described nor are the results of tests used to screen for reactivity with similar or shared antigenic determinants in normal serum. Therefore, the reviewer must take for granted that the antibody used for tumor localization has no cross-reactivity for other normal tissue components which may be present. This is open to question. If there was, in fact, such cross-reactivity with a serum component, results identical to those described would be obtained for reasons quite different from those proposed. The statement that imaging was possible with tumor grown in nude mice does not guard against this possibility, since the interfering component might well be species-specific. For an article depending so heavily on the characteristics of a given monoclonal antibody, a more detailed description or definition of that antibody should be included. The monoclonal antibody to choriocarcinoma cells was prepared from nonseminomatous male choriocarcinoma (a teratoma). Gestational trophoblastic disease is folic acid-dependent whereas choriocarcinoma arising in a teratoma is folic acid-dependent. This accounts for some of the differences in the response to chemotherapy among these two seemingly different cancers. It is conceivable that a subtle change in the antibody may be identified in a larger series. By taking advantage of the extraordinary power of discrimination of the immune defense mechanism, the antibody produced against teratomatous choriocarcinoma may not always react against gestational trophoblastic choriocarcinoma cells as described in this article. There are discrepancies in the manuscript: The authors stated that no lesion could be identified by roentgenography, computerized axial tomography, or ultrasound. However, they later state that they scanned the area of the upper left lung field where a lesion had
Choriocarcinoma
1047
been identified on the computerized axial tomographic scan. In addition, the authors claimed that blocking factor correlates with the presence of tumor, yet they described Patient BarN who developed massive pulmonary metastases but say that "Blocking factor resolution occurred promptly ... , and the patient now has undetectable hCG and blocking factor levels." This is scientifically impossible to explain from the premise advanced in their paper. The figures and tables, although complicated, are good. The lack of more detailed characterization of the antibody and speculation, on the part of the authors, about a specific blocking factor that is circulating and blocks localization of the monoclonal 1311-labeled antibody raises more questions than have been answered. Since one case of monoclonal antibody 131 I imaging and blocking factor to choriocarcinoma is reported in detail and blocking factor activity is reported in 13 additional patients, there is "a paper within a paper." In conclusion, at best, I can say only that the results from the material presented are suggestive but not conclusive. DR. JAMES C. WARREN, St. Louis, Missouri. I wonder whether you could have studied the tumor in vitro and then obtained serum from the patient to determine if it was related to the uptake or binding of the antibody. Could you have done that test? DR. JOHN L. LEWIS,JR., New York, New York. I have a very brief comment, and it is a sideline to the paper. It concerns the woman who had an increase in blocking factor activity; the authors were concerned about a recurrence, but she was pregnant. Her situation indirectly raises the question of what the antibody is working against. More than 20 years ago Kaliss proposed that, in mice, antibodies generated to trophoblastic tissue had the effect of blocking antibodies, thus enabling pregnancy to proceed without immunologic rejection. This would suggest that the antibody is not directed against trophoblastic tissue. I think you will agree that it is not a specific antibody if it has been raised to one tumor of one sex and it is reacting with another tumor in the other sex, so I am interested in what you think the antibody has been responding to. What is the antigen or antigens? DR. PATTILLO (Closing). In response to Dr. Barber's questions, the procedures to identify the nature of the monoclonal antibody and its production are being published in the Journal of Nuclear Medicine. Controls for the monoclonal antibody are dealt with in this publication and could not be presented in the short time allotted here. The computerized axial tomographic scan identification of a chest lesion just prior to the imaging study is included in the paper and was noted in the presentation. This proved to be an asset because there was not sufficient isotope in the lesion to distinguish it from the blood pool and background. The fact that we knew there was a gross lesion permitted us to excise that lesion at thoracotomy and show that localization to
1048
Pattillo et al.
the tumor by the monoclonal antibody had occurred. With regard to Dr. Barber's final question concerning the BaRn patient, there was a typographical omission in the manuscript. It should have been stated that: The blocking factor and hCG became undetectable after appropriate chemotherapy. Dr. Warren's approach for evaluating the antibody by immunoprecipitation is a good suggestion. The in vitro system that we used, however, was to identify the monoclonal antibody's specific recognition of the patient's choriocarcinoma in an indirect fashion. The lymphocytes of the patient recognized the choriocarcinoma that was established in culture and the monoclonal antibody also showed cytotoxicity to it. We felt
April I, 1984 Am. J. Obstet. Gynecol.
that this was appropriate documentation for the specificity of the antibody. In answer to Dr. Lewis' comments with regard to pregnancy systems, the blocking factor assay was negative in normal pregnancy, which suggests that there is no cross-reactivity of the choriocarcinoma-specific antibody with normal pregnancy trophoblast, that is, patients with normal pregnancies do not show recognition of choriocarcinoma cells. Their lymphocytes do not show recognition of choriocarcinoma cells in culture; therefore, we presume that the antigen that we are dealing with in choriocarcinoma is a trophoblastic tumor-associated antigen.
Erratum John W. C. Johnson, M.D., was inadvertently omitted from the list of Consultants for the AMERICAN JOURNAL OF OBSTETRICS AND GYNECOLOGY, published in the December 15, 1983, issue, on page 857.