Radionuclide immunoglobulin lymphangiography: A case report

RADIONUCLIDE IMMUNOGLOBULIN LYMPHANGIOGRAPHY: A CASE REPORT STANLEY E. ORDER, WILLIAM D. BLOOMER, ALUNG. JONES, WILLIAM D. KAPLAN, MICHAEL A. DAVIS, S. JAMES ADELSTEIN, A N D SAMUEL HELLMAN

Iodine-13 1-labeled immunospecific gamma globulin derived from immunization of rabbits with F antigen, a tumor associated antigen in Hodgkin’s disease, has been utilized for intralymphatic infusion in a patient with known recurrent Hodgkin’s disease in the inguinofemoral and pelvic regions. Rectilinear scanning successfully delineated the tumor masses, and external monitoring showed retention of activity in the tumor sites over an 8-day period. Cancer 35:1487-1492, 1975.

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ANTIGENS H A V E BEEN R E P O R T E D IN of utilizing radiolabeled antiserum against tumorHodgkin’s disease and identified as F (fer- associated antigens as agents for tumor localizaritin) and S (an alpha-1-globulin) antigens, tion and therapy. This is because the scan obthrough the use of heterologous rabbit gamma tained can be compared with the radiographic globu[in,3.4.8.14-19 ’The ferritin was found to occur appearance of lymph nodes as demonstrated by both extracellularly and intracellularly in lymphangiography. The intralymphatic administration of antiferritin antiserum allows the lymphocytes of Hodgkin’s 18~19 Ferritin has also been identified as a tumor-associated Antibody to bind with the high concentration of antigen in leukemia, breast cancer, and pan- ferritin known to be in tumor-bearing sites creatic carcinoma.7.12*2eSome authors have prior to the antibody reaching the peripheral stated that different ferritins may be dis- circulation, where a high ferritin concentration tinguished depending on the disease and/or exists in the serum. Advantage is taken of this organ of origin by biochemical but not by im- fortunate circumstance of being able to infuse munologic procedures.eJ3 High titers of ferritin the tumor-bearing site without having to trahave been demonstrated in the serum of patients verse the high concentration of circulating antiwith Hodgkin’s disease.’J2J5 The concentration gen. The present case report concerns the results o f ferritin in Hodgkin’s-involved lymphoid tissue of an in vivo lymphatic infusion with iodinehas also been shown to be significantly in- 131-labeled immunospecific rabbit gamma globucreased.lg lin derived by immunization with the ferritin Lymphangiography performed in the course isolated from Hodgkin’s disease.‘.’ of determining the extent of Hodgkin’s disease affords an opportunity to evaluate the feasibility CASEREPORT

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A 44-year-old woman was hospitalized in January, 1973, because of a 3-week history of fatigue and pruritis. Three days prior to admission, she developed cough and fever and discovered a mass in the left supraclavicular fossa. This was biopsied and found to be mixed cellularity Hodgkin’s disease. She denied weight loss, anorexia, or night sweats. Past history included hospitalization for a manic-depressive reaction for which imi ramine hydrochloride and chlorpromazine ha{ been prescribed. Physical examination revealed a healthy appearing woman with a healing left supraclavicular incision. A small lymph node in the left neck and several small mobile nodes in the left axilla were palpable; the tonsillar fossae were unremarkable. The chest was clear to percussion and auscultation; the cardiac rhythm 1487

Presented at the 16th Annual Meeting of the American Society of ‘Therapeutic Radiologists, Key Biscayne, FL, October 30-November 3. 1974. From the Department of Radiation Therapy and Radiology, Harvard hledical School, Joint Center for Radiation ‘l’herapy, and New England Deaconess Hospital, Boston, hlA. Supported by Grants CA-12662, CA-05237, and GM18674 from the U.S. Public Health Service. Address for reprints: Stanley E. Order, MD, Joint Center lor Radiation Therapy, 50 Binney St., Boston, MA 021 15. The authors thank Dr. Melvin E. Clouse, Dr. Edward Sun. and M r . Louis H. Emond for expert assistance in performing the procedure; the Departments of Pathology and hledicine of the New England Deaconness Hospital for support of the research program and aid in establishing the clinical investigation; and Mr. Francis E. Chabot for technical assistance. Received for publication February 28, 1975.

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was regular and without murmurs. There was developed an abnormal right axillary lymph no hepatosplenomegaly nor palpable abdominal node. The remainder of the physical examinamasses. There was no significant inguinal tion and chest abdominal radiographs were lymphadenopathy. Neurologic examination was unremarkable. In August, 1974, while on normal. chemotherapy, a left inguinal and a left iliac Chest radiographs demonstrated widening node became palpable. Biopsy of these persisof the superior mediastinum with an anterior tent nodes demonstrated lymphocyte-depleted soft tissue mass consistent with Hodgkin's dis- Hodgkin's disease. She subsequently developed ease. Laminographic examination of the pe- recurrent pruritis, bladder discomfort, and right ripheral lung fields was normal. An intravenous inguinofermoral lymphadenopathy. Lymphanpyelogram demonstrated an extrinsic pressure giography with a small amount of Ethiodol defect in the right renal pelvis of undetermined (ethiodized oil) and iodine-131-labeled gamma etiology. Liver-spleen scan performed with 8 8 m T ~ globulin specific for F (ferritin) antigen ot sulfur colloid showed heterogeneous distribution Hodgkin's disease was performed on October of isotope within the liver and a prominent left 2, 1974. The patient had no adverse reactions to lobe; the spleen was normal. Bipedal lymphan- the procedure. She is currently receiving giography was normal. Bone marrow biopsy radiotherapy locally to gross disease to be foldemonstrated adequate cellularity and moderate lowed by further chemotherapy. eosinophilia, but no evidence of Hodgkin's disease. Percutaneous liver biopsy showed no evidence MATERIALS A N D METHODS of Hodgkin's disease. Admission hemoglobin was 11 g/lOO ml, hematocrit 22%, white blood Details of antigen isolation and purification as count 6800/pmms with 62% neutrophils, 18% well as antiserum preparation have been lymphocytes, 5% monocytes, 15% eosinophiles, ' ~ gamma platelets 572,000/pmms, and reticulocyte count previously d e s ~ r i b e d . ~ " . ~ . " -The globulin fraction of the antiserum was isolated 3.2%. The erythrocyte sedimentation rate was 31 mm/l hour, serum copper 214 &lo0 mg, by one-third saturated ammonium sulfate alkaline phosphatase 112 IU (normal 11-86 IU), precipitation, dialysis, and concentration with and leukocyte alkaline phosphatase 195 U (nor- ultra-filtration utilizing a UM-10 membrane mal 75-133 U). Direct and indirect Coomb's (Amicon). Enzymatic radioiodination was pertests and cold agglutinins were negative. The formed utilizing the technique described by serum iron, iron binding capacity, LDH, Marchalonis. SGOT, SGPT, and total bilirubin were normal. Clinically, the patient was felt to have Stage IIB disease; however, because of the substantcal Lymphangiographic Technique mediastinal mass, mantle irradiation was inUsing aspectic conditions, 5 mg gamma itiated without further staging. From January 24 globulin specific for F (ferritin) antigen of to February 21, 1973, the patient received 4000 rads in 20 fractions to the cervical, axillary, Hodgkin's disease in 2 ml of 0.1 M phosphate supraclavicular, and mediastinal regions, using buffer (pH 7.4) were combined with 50 p g laca 4-Mev linear accelerator. Small bilateral toperoxidase (Calbiochem) and 1 mCi carrierpleural effusions developed early in the course of free NaI'*l (New England Nuclear). The reacirradiation and were considered to be related to tion was initiated by the addition of 10 pl freshly Ethiodol embolization following lymphangi- prepared 0.5 M hydrogen peroxide and allowed ography. These resolved over a period of days. to proceed for 30 minutes at room temperature Following mantle irradiation, the patient became severely depressed and required with vigorous mixing. Unbound iodide was electroconvulsive therapy. Although there was removed by successive cycles of buffer addition no evidence of active disease, persistent fever and vacuum dialysis until chromatographic and malaise of increasing severity prompted the determinations showed less then 3% unbound initiation of chemotherapy consisting of nitrogen iodide. Specific activity of the iodinated gamma mustard, vinblastine, prednisone, and procar- globulin was 0.47 Ci/g. The entire sample was bazine. Rapid improvement in symptoms per- passed through a sterile 0.22-pm Millipore mitted exploratory laparotomy with splenec- membrane filter. Pyrogen testing in three rabtomy, liver, and para-aortic node biopsies to be bits showed a total temperature rise of 0.5' C, performed on March 8, 1973; all histologic well within the U.S.P. limits for pyrogenicity. specimens were free of Hodgkin's disease. FolDermal and conjunctival tests for sensitivity to lowing splenectomy, persistent fever and malaise led to the continued use of chemotherapy with- rabbit gamma globulin in the patient were negative. out further irradiation. The patient received Lugol's solution prior to T h e patient became asymptomatic and remained so until May, 1974, when she the study. Standard technique was employed in

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cannulating the lower extremity lymphatics. Each lower extremity was infused with 2 ml Ethiodol a t a flow rate of 0.13 ml/minute to ensure intralymphatic infusion prior to the introduction of 250 yCi antiserum. Total body scanning from the knees to the mandible was carried out with a dual probe rectilinear scanner (Ohio Nuclear) using a 3.5-inch focused medium energy collimator. Scanning was performed daily for 4 days and on the 7th day following infusion. Activity over the pelvis, right groin, and heart was monitored with a scintillation detector for the first 8 postinfusion days. RESULTS Intralymphatic administration of Ethiodol 19 months after splenectomy demonstrated a large left pelvic mass (Fig. 1). Ethiodol entered both inguinal-femoral nodes, pelvic nodes, and paraaortic nodes, and demonstrated the outline of these lymphatic structures. Rectilinear total body scanning 90 minutes following infusion (Fig. 2A) demonstrated significantly increased activity in both inguinofemoral regions and the pelvis, areas corresponding to palpable masses and radiographically demonstrated disease. Twenty-one hours following infusion (Fig. 2B),

Frc. 1. Pelvic radiograph following Ethiodol infusion dernonstrating left pelvic (LP) mass, bilateral inguinal-femoral (IF) nodes, and right pelvic mass.

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the left pelvic mass was completely delineated by radioactivity, as were the right pelvic and bilateral inguinofemoral masses. Radioactivity persisted in the right lower extremity despite the absence of Ethiodol in the area. Persistence of activity over areas of known recurrent Hodgkin's disease was noted through 52 hours, but no new sites of localization, such as the liver, developed. External monitoring during radionuclide lymphangiography demonstrated activity over the knees at 2 minutes, pelvis at 5 minutes, and para-aortic region at 8 minutes. At 50 minutes, there was no significant activity over the thoracic duct or supraclavicular fossae. Radioactivity over tumor-bearing regions exceeded that of the blood pool, and a n effective half-life of 4.5 days was estimated for both (Fig. 3). The biological half-life was calculated to be 10 days; disappearance of activity over tumor bearing regions and the blood pool was parallel. DISCUSSION Current efforts directed toward the utilization of tumor-associated antigens or markers for diagnostic and therapeutic purposes have demonstrated that 'SII-labeled antiserum against carcinoembryonic antigen (CEA) could be lo-

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FIG. 2. Rectilinear total body scans, anterior views, following infusion 01 ‘3’1-labeled antiferritin antiserum. L P = left pelvic mass. IF = inguinal-femoral nodes (biopsy positive for disease). Arrow delineate right inguinal-femoral and pelvic nodes. A (left). Scan 90 mi n u t es following infusion. €3 (right). Scan 21 hours following infusion.

calized in a CEA-producing human colonic carcinoma when transplanted into either xenogeneic immune-deficient recipients or immunologically privileged Although scintigraphic detection of tumors in these animal models was possible under idealized conditions, tumor/nontumor ratios in the torso region were disappointing. Using a syngeneic experimental murine ovarian carcinoma in this laboratory, it has been possible to demonstrate therapeutic efficacy from antiserum directed against tumor-associated antigens.20J1 Positive scans have also been obtained with radiolabeled antiserum when the tumor was grown subcutaneously in the thigh. Very few efforts have been made to employ this approach to patients with cancer. However, radiolabeled antiserum to human fibrinogen, when administered intravenously, has been shown to localize in a variety of selected human neoplasms with limited s ~ c c e s s . ’ ~ ~ ~ ~ It was hoped that the isolation of tumorassociated antigens in Hodgkin’s disease would

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DA YS Fro. 3. Effective half-life of radioactivity as monitored over tumor-bearing left pelvis and heart.

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permit clinical investigation using similar techniques. lntralymphatic infusion is well suited for the evaluation of tumor-associated antibody in patients with Hodgkin’s disease. By its use, it was hoped that Hodgkin’s-positive nodal masses in the inguinofemoral, pelvic, and retroperitoneal regions could’ be exposed to a high titer of antibody, that the radioactivity could be concentrated for greater contrast, and that reaction with circulating antigen could be delayed if not prevented. In contrast to lymphangiographic infusion, intravenous administration offers the potential of demonstrating tumor masses not otherwise visualized; however, the possibility of cross-reactive binding with several target organs or with high levels of circulating antigen argues against the intravenous route for tumor localization or therapy. Although the antiserum used in this lymphangiographic study contained a high titer of anti-F antibody, other nonspecific rabbit gamma globulin was present and radiolabeled. Nonetheless, a rather clear delineation of radioactivity was apparent in tumor bearing regions. Despite the striking localization of radioactivity in this instance, many questions remain unanswered: 1. Was the concentration of ferritin in the normal lymphatic system elevated sufficiently for binding of specific antibody to occur in normal lymph nodes?

2. Did antibody bind outside the lymphatic system by diffusion or did antibody precipitate in the lymphatic walls causing

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obstruction or effusion? (Note the right lower extremity lymphatics, Fig. 2). 3. Was rabbit gamma globulin handled by the lymphatic system as an antibody or an antigen? Kabbit gamma globulin, a foreign protein, may be taken up by remaining lymphoid elements for reasons other than antibody specificity. 4 . What was the distribution of the radiolabeled antibody in relationship to the histologic infiltrate of Hodgkin’s disease?

5. Did the viscosity of Ethiodol preceding antiserum infusion aid or detract from tumor visualization? Ethiodol may prolong the time available for antibody binding by its increased viscosity causing slowing of lymph flow; conversely, by cell surface interference, it may inhibit binding that might otherwise have occurred. 6 . How would radiolabeled nonspecific rabbit gamma globulin or even albumin be handled when administered by intralymphatic infusion?

Despite a number of unanswered questions, the successful delineation of tumor-bearing nodes by intralymphatic infusion of radiolabeled antibody provides a new approach to tumor localization. T h e mechanism of localization and most useful routes of administration need clarification. Finally, it may be possible to use this antibody delivery system to deliver therapeutic doses of irradiation to some tumor deposits, i.e. antibody-directed radiotherapy; this must be investigated.

REFERENCES I Aungst, C . W . : Ferritin in body fluids. \led. 71:517-522, 1968.

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7. Castellino, K.A , . Silverman, J. F., Glastein, E., Blank, U.. and Wexler, L.: Splenic arteriography in Hodgkin’s t-lisease--PI roentgenologic-pathologic study of 33 con~ecutiveuntreated patients. A m . 3. R o d . 14:574-582, 1972. 3. Chisni. S., Order, S. E., and Hellman, S.: Tumor fetal .intigens associated with Hodgkin’s disease-An immunoelectrophoretic analysis. A m . J . Roenlgenol. 1 1 7 5 1 I , 1973. 4 . Eshhar, Z.,Order, S. E., and Katz, D.: Ferritin-A Hodgkin’s disease associated antigen. Proc. .Null. h a d . Sct. 1 :.GI7 1 :3956-3960, 1974. 5. Goldbenberg, D. IM., Preston, D. F., Primus, F. J.. and Hansen, H . J . : Photoscan localization of GW-39 tumors in hamsters using a radiolabeled anticarcinoembryonic antigen immunoglobulin G . Cancer Res. 34: 1-9, 1974.

6. Hoffer, P. B., Lathrop, K.. Bekerman, C., Fang, V. S., and Kefetoff, S. : Use of CEA antibody as a tumor scanning agent. 3. Nucl. M e d . 15:323-327, 1974. 7. Jones, P. A. E., Miller. I;. .M., Worwood, h1.. and Jacobs, A . : Ferritinemia in leukemia and Hodgkin’s disease Br. 3. Cancer 27:2 12-2 17, 1973. 8. Katz, D. H., Order, S. E., Graves, M., and Benacerraf, B . : Purification of Hodgkin’s disease tumor associated antigens. Proc. Nutl. Acud. Scz. C’SA 70:396-400, 1973. I). Linder, M. C., and hlunro, H. N.: Metabolic and chemical features of ferritin a series of iron inducible tissue proteins. A m . 3. Pathol. 2:263-282, 1973. 10. Mach, F. P., Carrel, S.. hlerenda, C., Sordat, B., and Cerottini, J. C.: In vitro localization of radiolabeled antibodies to carcinoembryonic antigen to human colon carcinoma grafted into nude mice. Nature 248:704-706, 1974. I I . Marchalonis, J . J . : A n emzymatic method for the

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antigens. Cancer Res. 34:1182-1186, 1974. trace iodination of immunoglobulin and other proteins. 10. Order, S. E.: Antigenic analysis of the lymphomata. Bmchrm. 3. 113:299-305, 1969. Br. J . Cancer 31:128-139, 1975. 12 hlarcus, I). X I . , and Zinberg, N.: Isolation of ferritin 20. Order, S E., Donahue, V., and Knapp, R . : Imfrom human mammary and pancreatic carcinoma by means ol antibody immunoadsorbents. Arch. Biochem. Btophys. munotherapy of ovarian carcinoma-An experimental model. Cancer 32:573-579, 1973. 162:493-501, 1974. 21. Order, S. E., Kirkman, R., and Knapp, R.: Serologic 13. McCardle, R. J., Harper, P. V., Spar, I . L., Bale, W. F., Andros, G., and Jiminez, F.: Studies with iodine-131 immunotherapy-Results and probable mechanism of action. Concer 34:163-171, 1974. labeled antibody to human fibrinogen for diagnosis and 22. Reissman, K. K., and Dietrich, M. R.: O n the therapy of tumors. 3. Nucl. Med. 7~837-847, 1966. 14. Order, S. E., Porter, M., and Hellman, S.: Hodgkin's prescence of ferritin in the peripheral blood of patients with disease-Evidence for a tumor associated antigen. N Engl. 3. hepatocellular disease. 3. Clin. Invest. 35:588-595. 1956. bled. 285:471-474, 1971. 23. Richter, G . W . : Comparison of ferritins from 15. Order, S. E., Chism, S. E., and Hellman, S.: Studies neoplastic and non-neoplastic human cells. Nature 207 :6 16-618, of antigens associated with Hodgkin's disease. Blood 1965. 40:621-633, 1972. 24. Spar, I . L., Bale, W. F.. Marrack, D., Dewey, M'. C., 16. Order, S. E . , and Hellman, S.: Tumor associated anMcCardle, K. J., and Harper, P. V.: I"' labeled antibodies tigens in Hodgkin's disease. Fronf. Radial. Ther. Oncol. 7 : to human fibrinogen-Diagnostic studies and therapeutic trails. Cancer 20:865-870, 1967. 155-167, 1972. 25. M'ohler, F., and Schonlau, F.: Uber das Vorkarmmen 17. Order, S. E., Chism, S. E., and Hellman, S . : Hodgkin's disease associated antigens-Studies on segrega- van Ferritin im Serum. Klin. Wochenschr. 37:445-451, 1959. tion and specificities. Null. Cancer I n d . Mongr. 36: 139-145, 26. M'orwood, M., Summers, M . , Miller, F., Jacobs, A., 1973. and Whittaker. J. A . : Ferritin in blood cells from normal 18. Order, S. E., Colgan, J., and Hellman, S.: Distribu- subjects and patients with leukemia. Br. 3. Hematol. tion of fast and slow migrating Hodgkin's tumor associated 28:27-35. 1974.