Journal of Surgical Oncology Supplement 2%-91
(1991)
Perioperative Radioimmunolocalization of Colorectal Cancer by Radiolabeled Monoclonal Antibodies FAUSTO BADELLINO, MD, PlERLUlGl PERCIVALE, MD, PAOLO MESZAROS, MD, MARC0 GIPPONI, MD, STEFAN0 BARTOLOMEO, MD, GUlDO NICOLO, MD, BRUNO SPINA, MD, FEDERICO SCHENONE, MD, AND SERGlO BERTOGLIO, MD From the Division of Surgical Oncology (F.B., P.M., M.C., S.Ba.), Service of Anatomy and Pathologic Cytohistology (C.N., B.S.), Service of Nuclear Medicine (F.S.), National lnstitute for Cancer Research (/ST) and the lnstitute of Clinical and Experimental Oncology, The University of Cenoa (P.P., S.Be.), Cenoa, ltaly
KEYWORDS:murine monoclonal antibodies, tumor associates antigens, gamma-emitting radionuclides
INTRODUCTION antigen, leading to the detection of subclinical tumor The hybridoma technology for manufacturing mono- deposits that may alter surgical approach in approxiclonal antibodies (MAbs) has made available a large mately one third of patients. This technique has been number of biocompatible murine MAbs that can be used expected to enhance intraoperative staging and surgical to target specific tumor associates antigens (TAA) for radicality, therefore improving long term prognosis in both diagnostic and therapeutic purposes. The potential colorectal cancer patients [&13]. The authors present their experience on the use of diagnostic utility of MAbs has been facilitated by techintraoperative immunodetection and staging of colorectal nological advances that permit their coupling with diftumors by RIGS and the use of two different murine ferent gamma-emitting radionuclides. Tumor-localizing "' 1 B72.3 and IgGl and I2'-I F023C5 FAb'2, a MAbs: techniques by external gamma camera imaging equipMAb that reacts with CEA antigen. ment, the so-called immunoscintigraphy , have been expanding and extensively investigated in presurgical MATERIALS AND METHODS patients with colorectal carcinoma. Many reports have Patients shown that immunoscintigraphy , using several polyThe study, which was carried out started in October clonal MAbs such as those directed against the carcino1988 and is to be completed in July 199 1. Patients were embryonic antigen (CEA) or the TAG-72 antigen (MAb divided into 2 series and were respectively injected with B72.3), may preoperatively detect 70435% of primary the 2 mentioned MAbs. Preoperative diagnosis and and recurrent tumor sites [ 1-41. Provided this encouragstaging of the patients included colonoscopy with biopsy ing diagnostic results, the preoperative detection of and/or double contrast barium enema, abdomino-pelvic unsuspected lymph node involvement or synchronous computerized tomography (CT), and ultrasonography lesions in patients with colon cancer, therefore altering (US) scan and measurement of tumor marker levels; extra surgical approach and improving long term prognosis, is abdominal disease was excluded by conventional diagstill controversial [ 5 ] . nostic procedures. Intraoperative detection and staging of colorectal cancer by radiolabeled MAbs and the use of a hand-held SERIES A gamma detecting probe (GDP) is a new technique, named From October 1988 to December 1989 MAb B72.3 radioimmunoguided surgery (RIGS 'rh'), proposed in 1986 was injected in 15 surgical patients with primary or by Martin et al. [6]. This method is based on a preoperrecurrent colorectal cancer. Eight patients had primary MAbs which selectively bind a ative injection of 125-1 TAA at the site of tumor. At surgery, the abdominal Accepted for publication May 30, 1991. cavity is searched for selective radioactivity emission Address reprint requests to Dr. Fausto Badellino, Division of Surgical Oncology, National Institute for Cancer Research, IST Genoa, Viale from radiolabeled tumor tissue. Encouraging results have Benedetto XV 10, 16132 Genoa, Italy. been obtained in colorectal surgery with the use of "'-1 The research in this manuscript was supported by grants of AssociaMAb B72.3, a murine IgG1 that reacts with TAG-72 zione Italiana per la Ricerca sul Cancro (A.I.R.C.). 0 1991 Wiley-Liss, Inc.
Radioimmunolocalizationof Colorectal Cancer
87
tumors and 7 had a recurrence; 7 patients were male with TABLE I. Intervals to Surgery in 28 Patients Operated a mean age of 62.6 years (range 32-72) and 8 were for Primary and RecurrentColorectal Cancer With RIGS female with a mean age of 59.3 years (range 41-73). and I2'-I Anti-CEA FAb2 Patients were eligible for surgery as soon as external Primary Recurrent tumors tumors Overall precordial counts performed with the GDP were less than 20 counts/2 seconds. 6 Days 2 4 6
SERIES B
9 Days 12 Days 15 Davs
4 3 3
6 3 3
10 6 6
The aims of study series were to define the best interval from the preoperative injection of anti-CEA FAb'2 (F023C5) to surgery, evaluate the potential diagnostic and staging utility, and to assess the patterns of biodisThree days before the injection series A and B patients tribution of the antibody for its possible future use for therapy. Being FAb'2 proteins with a lower molecular received a supersaturated solution of potassium iodide weight they should be expected to target tumors in a (SSKI) 10 drops/3 times per day (500 mg of potassium shorter period of time and clear faster from the blood pool iodide) to prevent thyroid uptake of 125-1. Infusion was [ 1&16]. These specific features should allow surgery to performed in 5 minutes, checking vital signs for possible be performed in a shorter period of time from the anaphylactic reaction. injection of the MAb. The phase 1-11 study design GDP and Microprocessor Control Unit provided single series that included at least 6 patients who were operated on at different intervals; in particular, 6, 9, GDP and Control Unit (Neoprobe 1000 SYSTEM) 12, and 15 days from the preoperative injection of the were provided by Neoprobe Co. (Columbus, Ohio, radiolabeled F023C5. USA). The probe is stainless steel and gas sterilizable, From January 1990 to July 1991 at the Division of measuring 18 cm in length with the tip angled at 120" Surgical Oncology of our Institution, MAb F023C5 was for optimal intraoperative use. It contains a 1 cm diampreoperatively injected in 28 patients with colorectal eter cadmium-telluride crystal as detector and a preamcancer. Twelve had primary cancer while 16 were plifier. Gamma emissions from a radioactive source are operated on for recurrent or metastatic tumors. Fourteen converted by the preamplifier to a digital read out on the patients were males with a mean age of 59.6 years (range portable control unit; the software of the control unit can 43-74) and 14 were females with a mean age of 56.8 also convert counts/seconds into audible signals. The years (range 41-78). Table I analyzes patterns of the sound volume can be controlled and a threshold count series related to preoperative diagnosis and timing of rate, below which no sound is produced, can be easily set. Its intraoperative use is based upon the fact that as surgery. each normal tissue is probed, a background count threshMonoclonal Antibody old can be selected that will not give auditory signals to Series A. MAb B72.3, a murine IgGl that reacts the surgeon. As the probe passes to an area of concenwith a high molecular weight glycoprotein with the tration of gamma emissions, the count rates and the sound mucin characteristic named TAG-72, was used intraop- increase, thus allowing the surgeon to quickly and eratively to detect cancer sites [17]. B72.3 was radiola- accurately locate the source of radiation. The absence of beled with 125-1 by the Iodo-Gen'" method. Unbound a bulky collimator, due to the low energy isotope used iodine was removed by gel filtration chromatography on (125-I), is a favorable feature of the technique for better Sephadex column and filtrated through a 0.22 micron and faster scanning of the whole abdomen during sursterile filter before preparation for clinical use. Each gery. patient received before surgery an intravenous (i.v.) dose Method of RIGS of 1 mg MAb/l.5 mCi of 125-1 (Surgimab R3@,Sorin At surgery, after the assessment of levels of residual Biomedica, Italy). Series B. MAb F023C5, a murine FAb'2 that reacts background radioactivity, the GDP was used to search for with CEA antigen, was injected to intraoperatively detect primary, metastatic, or recurrent tumor location, and to cancer sites [ 181. F023C5 was radiolabeled with 125-1by survey the entire abdomen with particular attention to the the Iodo-Gen method. Unbound iodine was removed by tissues adjacent to the tumor, the lymph nodes and the gel filtration chromatography on Sephadex column and liver. All tissues with a tumor-to-normal tissue (Tu/NT) filtrated through a 0.22 micron sterile filter before count ratio > 1.75: 1 were considered to be positive for preparation for clinical use. Each patient received before tumor location and resected, if possible, or biopsied. surgery an i.v. dose of 1 mg MAW1.5 mCi of 125-1 Tumor resected specimens as well as similar non malignant control tissues, whenever available, were given to (Surgimab R 2 9 .
88
Badellino et al. TABLE 11. RIGS Diagnostic Results of Primary Tumors Operated After the Injection of 125-1B72.3 MAb
Site Primary cancer Stage 1-11 Stage 111-IV Liver metastases Lymph node metastases Total
True Positive (%)
False Positive
112 (50) 517 (71.4) 7/10 (70) 517 (71.4) 18/26 (69.23)
0 0 0 0 0126
False negative Tag AgSa
(%I
False negative Tag Ag-a (%)
0 1/7 (14.28) 1/10 (10) 2/7 (28.57) 4/26 (15.38)
1/2 (50) 117 (14.28) 2/ I0 (20) 0 4/26 (15.38)
"Tag Ag+ and - indicate tumor sites staining positive and negative for TAG-72 at immunohistochemical analysis.
TABLE 111. RIGS Diagnostic Results of Recurrent Tumors Operated After the Injection of lZ5-I B72.3 MAb
Site
True positive (%)
False positive
Local recurrence Liver metastases Lymph node metastases Other Total
11/13 (84.6) 811 1 (72.7) 6/8 (75) 3/4 (75) 28/36 (77.7)
0 0 0 0 0
False negative Tag Ag+a
(%I 2/13 (15.38) 3/11 (27.3) 2/8 (25.00) 1/4 (25.00) 8/36 (22.2)
False negative Tag Ag-a
0 0 0 0 0
aTag A g f and - indicate tumor sites staining positive and negative for TAG-72 at immunohistochemical analysis.
the pathologist for routine examination and immunohistochemical assay for TAG-72 and CEA antigen expression, respectively, for series A and B specimens. Preinfusion human antimouse monoclonal antibody (HAMA) titers levels were available for all patients; subsequent samples were obtained at 4 and 8 weeks after the injection of B72.3 and F023C5 MAbs.
RESULTS Series A (MAb B72.3 anti-TAG-72) No adverse reactions were observed in patients injected with B72.3 MAb. Patients underwent surgery 21.12 ? 6.48 and 24.2 2 7.8 days after MAb injection, respectively, for primary and recurrent colorectal tumors. Sixty-two suspected tumor sites were intraoperatively recognized by RIGS; 26 were detected in the 8 patients with primary tumor and 36 in the 7 patients with recurrences. All sites were confirmed to be neoplastic tissue by histological examination. Tables I1 and 111 analyze the clinical results of intraoperative radioimmunodetection for primary and recurrent tumors. Results of patients with primary tumors were divided into 2 stage groups based on the 4th Ed. TNM classification: Stage 1-11, and stage 111-IV.
RIGS was able to detect 50% of sites in Stage 1-11 primary cancer stage, and (71.4%) in stage 111-IV: 4/26 (15.38%) sites expressing TAG-72 have failed to be detected by RIGS. Sites not expressing the antigen failed to be detected with a similar average of 15.38%. It is important to note that 2 out of these 4 lesions (50%) not expressing the antigen were observed at the site of the primary colonic lesion. In recurrent cancer we were able to localize 28/36 (77.7%) of cancer sites. All the false negative sites in this group expressed TAG-72 antigen. We finally evaluated the utility of the RIGS system in surgical decision making changes. In 217 patients with recurrent or metastatic disease the probe was able to identify subclinical deposits of tumor that were not otherwise evident either preoperatively or intraoperatively; 1 of these localizations was a perianastomotic recurrence and 1 was a peri-aortic lymph node metastasis. As a summary of this additional information, RIGS system modified the surgical approach in 1/15 (6.6%) patients; in particular, surgical decision making was changed in 1/7 (14.28%) patients with recurrent tumors and never during surgery for primary colonic lesions. Both patients underwent laparotomy only on the basis of preoperative high CEA levels. Serum
Radioimmunolocalization of Colorectal Cancer
HAMA levels increased at least twice the baseline within 8 weeks from the MAb injection in 7 (46%) patients.
Series B (MAb F023C5 anti-CEA) No adverse reactions were observed in patients injected with anti-CEA MAb. Table IV shows potential diagnostic features of RIGS in patients operated at different intervals. Forty-nine suspected tumor sites were intraoperatively recognized; 42 of them (85.7%) were confirmed to be tumor by histology. All recurrent and metastatic sites as well as 92% of primary resected tumor stained positively for CEA antigen at immunohistochemical examination. We correctly localized 5/7 (71.4%) sites in patients operated at 6 days, 18/22 (81.8%) in patients operated at 9 days, 416 (66.6%) in patients operated at 12 days, and 2/7 (28.6%) in patients operated at 15 days interval from MAb injection. In patients operated at 9 days we localized 2 subclinical tumors out of 22 detected sites (9.1%). One was a lymph node in 1 patient operated for a local recurrence while the second was a liver metastasis of 15 mm of diameter that was resected following RIGS indication, thus altering surgical decision making. We observed 7 (14.29%) false positive localizations; 3 were enlarged reactive non neoplastic lymph nodes expressing CEA antigen while 4 sites resulted to be granulomatous nodules located in the pelvic peritoneum. Three localizations occurred in patients operated at 6 days (30%) and 4 in patients operated at 12 days (40%) from the MAb injection. Table V analyzes patterns of biodistribution of the antibody. As a confirmation of diagnostic findings, the best intraoperative tumor/normal tissue gamma emittence ratio and radiolocalization index were observed 9 days after the MAb injection with values of 3.89/1 and 7.01/1, respectively. From these data it appears that before 9 days from the MAb injection, the blood pool radioactivity is too high covering probably the MAb related radioactivity at tumor sites. Opposite, after 12 days, difference on radioactivity from tumor and normal tissues may be weak to be certainly recognizable by RIGS. Table VI shows clinical diagnostic results as well as biodistribution patterns observed for differentiated tumor tissues in patients operated at 9 days when the best results were obtained. Anti-CEA F023C5 MAb made recognizable by RIGS recurrent tumors in 88.8% sites, liver metastases in 100% sites, lymph node metastases in 87.5% sites, while primary tumor sites, regardless of stage, were localized in 62.5% of cases. Thus, a high intraoperative diagnostic and staging potential of RIGS for advanced stages of colorectal cancer was demonstrated. Postinjection HAMA titers increased in 4 (14%) pa-
89
TABLE IV. Diagnositc Results of RIGS in 28 Patients Operated at Different Intervals After Injection of lz5-I F023C5 Anti-CEA FAbZ MAb Interval to surgery
RIGS positive detections RIGS negative detections False positive detections Subclinical tumor detections Surgical decision making changes
6 Days
9 Days
12 Days
15 Days
(%)
(%)
(%)
(%)
511
18/22 (81.8) 4/22 (1 8.2) 0122
416 (66.6) 216 (33.3) 4/10
211 (28.6)
(71.4)
211 (29.6) 3/10 (30) 0 0
517
(71.4) 017
(40)
2/22 (9.1) 1/22 (4.6)
0
0
0
0
tients; only 3 (10.2%) had levels higher than at least twice the baseline.
DISCUSSION The encouraging findings of the early investigational reports on the use of radiolabeled MAbs in the preoperative evaluation and follow-up in patients with colorectal cancers suggested that their intraoperative use by means of RIGS may alter surgical approach, therefore increasing patient's overall survival [&9, 12-13]. Results reported by Martin et al. with this technique concluded that RIGS was more accurate in localizing clinical obscure metastases than other methods. In patients with recurrent colorectal cancer, unknown tumor sites were detected in almost 20% of the cases thus altering surgical approach in nearly all of them [6-11]. B72.3 The results of our first study with the use of 125-1 MAb demonstrated the safely of i.v. administration to humans of murine antibody. Primary colorectal carcinomas were detected with an average of 50% and 7 1.4% in Stages 1-11 and 111-IV group, respectively. In recurrent cancers tumor sites were located in 8 1.9% of cases. Even the localization of subclinical occult tumor sites leading to intraoperative changes on surgical decision making occurred only for recurrent tumors (28.5%). Finally, we were able to demonstrate that RIGS with 125-1 B72.3 MAb was very encouraging in terms of clinical utility for advanced and recurrent tumors. Opposite, for primary cancer the non homogeneous patterns of TAG-72 antigen expression, 30% of which stained negatively at the immunohistochemical assay, may be responsible for the worse obtained results [19,20]. Nevertheless, it has to be pointed out that postinjection elevated HAMA titers were found in 46% of patients. Another controversial finding, depending on the use of a IgGl MAb, may be the adequate period of time needed to obtain an optimal concentration of the radioimmuno-
90
Badellino et al. TABLE V. Biodistribution Patterns of lz5-I Conjugated F023C5 Anti-CEA FAbZ MAb Injected in 28 Patients herated With RIGS at Different Intervals From the MAb Injection ~~
6 Days
Interval to surgery Intraoperative Tu/NT ratios (GDP counts 2”) Radiolocalization index Mean radioactivity NT (cpm/O.l g) Mean radioactivity Tu (cpm/O.l g) Whole blood pool radioactivit; (cpm/O.l ml)
9 Days
12 Days
15 Days
2.42
3.89
3.47
1.45
6.28 651.9
7.0 1 251.0
3.49 204.2
1.93 58.97
1,473.8
1,730.5
708.6
179.6
1,711.1
892. I
698.2
163.3
Abbreviations: Tu, tumor tissue; NT, adjacent normal tissue; cpm, gamma counts per minute.
TABLE VI. Clinical Results and Tissue Radioactivity of Different Tumor Sites in Patients Operated 9 days After Anti-CEA lZ5-IF023C5 MAb Primary cancer
Local recurrence
Liver metastases
Lymph node metastases
62.5% 1.75
88.8% 4.92
100% 2.26
87.5% 2.82
2.5 1
4.75
8.02
5.88
~
% of detections Intraoperative Tu/NT ratio (2” counts) Radiolocalization index (0.1 g Tu/NT cpm)
Abbreviations: Tu, tumor tissue; NT, adjacent normal tissues; cpm, gamma counts per minute.
conjugate at the site of tumor. Previous reports showed that 16-20 days from the preoperative MAb injection were required to reach this concentration. In our series 21 and 24 days were needed for primary and recurrent tumors, respectively. The use of FAb’2 anti-CEA MAb represented a significant technical improvement for RIGS. A high concentration of the antibody was taken up into the tumors within 9 days with a high Tu/NT ratio so that small quantities of tumor could be intraoperatively recognized. Anti-CEA MAb was taken up by nearly all primary and recurrent resected tissues with a good differentiation in as little as 9 days. At this interval we localized nearly all metastatic and recurrent sites while one third of primary tumors failed to be correctly detected by RIGS. Here again, as suggested for MAb B72.3 and TAG-72 antigen, the non-homogeneous antigenic expression of primary tumors may be claimed to be responsible for the RIGS detection failures. Opposite, it has to be pointed out that intraoperatively localizing primary tumor sites is far less important than the detection of occult liver or lymph node metastases and subclinical occult recurrences. By using anti-CEA MAb surgical decision making changes, based on the localiza-
tion of occult tumor sites, was shown to be possible in 1 patient operated for a recurrent tumor. False positive results observed in patients operated at 6 and 12 days from the MAb injection were always related to lymph node or granulomatous tissue; detailed long term followup of these patients has been undertaken in order to better assess the clinical meaning of this information. The relative low incidence of HAMA formation in these patients also favors the use of FAb’2 instead of entire IgGl in clinical trials with murine antibodies, especially when planning further immunotherapy .
CONCLUSIONS RIGS technique was validated to be a safe and useful diagnostic technique. The encouraging findings of these early investigations show that using 125-1MAbs reactive with TAA provides useful information for the surgical management of colorectal cancer. Results to date indicated that this approach may provide new and early diagnostic information therefore enhancing surgical radicality . While MAb B72.3 appears to be of great utility in patients with recurrent tumors, we believe that a preop-
Radioimmunolocalization of Colorectal Cancer
erative bioptic staining for TAG-72 antigen expression should be mandatory for primary tumors, especially for Stages 1-11. The delay of surgery of up to 3 weeks may be a serious disadvantage particularly for primary tumors or there may be a risk of intestinal obstruction. The use of anti-CEA fragments confirms previous immunoscintigraphic reports on their effect on increasing diagnostic utility both in primary and recurrent tumors. RIGS tumor detection reached high levels. Their use reduced to 9 days the interval from injection to surgery. Finally, we could demonstrate a lower immunogenic effect of fragments on HAMA titers increase and biodistribution data may suggest a helpful use of MAb F023C.5 for therapy because of their selective targeting of recurrent and metastatic tumor sites. The accuracy of both antibodies in localizing recurrent tumor tissues may also be of great importance in preventing unnecessary aggressive enlarged procedures and in recognizing inflammatory rather than neoplastic tissue. Therefore the benefits of RIGS in increasing long term survival and in reducing local recurrence may only be assessed by long term follow-up protocols.
REFERENCES I . Fritzberg AR, Beminger RW, Hadley SW, Webster DW: Approaches to radiolabeling of antibodies for diagnosis and therapy of cancer. Pharm Res 5:325-332, 1988. 2. Mach JP, Carrel S , Forni M, Ritschard J, Donath A, Alberto P: Tumor localization of radiolabeled antibodies against carcinoembryonic antigen in patients with carcinoma. N Engl J Med 3 0 3 5 1 0 , 1980. 3. Patt YZ, Lamki LM, Haynie TP, Unger MW, Rosenblum MG, Shirkoda A, Murray JL: Improved tumor localization with increasing dose of Indium- 111-labeled anti-carcinoembryonic antigen monoclonal antibody ZCZ-025 in metastatic colorectal cancer. J Clin Oncol 6:1220-1230, 1988. 4. Maguire RT, Schmelter RF, Pascucci VL, Conklin JJ: Immunoscintigraphy of colorectal adenocarcinoma: results with sitespecifically radiolabeled B72.3 (”’-In-CYT-103). Antibody Immunoconj Radiopharm 2:257-269, 1989. 5. Holying T, Schlag P, Georgi P: Current status of immunoscintigraphy in colorectal cancer; results of 5 years’ clinical experiences. Eur J Surg Oncol 16:312-318, 1990. 6. Martin EW, Tuttle SE, Rousseau M, Mojzisik CM, O’Dwyer PJ, Hinkle GH, Miller EA, Goodwin RA, Oredipe OA, Barth RF, Olsen JO, Houchens D, Jewell SD, Bucci DM, Adams D, Steplewski Z, Thurston MO: Radioimmunoguided surgery: intraoperative use of monoclonal antibody 17-IA in colorectal cancer Hybridoma 5:97-108, 1986. 7. Sikle-Sananello BJ, O’Dwyer PJ, Mojzisik C, Tuttle SE, Hinkle GH, Russeau M, Schlom J, Colcher D, Thuston MO, Nieroda C,
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Sardi A, F a r m W, Martin EW: Radioimmunoguided surgery using the monoclonal antibody B72.3 in colorectal tumors. Dis Colon Rectum 30:761-764, 1988. O’Dwyer PJ, Mojzisik C, Hinkle GH, Russeau M, Olsen J, Tuttle SE, Barth RF, Thurston MO, McCabed DP, Farrar WB, Martin EW: Intraoperative probe-directed radioimmunodetection using a monoclonal antibody. Arch Surg 121:1391-1394, 1986. Martin EW, Mojzisik C, Hinkle GH, Sampsel J, Siddiqi A, Tuttle S, Sikle-Santanello B, Colcher D, Thurston MO, Bell JG, Farrar W, Schlom J: Radioimmunoguided surgery using monoclonal antibody B72.3. Am J Surg 156:38&392, 1988. Oredipe OA, Barth RE, Tuttle SE, Adams DM, Sautis 1, Bucci DM, Mojzisik CM, Hinkle GH, Jewell S , Steplewski Z, Thurston MO, Martin EW: Limits of sensitivity for the radioimmunodetection of colon cancer by means of a hand held gamma probe. Nucleic Med Biol 15595-603, 1988. Tuttle SE, Jewell SD, Mojzisik C, Hinkle GH, Colcher D, Schlom J, Martin EW: Intraoperative radioimmunolocalization of colorectal carcinoma with a hand held gamma probe and MAb B72.3: comparison of in vivo gamma probe counts with in vitro MAb radiolocalization. Int J Cancer 42:352-358, 1988. Badellino F, Bertoglio S , Nicolo G, Aste H, Schenone F, Catturich A, Canavese G, Percivale P: The intraoperative detection of colorectal cancer by radiolabeled B72.3 125-1 monoclonal Antibody (MAb) and radioimmunoguided (RIGS-T‘’)surgery. J Exp Cancer Res 8(s):124-125, 1989. Bertoglio S , Percivale P, Bartolomeo S, Schenone F, Meszaros P, Conzi GF, Spina B, Nicolo G, Spagnolo W, Badellino F: Radioimmunoguided surgery (RIGS-’”) for colorectal cancer with B72.3 125-1 monoclonal antibody. In Klapdor R (ed): “Recent Results in Tumor Diagnosis and Therapy.” Munchen, W. Zuckschwerdt: Verlag, 1990, 437443. Sutherland R, Buchegger F, Schreyer M, Vacca A, Mach JP: Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids. Cancer Res 47: 1627-1 633, 1987. Jain RK, Baxter LT: Mechanisms of heterogeneous distribution of monoclonal antibodies and other macromolecules in tumors: significance of elevated interstitial pressure. Cancer Res 48:70227032, 1988. Goldemberg DM, Goldemberg H, Sharkey RM, Higginbotham Ford E, Lee RE, Swyne LC, Burger KA, Tsai D, Horowitz JA, Hall TC, et al: Clinical studies of cancer radioimmunodetection with carcinoembryonic antigen monoclonal antibody fragments labeled with 123-1 or 99nTc. Cancer Res 50:909~-921s, 1990. Colcher D, Horan Hand P, Nuti M, Schlom J: A spectrum of monoclonal antibodies reactive with human mammary tumor cells. Proc Natl Acad Sci USA 78:3199-3203, 1981. Riva P, Paganelli G , Cacciaguerra G , Gentili G, Marri L, Tison V, Marangolo M, Amadori D: Antibody guided tumor detection using 131-1 labeled F(ab’)2 fragments of an anti-CEA MoAb. Br J Cancer 52:649, 1985. Thor A, Ouchi N, Szatack C: Distribution of oncofetal antigen tumor associated glycoprotein 72 defined by monoclonal antibody B72.3. Cancer Res 46:3118-3124, 1986. Listrom MB, Little JV, McKinley M, Fenoglio-Preiser CM: Immunoreactivity of tumor associated glycoprotein (TAG-72) in normal, hyperplastic and neoplastic colon. Hum Pathol 2 0 : 9 9 4 1000, 1989.
(1991)
Perioperative Radioimmunolocalization of Colorectal Cancer by Radiolabeled Monoclonal Antibodies FAUSTO BADELLINO, MD, PlERLUlGl PERCIVALE, MD, PAOLO MESZAROS, MD, MARC0 GIPPONI, MD, STEFAN0 BARTOLOMEO, MD, GUlDO NICOLO, MD, BRUNO SPINA, MD, FEDERICO SCHENONE, MD, AND SERGlO BERTOGLIO, MD From the Division of Surgical Oncology (F.B., P.M., M.C., S.Ba.), Service of Anatomy and Pathologic Cytohistology (C.N., B.S.), Service of Nuclear Medicine (F.S.), National lnstitute for Cancer Research (/ST) and the lnstitute of Clinical and Experimental Oncology, The University of Cenoa (P.P., S.Be.), Cenoa, ltaly
KEYWORDS:murine monoclonal antibodies, tumor associates antigens, gamma-emitting radionuclides
INTRODUCTION antigen, leading to the detection of subclinical tumor The hybridoma technology for manufacturing mono- deposits that may alter surgical approach in approxiclonal antibodies (MAbs) has made available a large mately one third of patients. This technique has been number of biocompatible murine MAbs that can be used expected to enhance intraoperative staging and surgical to target specific tumor associates antigens (TAA) for radicality, therefore improving long term prognosis in both diagnostic and therapeutic purposes. The potential colorectal cancer patients [&13]. The authors present their experience on the use of diagnostic utility of MAbs has been facilitated by techintraoperative immunodetection and staging of colorectal nological advances that permit their coupling with diftumors by RIGS and the use of two different murine ferent gamma-emitting radionuclides. Tumor-localizing "' 1 B72.3 and IgGl and I2'-I F023C5 FAb'2, a MAbs: techniques by external gamma camera imaging equipMAb that reacts with CEA antigen. ment, the so-called immunoscintigraphy , have been expanding and extensively investigated in presurgical MATERIALS AND METHODS patients with colorectal carcinoma. Many reports have Patients shown that immunoscintigraphy , using several polyThe study, which was carried out started in October clonal MAbs such as those directed against the carcino1988 and is to be completed in July 199 1. Patients were embryonic antigen (CEA) or the TAG-72 antigen (MAb divided into 2 series and were respectively injected with B72.3), may preoperatively detect 70435% of primary the 2 mentioned MAbs. Preoperative diagnosis and and recurrent tumor sites [ 1-41. Provided this encouragstaging of the patients included colonoscopy with biopsy ing diagnostic results, the preoperative detection of and/or double contrast barium enema, abdomino-pelvic unsuspected lymph node involvement or synchronous computerized tomography (CT), and ultrasonography lesions in patients with colon cancer, therefore altering (US) scan and measurement of tumor marker levels; extra surgical approach and improving long term prognosis, is abdominal disease was excluded by conventional diagstill controversial [ 5 ] . nostic procedures. Intraoperative detection and staging of colorectal cancer by radiolabeled MAbs and the use of a hand-held SERIES A gamma detecting probe (GDP) is a new technique, named From October 1988 to December 1989 MAb B72.3 radioimmunoguided surgery (RIGS 'rh'), proposed in 1986 was injected in 15 surgical patients with primary or by Martin et al. [6]. This method is based on a preoperrecurrent colorectal cancer. Eight patients had primary MAbs which selectively bind a ative injection of 125-1 TAA at the site of tumor. At surgery, the abdominal Accepted for publication May 30, 1991. cavity is searched for selective radioactivity emission Address reprint requests to Dr. Fausto Badellino, Division of Surgical Oncology, National Institute for Cancer Research, IST Genoa, Viale from radiolabeled tumor tissue. Encouraging results have Benedetto XV 10, 16132 Genoa, Italy. been obtained in colorectal surgery with the use of "'-1 The research in this manuscript was supported by grants of AssociaMAb B72.3, a murine IgG1 that reacts with TAG-72 zione Italiana per la Ricerca sul Cancro (A.I.R.C.). 0 1991 Wiley-Liss, Inc.
Radioimmunolocalizationof Colorectal Cancer
87
tumors and 7 had a recurrence; 7 patients were male with TABLE I. Intervals to Surgery in 28 Patients Operated a mean age of 62.6 years (range 32-72) and 8 were for Primary and RecurrentColorectal Cancer With RIGS female with a mean age of 59.3 years (range 41-73). and I2'-I Anti-CEA FAb2 Patients were eligible for surgery as soon as external Primary Recurrent tumors tumors Overall precordial counts performed with the GDP were less than 20 counts/2 seconds. 6 Days 2 4 6
SERIES B
9 Days 12 Days 15 Davs
4 3 3
6 3 3
10 6 6
The aims of study series were to define the best interval from the preoperative injection of anti-CEA FAb'2 (F023C5) to surgery, evaluate the potential diagnostic and staging utility, and to assess the patterns of biodisThree days before the injection series A and B patients tribution of the antibody for its possible future use for therapy. Being FAb'2 proteins with a lower molecular received a supersaturated solution of potassium iodide weight they should be expected to target tumors in a (SSKI) 10 drops/3 times per day (500 mg of potassium shorter period of time and clear faster from the blood pool iodide) to prevent thyroid uptake of 125-1. Infusion was [ 1&16]. These specific features should allow surgery to performed in 5 minutes, checking vital signs for possible be performed in a shorter period of time from the anaphylactic reaction. injection of the MAb. The phase 1-11 study design GDP and Microprocessor Control Unit provided single series that included at least 6 patients who were operated on at different intervals; in particular, 6, 9, GDP and Control Unit (Neoprobe 1000 SYSTEM) 12, and 15 days from the preoperative injection of the were provided by Neoprobe Co. (Columbus, Ohio, radiolabeled F023C5. USA). The probe is stainless steel and gas sterilizable, From January 1990 to July 1991 at the Division of measuring 18 cm in length with the tip angled at 120" Surgical Oncology of our Institution, MAb F023C5 was for optimal intraoperative use. It contains a 1 cm diampreoperatively injected in 28 patients with colorectal eter cadmium-telluride crystal as detector and a preamcancer. Twelve had primary cancer while 16 were plifier. Gamma emissions from a radioactive source are operated on for recurrent or metastatic tumors. Fourteen converted by the preamplifier to a digital read out on the patients were males with a mean age of 59.6 years (range portable control unit; the software of the control unit can 43-74) and 14 were females with a mean age of 56.8 also convert counts/seconds into audible signals. The years (range 41-78). Table I analyzes patterns of the sound volume can be controlled and a threshold count series related to preoperative diagnosis and timing of rate, below which no sound is produced, can be easily set. Its intraoperative use is based upon the fact that as surgery. each normal tissue is probed, a background count threshMonoclonal Antibody old can be selected that will not give auditory signals to Series A. MAb B72.3, a murine IgGl that reacts the surgeon. As the probe passes to an area of concenwith a high molecular weight glycoprotein with the tration of gamma emissions, the count rates and the sound mucin characteristic named TAG-72, was used intraop- increase, thus allowing the surgeon to quickly and eratively to detect cancer sites [17]. B72.3 was radiola- accurately locate the source of radiation. The absence of beled with 125-1 by the Iodo-Gen'" method. Unbound a bulky collimator, due to the low energy isotope used iodine was removed by gel filtration chromatography on (125-I), is a favorable feature of the technique for better Sephadex column and filtrated through a 0.22 micron and faster scanning of the whole abdomen during sursterile filter before preparation for clinical use. Each gery. patient received before surgery an intravenous (i.v.) dose Method of RIGS of 1 mg MAb/l.5 mCi of 125-1 (Surgimab R3@,Sorin At surgery, after the assessment of levels of residual Biomedica, Italy). Series B. MAb F023C5, a murine FAb'2 that reacts background radioactivity, the GDP was used to search for with CEA antigen, was injected to intraoperatively detect primary, metastatic, or recurrent tumor location, and to cancer sites [ 181. F023C5 was radiolabeled with 125-1by survey the entire abdomen with particular attention to the the Iodo-Gen method. Unbound iodine was removed by tissues adjacent to the tumor, the lymph nodes and the gel filtration chromatography on Sephadex column and liver. All tissues with a tumor-to-normal tissue (Tu/NT) filtrated through a 0.22 micron sterile filter before count ratio > 1.75: 1 were considered to be positive for preparation for clinical use. Each patient received before tumor location and resected, if possible, or biopsied. surgery an i.v. dose of 1 mg MAW1.5 mCi of 125-1 Tumor resected specimens as well as similar non malignant control tissues, whenever available, were given to (Surgimab R 2 9 .
88
Badellino et al. TABLE 11. RIGS Diagnostic Results of Primary Tumors Operated After the Injection of 125-1B72.3 MAb
Site Primary cancer Stage 1-11 Stage 111-IV Liver metastases Lymph node metastases Total
True Positive (%)
False Positive
112 (50) 517 (71.4) 7/10 (70) 517 (71.4) 18/26 (69.23)
0 0 0 0 0126
False negative Tag AgSa
(%I
False negative Tag Ag-a (%)
0 1/7 (14.28) 1/10 (10) 2/7 (28.57) 4/26 (15.38)
1/2 (50) 117 (14.28) 2/ I0 (20) 0 4/26 (15.38)
"Tag Ag+ and - indicate tumor sites staining positive and negative for TAG-72 at immunohistochemical analysis.
TABLE 111. RIGS Diagnostic Results of Recurrent Tumors Operated After the Injection of lZ5-I B72.3 MAb
Site
True positive (%)
False positive
Local recurrence Liver metastases Lymph node metastases Other Total
11/13 (84.6) 811 1 (72.7) 6/8 (75) 3/4 (75) 28/36 (77.7)
0 0 0 0 0
False negative Tag Ag+a
(%I 2/13 (15.38) 3/11 (27.3) 2/8 (25.00) 1/4 (25.00) 8/36 (22.2)
False negative Tag Ag-a
0 0 0 0 0
aTag A g f and - indicate tumor sites staining positive and negative for TAG-72 at immunohistochemical analysis.
the pathologist for routine examination and immunohistochemical assay for TAG-72 and CEA antigen expression, respectively, for series A and B specimens. Preinfusion human antimouse monoclonal antibody (HAMA) titers levels were available for all patients; subsequent samples were obtained at 4 and 8 weeks after the injection of B72.3 and F023C5 MAbs.
RESULTS Series A (MAb B72.3 anti-TAG-72) No adverse reactions were observed in patients injected with B72.3 MAb. Patients underwent surgery 21.12 ? 6.48 and 24.2 2 7.8 days after MAb injection, respectively, for primary and recurrent colorectal tumors. Sixty-two suspected tumor sites were intraoperatively recognized by RIGS; 26 were detected in the 8 patients with primary tumor and 36 in the 7 patients with recurrences. All sites were confirmed to be neoplastic tissue by histological examination. Tables I1 and 111 analyze the clinical results of intraoperative radioimmunodetection for primary and recurrent tumors. Results of patients with primary tumors were divided into 2 stage groups based on the 4th Ed. TNM classification: Stage 1-11, and stage 111-IV.
RIGS was able to detect 50% of sites in Stage 1-11 primary cancer stage, and (71.4%) in stage 111-IV: 4/26 (15.38%) sites expressing TAG-72 have failed to be detected by RIGS. Sites not expressing the antigen failed to be detected with a similar average of 15.38%. It is important to note that 2 out of these 4 lesions (50%) not expressing the antigen were observed at the site of the primary colonic lesion. In recurrent cancer we were able to localize 28/36 (77.7%) of cancer sites. All the false negative sites in this group expressed TAG-72 antigen. We finally evaluated the utility of the RIGS system in surgical decision making changes. In 217 patients with recurrent or metastatic disease the probe was able to identify subclinical deposits of tumor that were not otherwise evident either preoperatively or intraoperatively; 1 of these localizations was a perianastomotic recurrence and 1 was a peri-aortic lymph node metastasis. As a summary of this additional information, RIGS system modified the surgical approach in 1/15 (6.6%) patients; in particular, surgical decision making was changed in 1/7 (14.28%) patients with recurrent tumors and never during surgery for primary colonic lesions. Both patients underwent laparotomy only on the basis of preoperative high CEA levels. Serum
Radioimmunolocalization of Colorectal Cancer
HAMA levels increased at least twice the baseline within 8 weeks from the MAb injection in 7 (46%) patients.
Series B (MAb F023C5 anti-CEA) No adverse reactions were observed in patients injected with anti-CEA MAb. Table IV shows potential diagnostic features of RIGS in patients operated at different intervals. Forty-nine suspected tumor sites were intraoperatively recognized; 42 of them (85.7%) were confirmed to be tumor by histology. All recurrent and metastatic sites as well as 92% of primary resected tumor stained positively for CEA antigen at immunohistochemical examination. We correctly localized 5/7 (71.4%) sites in patients operated at 6 days, 18/22 (81.8%) in patients operated at 9 days, 416 (66.6%) in patients operated at 12 days, and 2/7 (28.6%) in patients operated at 15 days interval from MAb injection. In patients operated at 9 days we localized 2 subclinical tumors out of 22 detected sites (9.1%). One was a lymph node in 1 patient operated for a local recurrence while the second was a liver metastasis of 15 mm of diameter that was resected following RIGS indication, thus altering surgical decision making. We observed 7 (14.29%) false positive localizations; 3 were enlarged reactive non neoplastic lymph nodes expressing CEA antigen while 4 sites resulted to be granulomatous nodules located in the pelvic peritoneum. Three localizations occurred in patients operated at 6 days (30%) and 4 in patients operated at 12 days (40%) from the MAb injection. Table V analyzes patterns of biodistribution of the antibody. As a confirmation of diagnostic findings, the best intraoperative tumor/normal tissue gamma emittence ratio and radiolocalization index were observed 9 days after the MAb injection with values of 3.89/1 and 7.01/1, respectively. From these data it appears that before 9 days from the MAb injection, the blood pool radioactivity is too high covering probably the MAb related radioactivity at tumor sites. Opposite, after 12 days, difference on radioactivity from tumor and normal tissues may be weak to be certainly recognizable by RIGS. Table VI shows clinical diagnostic results as well as biodistribution patterns observed for differentiated tumor tissues in patients operated at 9 days when the best results were obtained. Anti-CEA F023C5 MAb made recognizable by RIGS recurrent tumors in 88.8% sites, liver metastases in 100% sites, lymph node metastases in 87.5% sites, while primary tumor sites, regardless of stage, were localized in 62.5% of cases. Thus, a high intraoperative diagnostic and staging potential of RIGS for advanced stages of colorectal cancer was demonstrated. Postinjection HAMA titers increased in 4 (14%) pa-
89
TABLE IV. Diagnositc Results of RIGS in 28 Patients Operated at Different Intervals After Injection of lz5-I F023C5 Anti-CEA FAbZ MAb Interval to surgery
RIGS positive detections RIGS negative detections False positive detections Subclinical tumor detections Surgical decision making changes
6 Days
9 Days
12 Days
15 Days
(%)
(%)
(%)
(%)
511
18/22 (81.8) 4/22 (1 8.2) 0122
416 (66.6) 216 (33.3) 4/10
211 (28.6)
(71.4)
211 (29.6) 3/10 (30) 0 0
517
(71.4) 017
(40)
2/22 (9.1) 1/22 (4.6)
0
0
0
0
tients; only 3 (10.2%) had levels higher than at least twice the baseline.
DISCUSSION The encouraging findings of the early investigational reports on the use of radiolabeled MAbs in the preoperative evaluation and follow-up in patients with colorectal cancers suggested that their intraoperative use by means of RIGS may alter surgical approach, therefore increasing patient's overall survival [&9, 12-13]. Results reported by Martin et al. with this technique concluded that RIGS was more accurate in localizing clinical obscure metastases than other methods. In patients with recurrent colorectal cancer, unknown tumor sites were detected in almost 20% of the cases thus altering surgical approach in nearly all of them [6-11]. B72.3 The results of our first study with the use of 125-1 MAb demonstrated the safely of i.v. administration to humans of murine antibody. Primary colorectal carcinomas were detected with an average of 50% and 7 1.4% in Stages 1-11 and 111-IV group, respectively. In recurrent cancers tumor sites were located in 8 1.9% of cases. Even the localization of subclinical occult tumor sites leading to intraoperative changes on surgical decision making occurred only for recurrent tumors (28.5%). Finally, we were able to demonstrate that RIGS with 125-1 B72.3 MAb was very encouraging in terms of clinical utility for advanced and recurrent tumors. Opposite, for primary cancer the non homogeneous patterns of TAG-72 antigen expression, 30% of which stained negatively at the immunohistochemical assay, may be responsible for the worse obtained results [19,20]. Nevertheless, it has to be pointed out that postinjection elevated HAMA titers were found in 46% of patients. Another controversial finding, depending on the use of a IgGl MAb, may be the adequate period of time needed to obtain an optimal concentration of the radioimmuno-
90
Badellino et al. TABLE V. Biodistribution Patterns of lz5-I Conjugated F023C5 Anti-CEA FAbZ MAb Injected in 28 Patients herated With RIGS at Different Intervals From the MAb Injection ~~
6 Days
Interval to surgery Intraoperative Tu/NT ratios (GDP counts 2”) Radiolocalization index Mean radioactivity NT (cpm/O.l g) Mean radioactivity Tu (cpm/O.l g) Whole blood pool radioactivit; (cpm/O.l ml)
9 Days
12 Days
15 Days
2.42
3.89
3.47
1.45
6.28 651.9
7.0 1 251.0
3.49 204.2
1.93 58.97
1,473.8
1,730.5
708.6
179.6
1,711.1
892. I
698.2
163.3
Abbreviations: Tu, tumor tissue; NT, adjacent normal tissue; cpm, gamma counts per minute.
TABLE VI. Clinical Results and Tissue Radioactivity of Different Tumor Sites in Patients Operated 9 days After Anti-CEA lZ5-IF023C5 MAb Primary cancer
Local recurrence
Liver metastases
Lymph node metastases
62.5% 1.75
88.8% 4.92
100% 2.26
87.5% 2.82
2.5 1
4.75
8.02
5.88
~
% of detections Intraoperative Tu/NT ratio (2” counts) Radiolocalization index (0.1 g Tu/NT cpm)
Abbreviations: Tu, tumor tissue; NT, adjacent normal tissues; cpm, gamma counts per minute.
conjugate at the site of tumor. Previous reports showed that 16-20 days from the preoperative MAb injection were required to reach this concentration. In our series 21 and 24 days were needed for primary and recurrent tumors, respectively. The use of FAb’2 anti-CEA MAb represented a significant technical improvement for RIGS. A high concentration of the antibody was taken up into the tumors within 9 days with a high Tu/NT ratio so that small quantities of tumor could be intraoperatively recognized. Anti-CEA MAb was taken up by nearly all primary and recurrent resected tissues with a good differentiation in as little as 9 days. At this interval we localized nearly all metastatic and recurrent sites while one third of primary tumors failed to be correctly detected by RIGS. Here again, as suggested for MAb B72.3 and TAG-72 antigen, the non-homogeneous antigenic expression of primary tumors may be claimed to be responsible for the RIGS detection failures. Opposite, it has to be pointed out that intraoperatively localizing primary tumor sites is far less important than the detection of occult liver or lymph node metastases and subclinical occult recurrences. By using anti-CEA MAb surgical decision making changes, based on the localiza-
tion of occult tumor sites, was shown to be possible in 1 patient operated for a recurrent tumor. False positive results observed in patients operated at 6 and 12 days from the MAb injection were always related to lymph node or granulomatous tissue; detailed long term followup of these patients has been undertaken in order to better assess the clinical meaning of this information. The relative low incidence of HAMA formation in these patients also favors the use of FAb’2 instead of entire IgGl in clinical trials with murine antibodies, especially when planning further immunotherapy .
CONCLUSIONS RIGS technique was validated to be a safe and useful diagnostic technique. The encouraging findings of these early investigations show that using 125-1MAbs reactive with TAA provides useful information for the surgical management of colorectal cancer. Results to date indicated that this approach may provide new and early diagnostic information therefore enhancing surgical radicality . While MAb B72.3 appears to be of great utility in patients with recurrent tumors, we believe that a preop-
Radioimmunolocalization of Colorectal Cancer
erative bioptic staining for TAG-72 antigen expression should be mandatory for primary tumors, especially for Stages 1-11. The delay of surgery of up to 3 weeks may be a serious disadvantage particularly for primary tumors or there may be a risk of intestinal obstruction. The use of anti-CEA fragments confirms previous immunoscintigraphic reports on their effect on increasing diagnostic utility both in primary and recurrent tumors. RIGS tumor detection reached high levels. Their use reduced to 9 days the interval from injection to surgery. Finally, we could demonstrate a lower immunogenic effect of fragments on HAMA titers increase and biodistribution data may suggest a helpful use of MAb F023C.5 for therapy because of their selective targeting of recurrent and metastatic tumor sites. The accuracy of both antibodies in localizing recurrent tumor tissues may also be of great importance in preventing unnecessary aggressive enlarged procedures and in recognizing inflammatory rather than neoplastic tissue. Therefore the benefits of RIGS in increasing long term survival and in reducing local recurrence may only be assessed by long term follow-up protocols.
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