Immunoscintigraphy of melanoma using radiolabelled antibodies: Pilot, prospective studies and Italian multicenter clinical trials

Nucl. Med. Biol. Vol. 16, No. 6, pp. 629-632, 1989 Inr. J. Radiat. Appl. Instrum. Part B Printed in Great Britain. All rights reserved

0883-2897/89 $3.043+ 0.00 Copyright 0 1989 Pergamon Press plc

Immunoscintigraphy of Melanoma Using Radiolabelled Antibodies: Pilot, Prospective Studies and Italian Multicenter Clinical Trials G. L. Nuclear Medicine

BURAGGI Division,

and M. GASPARINI

Istituto

Nazionale

Tumori, Milano, Italy

The studies on radioimmunodetection of malignant melanoma reported are based on the use of the 225.283 monoclonal antibody against a high molecular weight melanoma associated antigen, an IgG2a isolated and characterized by Ferrone et al. The strategy followed by us to check the characteristics and the behaviour of this antibody and to verify its possible use in clinical praxis, includes three groups of trials performed from 1982 to 1987. A pilot study was performed on 42 patients with 74 melanoma lesions, a multicenter study was realized, with the collaboration of 10 Italian nuclear medicine centers, on 254 patients with 539 lesions and prospective study performed on 44 patients with 54 suspected regional lymph nodes involvements. High sensitivity and specificity were obtained especially with the use of technetium compound which also allowed the attainment of very high predictive values of positive tests. No adverse reactions were noticed.

Introduction of malignant melanoma represents a consolidated approach in clinical oncology since various antigen-antibody systems and different techniques were proposed to apply the method in clinical practice. A great variety of melanoma associated antigens have been described (Hellstrom, 1984). Among the most-used to perform radioimmunodetection we can point out the p97 and two high molecular weight antigens (HMW-MAA) described by Reisfeld (1987) and Ruberto (1982). p97 is a 97,000 molecular weight glycoprotein, synthesized by melanoma cells; it appears to form an integral part of cell membrane (Brown et al., 1981a). It is expressed at very low concentrations in normal adult tissues but has been detected in substantial amounts in fetal colon (Brown et al., 1981b). The high molecular weight melanoma antigen described by Reisfeld et al. is a glycoprotein with a molecular weight of 250 kDa recognized by the 9.2.27 monoclonal antibody. This antigen is associated with a chondroitin sulfate-like proteoglycan in melanoma cells (Bumol, 1982). The HMW-MAA described by Ferrone et al. (1989) is made up of two non covalently-associated glycopolypeptides with apparent molecular weights of 280 kDa and more than 440 kDa respectively. It is Immunoscintigraphy

expressed by proliferating melanocytes, mevus cells and malignant melanoma cells. The trials on radioimmunoimaging of malignant melanoma reported in this paper are based on the use of 225.28s antibody against the HMW-MAA, an IGg2a isolated and characterized by Ferrone et al. (1982). A pilot study (Buraggi et al. 1984a,b, 1985, 1986) was performed with the aim of verifying the behaviour of the 225.288 MoAb in tumor detection. A further step was to confirm our results and to compare by means of a multicenter clinical trial (Siccardi et al., 1986) different experiences in a large series of patients from several Italian nuclear medicine centers. Finally, a prospective study was performed to control the required and appropriate characteristics of the antigen-antibody system in clinical practice (Buraggi et al., 1986b, 1987).

Materials and Methods The anti-HMW-MAA MoAb 225.288 was purified from ascitic fluid and F(ab’), fragments were generated by digestion of purified IgG using pepsin. F(ab’), fragments were separated from undigested IgG and other digestion products by Sephadex G200 SF chromatography. 629

G. L. BURAGCI and M. GASPARINI

630

In the pilot study five different immunoreagents were employed as radiolabelled IgG and F(ab’), and namely 13’1-MoAb, ‘3’I-F(ab’),, “‘In-F(ab’), ““Tc-F(ab’),. The ,purified MoAb 225.288 and F(ab’), fragments were radiolabelled with “‘I (ORISCEA, Saclay, France) or with “‘I (IRE, Fleurus, Belgium) at 148-185 GBq/mg using the iodogen method (Buraggi et al., 1985). “‘In (ORIS-CEA, Saclay, France) was bound to F(ab’), fragments by means of the cyclic anhydride of diethylenetriamine pentaacetic acid (DTPA) (Buraggi et al., 1984a). The DTPA-bound antibodies were separated from unbound ones using gel filtration and then incubated with “‘In chloride (185 MBq). Unbound indium was separated from the conjugated compound by gel filtration on Biogel P6. F(ab’), fragments were labelled with WmT~ after their lyophilization. Thirty minutes before injection lyophilized F(ab’), fragments (350 pg) were dissolved in 3-5 mL of a sterile solution of sodium [9ymTc]pertechnetate (ORIS-CEA, Saclay, France). After 15 min incubation, free 99mTc was separated from the antibody-bound 99mTc by ion exchange chromatography through a sterile Sephadex DEAEA 25 column (Callegaro et al., 1983). The radiolabelled antibodies were filtered through a 0.22pm filter. Sterility and pyrogen tests were performed by standard techniques (Buraggi et al., 1984a). Before iv. injection of monoclonal antibodies, patients were tested for hypersensitivity to mouse IgG with an intradermal injection of 2-3 pg antibody. Routine clinical analyses to evaluate hematopoietic, hepatic, pancreatic and renal functions were performed before and after injection. In patients injected with 13’1-or ‘231-labelled antibodies the thyroid was previously blocked by potassium iodide administration (120 mg/per day for 10 days, starting 3 days before injection). Patients injected with y9”Tc-labelled antibodies were treated with 400mg potassium perchlorate on the day of F(ab’), injection. A high specific activity was used, as a rule reaching 1900 MBq/mg or more. In most patients, 15-100 pg of antibody were administered, and no more than 1.5 mg used in any single case. In the multicenter trial only the indium and the technetium compounds were used. The prospective study was performed with the technetium compound.

Results The pilot study, performed on 42 patients with 74 melanoma lesions, permitted us to evaluate the principal parameters involved in radioimmunodetection such as the behaviour of immunoreagents in the tumor, the dosimetry, the choice of the characteristics of the reagent itself.

For example, the kinetic studies of various radiocompounds in the whole body and in the most important organs showed a differentiated pattern between the antibody labelled with iodine and the other compounds. Indium showed, for instance, a high liver and bone marrow uptake, whereas technetium showed the most rapid disappearance from the whole body and from liver, spleen, bone marrow and kidneys. This behaviour was explained by successive studies which proved that the labelling method utilized could break the disulphide bridges of the fragments and that WmTclabelled Fab’ fragments were produced to a greatest extent. These differences affected the doses absorbed by the patients. The most favourable dosimetric conditions were obtained with the technetium compound. No difference in tumor uptake were observed among the five different tracers used. In the positive cases, tumor uptake appeared immediately in the first minutes after the injection. This rapid accumulation probably correlates with a high degree of vascularization of lesions. The specificity of the antibody uptake was investigated by studying the tumor/background ratio of the radioactivity in four patients simultaneously injected with two different F(ab’),: the specific one radiolabelled with “‘In or 99mTc,and an irrelevant F(ab’), labelled with iodine. A clear difference was obtained between the uptake of the two antibodies, measured as tumor/background (T/B) ratio in each patient. The maximum value of T/B was reached at different times after injection in each patient. This ratio often showed differences in different tumor localizations. The kinetic studies showed that the best conditions for performing immunoscintigraphy, namely the highest T/B ratio values, were reached, as a rule, 6-24 h after injection. Evaluation of the antibody distribution and consideration of the dosimetric measurements indicated that WmT~was the most reliable tracer for immunoscintigraphy. Moreover, the 99mTc emission energy is easily detectable by y camera. Therefore, a lyophilized preparation of F(ab’), fragments was studied to assemble a labelling procedure kit for every nuclear medicine department. The results obtained by this pilot study are summarized in Table I. Seventy-three percent of all examined lesions were imaged; higher sensitivity was obtained with the technetium (85.7%) and indium compounds (76.7%), whereas with the different preparations of iodine poor results were observed. In every case no false positive results were noted. These data gave us the possibility to extend the study to other nuclear medicine centers adopting a well defined protocol. From November 1983 to April 1985, 254 melanoma patients with 539 lesions were investigated in 10 Italian nuclear medicine departments (see Siccardi er al., 1986), utilizing only technetium and

Immunoscintigraphy of melanoma

631

Table I. Incidence of positivity obtained in 42 patients with 74 lesions by immunoscintigraphy with 225.28s antimelanoma MoAb and F(ab’), fragments labelled with different radioisototxs Lesions Liver Lungs Bone Lymphatic nodes Skin Abdomen Total lesions *“‘I-MoAb,

“‘I-F(ab’),,

Iodine* pas/tot

RmTc pas/tot

“‘In pas/tot

Total pas/tot

216 -

l/2

415 l/l2 I3123 (56.5%)

,373 415 O/l 18/21 (85.7%)

O/3 317 l/l 7’l7 12112 23/30(76.7%)

O/3 6115 l/l 23/25 23129 O/l 54/74(73%)

‘*‘I-F(ab’),

indium compounds. The results of this multicenter clinical study are reported in Table 2. The overall sensitivity obtained was 70%, with a significant difference between the two immunoreagents: 59% for indium and 74% for technetium. Furthermore, the higher positivities were obtained for localizations in lymph nodes, abdomen, brain and bone, whereas skin and especially liver localizations showed the lowest sensitivity. In order to assess the clinical usefulness of melanoma immunoscintigraphy, a prospective study was started with patients with early stage disease. The method could be very useful clinically if applicable in patients with a good chance of efficacious therapy. The regional extension of primary melanoma of the trunk and arms may be difficult to evaluate, axillary lymphnodes are frequently swollen without being involved in the invasive process. Forty-four patients with, or operated for, primary melanoma on the trunk and arms with axillary or cervical lymph node enlargement were investigated. The results are described in Table 3. The sensitivity of immunoscintigraphy was 76%; the specificity was 100%. The predictive value was 100% for the positive and 78% for the negative test. Comparing the results of the clinical examination we obtained 86% for the positive test and 84% for the negative test.

Table 2. Results of Italian multicenter studv of immunoscintigraphy with radiolabelled monoclonal antibodies in 254 patients with 539 melanoma lesions (Siccardi er 01.. 1986)

Isototxs Anatomic site

*-k

Liver Lungs Bones Brain Superficial lymph nodes Deep lymph nodes Skin Others Total %ISG-positive lesions

1l/18* 30146 39153 13/17 93/l 14 24130 55178 I8124 2831380 74

“‘In 3/10 S/l3 IO/IS 617 36/53 315 23149 517 94/l 59 j9

*Number of positive images/total number of tumor lesions.

Discussion These studies demonstrate that 225.283 MoAb is a suitable reagent for performing immunoscintigraphy in malignant melanomas. Of the five radiophannaceuticals obtained with this antibody, the whole MoAb shows a less favourable distribution than the F(ab’& fragments. In fact, a higher uptake was evident in different abdominal organs and in bone marrow. This behaviour leads to an unfavourable dosimetry. Among the immunoreagents labelled with different radioisotopes, the 99mT~ compound gave the best results. The favourable dosimetry due to this compound allows the administration of higher activities. 99mTcenergy is very suitable for detection with a y camera and for single photon emission tomography. These factors explain the rate of positivity which is higher with *Tc than with other compounds. The overall positive rates of imaging were 73% in the pilot study and 70% in the multicenter study with a maximum for the 99mTccompound, corresponding to 85.7 and 74% respectively. With the prospective trial a very high specificity and predictive value of the positive test were achieved allowing a remarkable amelioration of the results obtained with clinical examinations (86%). Less favourable were the predictive values of the negative test (78% and 84% respectively). The possible clinical application of the described immunoscintigraphic method can be summarized as follows: -aid to evaluate the regional invasion of a primary melanoma -evaluation of metastatic spread in patients with a known locoregional extension -detection of unsuspected lesions -differentiation between malignant and benign lesions. Further possible applications may be the evaluation of the results of therapeutical procedures; lastly, the data obtained by this method can be used as a first approach to the development of radioimmunotherapeutical techniques.

G. L. BURAGGIand M. GASPARINI

632

Table 3. Scintigraphic results in 44 patients with 54 suspected regional lymph nodes involvement

N

Patients

Lesions

‘I-P

TN

FP

FN

44

54

22

25

0

7

Predictive value

Percent

Sensitivity

Specificity

Accuracy

Pos. test

Neg. test

76

100

87

100

78

Acknowledgements-This

study was partially supported by C.N.R. Special Project on Biomedical and Clinical Engineering and Special Project on Oncology.

Buraggi, G. L.; Turrin, A.; Cascinelli, N.; Attili, A.; Gasparini, M.; Callegaro, L.; Ferrone, S.; Seregni, E.; Bombardieri, E.; Belli, F. Radioimmunodetection of melanoma: preliminary results of a prospective study. Int.

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