European Journal of

Nuclear Medicine

Original article

European multicentre study on melanoma immunoscintigraphy by means of 99mTc-labelled monoclonal antibody fragments Antonio G. Siccardi 1, Gian Luigi Buraggi 2, Pier Giorgio Natali 3, Gian Alfredo Scassellati 4, Giovanna Viale 1, Soldano Ferrone 5, and the European Multicentre Study Group' 1 Dipartimento di Biologia e Genetica, Universitb. di Milano, via G.B. Viotti 3/5, 1-20133 Milano, Italy 2 Istituto Nazionale Tumori, via Venezian 1, 1-20130 Mi]ano, Italy 3 Istituto Tumori Regina Elena, viale Regina Elena 291, 1-00161 Roma, Italy 4 Centro Ricerche-SORIN Biomedica, Strada per Crescentino, 1-13040 Saluggia, Italy s Department of M i c r o b i o l o g y and Immunology, New York Medical College, Valhalla, NY 10595, USA 6 See Appendix Received August 16, 1989 and in revised form October 23, 1989

Abstract. A total of 493 melanoma patients were investi-

gated by 20 European nuclear medicine departments by means of the same 99mTc-labelled immunoradiopharmaceutical and the same immunoscintigraphy (ISG) protocol. (i) No chemical or clinical toxicity was detected during or following the studies. (ii) Positive results were obtained in 287/363 (79%) patients (321 carrying known lesions and 42 carrying previously occult lesions): in 231 (80%) of them, 402/402 lesions were imaged; in the remaining 56 ISG-positive patients, 108/204 lesions were imaged; in 76 patients 0/122 lesions were imaged. (iii) The fraction of melanoma lesions visualized by ISG was 510/728 (70.1%); 605 of these lesions were already documented at the time of the study, and 123 were previously occult. (iv) A total of 218 documented melanoma lesions (30%) were not visualized by ISG in 132 patients: about 70% of the ISG-negative lesions were of small size (less than 2 cm diameter). (v) The melanoma nature of 69/123 previously occult lesions was confirmed by clinical criteria and/or additional investigations in follow-up studies. The results obtained in this study are similar to those obtained in the Italian Multicentre Study which had previously been carried out with 258 melanoma patients.

Key words." Immunoscintigraphy study Eur J Nucl Med (1990) 16:317-323

Offprint requests to : A . G . Siccardi

99mTC -

multicentre

Introduction 99mTc-labelled F(ab')2 fragments of the murine monoclonal antibody (MoAb) 225.28S recognizing the human, high molecular weight, melanoma-associated antigen (HMW-MAA, expressed by more than 90% of melanoma neoplastic lesions) (Natali 1984) have been extensively used since 1983 in retrospective studies for melanoma radioimmunodetection (Buraggi et al. 1984a, b, 1985a; Mansi et al. 1984; Paganelli et al. 1986; Riva et al. 1985). Previously reported results have demonstrated fully the ability of this immunoreagent to localize melanoma metastatic foci with high specificity and sensitivity (Buraggi et al. 1984b, 1985b) and without any clinical side effects (Cascinelli et al. 1988). The results of prospective studies (Buraggi et al. 1986b, 1987) and of the Italian Multicentre Study (Siccardi et al. 1986) have confirmed the efficacy of the same immunoradiopharmaceutical. Moreover, the results of the Italian Multicentre Study have disclosed that although the performance of immunoscintigraphy (ISG) is influenced by a number of tumour variables such as anatomic localization, size, antigenic profile and clinical stage, the method is capable of detecting a significant number (about 24%) of clinically unknown lesions and, in several cases, of providing the first evidence of tumour recurrence. 99mTc-labelled 225.28S (F(ab')2 fragments have also been successfully employed in other European studies (Bockisch et al. 1989; Bfill et al. 1986; Cerny et al. 1987; Dudczak et al. 1987; Liewendahl et al. 1986; Lukac and Spaventi 1988; Rentsch etal. 1989; Scheidhauer etal. 1985) to perform ISG of primary and metastatic malig-

© Springer-Verlag 1990

318

nant melanoma lesions, with good sensitivity and specificity and without the occurrence of any adverse reactions. In particular, remarkable results have been reported for its use in malignant choroidal melanoma for diagnostic purposes (Scheidhauer et al. 1988) in comparison with ultrasound to provide "biological" confirmation of ultrasonography results (De Haro et al. 1988) and in the differential diagnosis between melanomas and other benign or non-melanomatous ocular lesions (Bomanij et al. 1987a, b, 1988; Schaling et al. 1989). These results prompted us to submit the experimental findings to another multi-evaluation analysis, by collecting the data from 20 European nuclear medicine departments involved in a multicentre study aimed at verifying the clinical usefulness of the ISG technique under the influence of different environmental circumstances in an ethnically heterogenous population of 493 patients in diagnostic centres of different academic background and employing different radiodetection equipment.

Materials and methods

the unreacted TcO4 . All these operations were performed under sterile conditions to obtain a sterile and pyrogen-free solution ready for injection. During the pretinning step, stannous chloride partially reduces the disulphide bridges between the two F(ab')2 chains, thus creating Fab' fragments with free SH residues (Rhodes et al. 1986). These residues are then preferentially and firmly bound by the reduced technetium once eluted from the generator. After the labelling step, protein-bound radioactivity was determined by high performance liquid chromatography (HPLC); 80% 85% was associated with monovalent Fab' and 15%-20% was associated with bivalent F(ab')z, while a significant amount of unlabelled F(ab')z was still present in the final radioactive preparation. No 99mTc-labelled HSA, which could be formed either by direct labelling or by transchelation, was detected. Quality control on the final product by gel filtration on Biogel P6 column (Biorad Laboratories Ltd., CA, USA) did not reveal the presence of colloidal 99mrc, while free pertechnetate was below 2% until 2 h after labelling. Immunoreactivity was determined by in vitro celt binding assay using antigen-positive COLO 38 human melanoma target cells, according to a standard protocol (Seccamani et al. 1989). The "clinical grade" of the radiolabelled preparation was assessed by standard controls according to European Pharmacopeia and ECC recommendations for murine MoAbs intended for human use.

Patients. A total of 493 informed patients, all having signed a writ-

Immunoscintigraphy protocol. The same immunoradiopharmaceuti-

ten consent form and selected according to the guidelines of Britton et al. (1989), were studied in 20 nuclear medicine departments. The clinical stage of the disease was defined according to the classification of Stehlin et al. (1963) and was distributed as follows: stage I + II, 40% ; stage III, 35% ; stage IV, 24%. Documented melanoma lesions (a total of 605, 56% of which was readily accessible, being located in the skin or in superficial lymph nodes) were present in 321 patients. In the remaining 172 "lesion-free" patients, known turnout lesions had been surgically removed.

cal and the same ISG protocol were used in all nuclear medicine departments. Routine clinical analyses to evaluate haematopoietic, hepatic, pancreatic and renal functions were performed before the administration of radioimmunopharmaceutical and were repeated 2-3 times during the subsequent 2 weeks. No adverse reactions were ever observed. The radiolabelled antibody solution (approximately 5 ml, containing 10-30 mCi of 99mTC)was injected intravenously over a 2rain period. Tissue distribution of MoAbs in vivo was analysed immediately after injection and at multiple time intervals up to 24 h by analogical and computerized scintigraphy. The optimal time for imaging, i.e. that giving the highest turnout/background ratio, was found to be between 6 and 12 h. Scans were taken by standard techniques of planar radioimaging and by single photon emission computerized tomography (SPECT). The imaging data were independently interpreted by two physicians in each nuclear medicine department. Confirmation of occult lesions detected by immunoscintigraphy was obtained by clinical examination, CT, ultrasound, planar radiography, biopsy or autopsy.

Immunoradiopharmaceutical. The radiolabelled antibody preparation used for this study derives from the murine MoAb 225.28S (IgG2a subtype) recognizing a HMW-MAA (Wilson et al. 1981). The MoAb purification from mouse ascites and the fragmentation of the purified IgG by pepsin digestion have been extensively described elsewhere (Buraggi et al. 1984b, 1985b; Callegaro et al. 1985; Siccardi et al. 1986). The purified F(ab')2 fragments were then labelled with 99mTC, using a direct labelling method (Rhodes et al. 1982), easily convertible in a kit procedure, with high labelling yield, practicability and reproducibility. Briefly, a 5 m M stannous chloride solution in 10 m M sodium tartrate and 40 m M potassium phthalate, pH 5.6, was mixed with a saline solution of F(ab')2 and allowed to stand for 21 h at room temperature under nitrogen atmosphere (pretinning step). After further dilution with tartratephthalate buffer and addition of human serum albumin (HSA) for a final concentration of 0.350 mg F(ab')2/ml and 0.5 mg HSA/ml, the solution was fiitered on 0.22 gm Millex filters (Millipore Corp., NJ, USA), dispensed into sterile vials and freeze-dried to obtain a ready-to-use reagent, stable for several months. 99mTc-labelling was carried out by introducing into the kit vial 20-30 mCi of a sterile sodium pertechnetate solution, eluted from a sterile Te generator, and left to react for 15-20 min at room temperature. Since the labelling yield ranges from 70% to 90%, a purification step by ion exchange chromatography through a sterile Sephadex DEAE-A 25 column was necessary to retain

Immunohistochem&try. Indirect immunoperoxidase testing was performed on frozen tissue sections as described (Buraggi et al. 1985 b) using the ABC detection system (Vectastain kit, Vector Labs., Burlingame or Immucolor Kit, Sorin Biomedica, Saluggia). Peroxidase activity was detected using 3-amino-9-ethylcarbazole solution (20 lag/ml) in 0.1 M sodium acetate, pH 4.9, containing 5% of N-Ndimethylformamide and 0.02% hydrogen peroxide. The sections were counterstained with Mayer's haematoxylin for 15min, mounted in buffered glycerol and sealed with nail polish.

Statistical analysis. The significance of the observed differences was evaluated by chi-square analysis. The preditive and diagnostic accuracy values (performance values) were calculated according to Galen and Gambino (1972).

319

Results Background radiolocalizations, specificity controls and reproducibility In all the patients investigated there was non-specific accumulation of radioactivity in the liver, spleen and kidneys. The detection of tumour lesions in these sites was then rather poor and could often only be achieved with the aid of subtraction methods or SPECT. Other, less marked, non-specific accumulations of radioactivity were observed in the cardiac area, lungs, urinary bladder and bone marrow. Non-specific accumulations of radioactivity were also occasionally detected in the caecum, scrotum, uterine fibromas and recent surgical scars; however, these did not affect the detection of specific uptake by known tumours. The localization of radiolabelled F(ab')2 fragments of HMW-MAA-specific MoAb 225.28S in melanoma lesions was demonstrated to be specific, since it was shown to be quite different from the distribution of radiolabelled F(ab')2 fragments of MoAb 4C4 (reactive with the irrelevant viral antigen HBsAg; Boniolo et al. 1982) in 15/15 patients investigated with dual-tracer immunoscintigraphy (Siccardi et al. 1986). Furthermore, a correlation was demonstrated with the immunohistochemical detection of H M W - M A A in 13/13 (Siccardi et al. 1986) and in 37/37 ISG-positive tumour lesions investigated during this study.

Radioimaging of melanoma lesions A total of 493 melanoma patients were investigated in the 20 nuclear medicine departments engaged in the European Multicentre Study (see Appendix). Table 1 reports the data of all patients and of all melanoma lesions, known and " u n e x p e c t e d " , both visualized and not visualized by ISG. No clinical or chemical toxicity was detected during or following the studies. Positive ISG results were obtained in 292/363 melanoma patients (321 carrying known lesions and 42 carrying occult lesions). In 231 (79%) of the 292 ISG-positive patients, classified as ISG-TP(A), 402/402 lesions were imaged; in 56 ISG-positive patients, classified as ISGTP(B), 108/204 (5;3%) lesions were imaged; in the remaining 5 patients, classified as ISG-FP, the radiolocalizations turned out to be false-positive (an inflammatory lymph node negative in immunohistochemistry, one hyperostosis frontalis, one granuloma, one recent surgical scar and a melanin-producing synovial sarcoma). In the 76 I S G - F N patients 0/122 lesions were imaged (Table 1 B). Some interdepartment variations in the distribution of patients into ISG classes were observed. However, taking into consideration the departments that contributed at least 20 patients each, their results (with a single

Table 1. Immunoscintigraphy (ISG) results from patients with melanoma lesions A Classification of patients into five ISG classes Patients"

No.

ISG results a Positive

Negative

(A)

(B)

FP

FN

TN

With known lesions Without known lesions

321 172

189 42

56 0

0 5

76 0

0 125

Total

493

231

56

5

76

125

B Classification and distribution of melanoma lesion Patients a

Known lesionsb

URL

Pos/total

Pos

Neg

iSG-positive ISG-P(A) ISG-P(B)

297 90

0 96

105 18

402/402 108/204

ISG-negative ISG-FN ISG-TN

0 0

122 0

0 0

0/122 0/0

387

218

123

510/728

Total

Patients in whom at least one lesion was visualized were classified as ISG-positive: ISG-P(A) if all lesions were visualized, ISG-P(B) if not all lesions were visualized, ISG-FP if the radiolocalization turned out to be false-positive. Patients in whom no lesions were visualized were classified as ISGnegative: ISG-FN (false negative) if they carried known lesions and ISG-TN (true negative) if they carried no lesions b Known lesions were classified as ISG-positive (Pos) or ISG-negative (Neg). ISG-positive occult lesions were classified as URL (unexpected radiolocalizations)

exception) did not show any significant difference in distribution and were not significantly different from those of the Italian section of the same multicentre study (Table 2 A). By classifying the patients according to the type of lesions carried, no significant difference in sensitivity ( P = 0 . 4 4 ) was observed among the three classes (Table 2 B). Table 3 reports the performance values of ISG, calculated from the results of the European section, and compares them with those calculated from the results of the Italian section of the same multicentre study. The difference between the two sets of results (ISG-TP, I S G - F N and ISG-FP; Table 2A) was not statistically significant (P=0.25). The overall fraction of melanoma lesions visualized by ISG was 510/728 (70.1%). In particular, 387 (64.0%)

320 Table 2. Summary of immunoscintigraphy (ISG) results obtained

Table 4. Influence of anatomic site on the outcome of immunoscin-

in the nuclear medicine departments of the European Multicentre Study. A Distribution of results among the different centres. The bottom line reports, for comparison purposes, the results obtained in the Italian Multicentre Study. B Distribution of results among ISG classes of patients according to different types of lesions

tigraphy

Dept.

No.

ISGresults" TP

FN

TN

FP

D2 D3 b GB1 GB2 CH A E3 E4 E5 Other c

85 38 41 22 37 23 59 56 33 99

55 18 33 15 21 14 17 31 23 60

17 12 4 3 4 5 5 4 2 20

12 8 4 4 10 4 37 21 8 17

1 0 0 0 2 0 0 0 0 2

Total IMCS d

493 258

287 174

76 32

125 48

5 4

B

Type

No.

ISG results a TP

FN

TN

FP

LF PO PM MO

172 48 25 248

42 34 21 190

0 14 4 58

125 0 0 0

5 0 0 0

Total

493

287

76

125

5

a As in Table 1 b For this department only, the distribution of TP and FN was significantly different (P=0.001) from that of the total population of patients c Cumulative data from 11 nuclear medicine departments which had less than 20 patients each a Italian Multicentre Study LF, lesion-free; PO, primary tumour only; PM, primary plus metastasis; MO, metastasis only

Anatomic site

Known lesions URL

Pos/Total

IMCS a Pos/Total

Pos

Neg

Liver Lungs Bones Brain Sup. L.N. b Deep L.N. Skin Others

12 28 37 10 125

11 31 18 9 53

1 24 8 4 39

13/24 52/83 45/63 14/23 164/217

14/28 38/59 49/68 19/24 129/167

26

8

11

37/45

27/35

100 49

58 30

28 8

128/186 79/87

78/127 23/31

Total

387

218

123

510/728

377/539

a Italian Multicentre Study b Superficial lymph nodes URL, unexpected radiolocalizations

lesions were i m a g e d a m o n g the 605 a l r e a d y d o c u m e n t e d at the time o f the s t u d y ; the r e m a i n i n g 123 were U R L ( u n e x p e c t e d r a d i o l o c a l i z a t i o n s ) , i.e. p r e v i o u s l y occult lesions r e v e a l e d b y I S G (Tables 1 B a n d 4). T h e m e l a n o m a n a t u r e o f 69/123 U R L was c o n f i r m e d b y clinical criteria a n d / o r a d d i t i o n a l l a b o r a t o r y investig a t i o n i m m e d i a t e l y after the I S G p r o c e d u r e ; thus, these lesions were u n k n o w n at the time o f the s t u d y o n l y because o f lack o f d o c u m e n t a t i o n . A n o t h e r 23 lesions which c o u l d n o t be d e t e c t e d b y v a r i o u s d i a g n o s t i c m e a n s were c o n f i r m e d o n l y in the f o l l o w - u p o f the p a t i e n t s (by the a p p e a r a n c e o f p l e u r a l o r p e r i t o n e a l effusions or b y the s u b s e q u e n t p o s i t i v i z a t i o n o f o t h e r d i a g n o s t i c tests, such as r a d i o l o g y , C T or u l t r a s o u n d ) . T h e n a t u r e o f the r e m a i n i n g 31 I S G - p o s i t i v e U R L c o u l d n o t be determined.

Variables influencing the outcome of ISG T h e large n u m b e r o f p a t i e n t s a n d lesions i n v e s t i g a t e d p r o v i d e d us w i t h an o p p o r t u n i t y to a n a l y s e the influence o f several v a r i a b l e s on the o u t c o m e o f I S G a n d to evaluate the statistical significance o f the o b s e r v e d differences.

Table 3. Comparison of the performance values of immunoscinti-

Anatomic site of tumour lesions. T h e f r a c t i o n o f lesions

graphy in the European and Italian Multicentre Studies

visualized s h o w e d significant differences ( P = 3 . 3 8 x 1 0 - 2 ) f r o m o r g a n to o r g a n (Table 4). I m a g i n g f r e q u e n c y was lower in o r g a n s in w h i c h non-specific l o c a l i z a t i o n o f r a d i o a c t i v i t y o c c u r r e d , for instance, o n l y 54% o f liver lesions were i m a g e d . O t h e r f a c t o r s also a p p e a r to be involved, since one o f the lowest p e r c e n t a g e s o f visuali z a t i o n was s h o w n b y superficial skin t u m o u r lesions, w h e r e non-specific a c c u m u l a t i o n o f r a d i o a c t i v i t y d o e s n o t occur.

Sensitivity Specificity Accuracy Predictive value negative result Predictive value positive result

European

Italian

79.06% 96.15 % 83.57% 62.19% 98.29%

84.47% 92.31% 86.05% 60.00% 97.75%

321

Size of melanoma lesions. Altogether, 218 documented melanoma lesions were not visualized by ISG in a total of 132 patients. About 70% of the ISG-negative lesions were of small size (less than 2 cm in diameter or extremely thin, superficial skin lesions). Clinical stage of patients. Standard staging was available for 236 patients, to whom 184 were classified as ISG-TP and 52 as ISG-FN. The stage distribution was as follows: 65 patients in stage I-II; 102 patients in stage III; 69 patients in stage IV. The fraction of false-negative results was significantly higher (P=2.68 x 10 -3) in stage IV patients (25/69) than in stage III patients (11/102). Expression of the HMW-MAA in lesions. Biopsy specimens from 48 melanoma lesions (37 ISG-positive and 11 ISG-negative) were analysed for their H M W - M A A content by immunostaining with MoAb 225.28S (immunoperoxidase method). All ISG-positive lesions were also positive in immunostaining, while 7/11 ISG-negative lesions were antigen-positive in immunostaining.

Discussion Previous studies (Buraggi et al. 1985b, 1986a) demonstrated that F(ab')2 fragments of the HMW-MAA-specific MoAb 225.28S have more favourable biodistribution than whole immunoglobulins. Comparative studies using different radioisotopes to label F(ab')2 fragments demonstrated that 99mTc is the label of choice for diagnostic imaging purposes (Buraggi et al. 1984b, 1986a; Siccardi et al. 1986). This paper reports the results of an European Multicentre Study carried out in 20 nuclear medicine departments using the same immunoradiopharmaceutical [99mTc-labelled F(ab')2 fragments of 225.28S] and the same immunoscintigraphy protocol to study a total of 493 melanoma patients. The results of this study, in terms of performance (sensitivity, specificity, accuracy and predictive value of positive and negative results), were compared with those previously reported (Siccardi et al. 1986) for the Italian Multicentre Study carried out with 258 patients from 10 nuclear medicine departments. No significant difference could be evidenced between the results obtained in the two studies. The overall sensitivity of the European trial and of the Italian Multicentre Study were 79% and 85%, respectively; 70% of total lesions were imaged in both studies. ISG of metastatic melanoma can refine and potentially extend the definition of the disease; 123 previously unsuspected lesions (URL) were identified, increasing the total number of lesions investigated by 20%. In 42 patients such identification was the first evidence of metastatic melanoma. ISG is helpful in assessing the clinical staging of the disease and often in modifying the prognosis and the therapeutic strategy. Two recent reports (Eary et at. 1989; Salk and the Multicentre Study

Group 1988) have confirmed that antibody imaging procedures for melanoma are safe and sensitive; moreover, in several cases, ISG results were decisive in assessing clinical staging and the choice of therapy or additional diagnostic procedures. More than half (56%) of the U R L detected in this study were corroborated by other detection modalities; the remainder had a high probability of being true melanoma lesions. Although some of the uncorroborated U R L might turn out to be false-positive, the same is true for lesions detected by other modalities but not visualized by ISG. Only five false-positive radiolocalizations were documented by ISG, and one of these, a melanin-producing synovial sarcoma, was truly antigenpositive, although not of melanomatous nature. Non-specific accumulation was a major cause of false-negative ISG results in organs (such as liver and bones) in which high background radiolocalization occurs. Conversely, the low percentage of visualization of superficial skin lesions (ca. 50%) can be explained in terms of the extremely low thickness, i.e. of the small volume of superficial lesions. Other variables were clearly identified as responsible for the poor outcome of ISG: level of expression of HMW-MAA in single lesions, size of lesions and finally clinical stage of the disease. As reported for the Italian Multicentre Study, the clinical stage highly influenced the outcome of ISG, the fraction of false-negative results being significantly higher in patients in stage IV than in patients in other stages. A possible biological explanation of this finding is the higher incidence of necrotic and/or poorly vascularized lesions in patients with advanced melanoma. In conclusion, the results of this European Multicentre Study are in good agreement with those previously reported for the Italian Multicentre Study and confirm that ISG with 99mTc-labelled fragments of MoAb 225.28S is a reliable and informative procedure for the visualization of human malignant melanoma lesions.

Acknowledgements. This work was supported by grants of the Special Project "Tecnologie Biomediche e Sanitarie" of the Italian National Research Council. The skilled assistance of Silvana Spedalini in data analysis is gratefully acknowledged.

Appendix The European Multicentre Study Group includes: Department of Nuclear Medicine, University of Aachen, F R G (U. Bfill, G. Weiler, F. Hofst/idter); Department of Radiology and Nuclear Medicine, Klinikum Groghadern, University of Miinchen, F R G (K. Scheidhauer, E. Moser, A. Marckl, G. Leinsinger); Department of Nuclear Medicine, University of Bonn, F R G (H.J. Biersack, A. Bockisch, P. Oehr); Department of Radiology and Nuclear Medicine, University of Heidelberg, F R G (P. Georgi, E. Pittelkow); Regional Department of Medical Physics and Bioengineering and CRC Department

322

of Medical Oncology, Christie Hospital and Holt Radium Institute, Manchester, UK (N. Thatcher, T. Cerny, S.E. Owens, S.A. MacKenzie, P.M. Nuttall); Department of Nuclear Medicine, St. Bartholomew's Hospital, London, UK (K.E. Britton, M. Granowska, J. Bomanji, K. Solanki); Department of Nuclear Medicine, Royal Marsden Hospital, London, UK (A. Irvine); Service de M6dicine Nucl4aire, Centre Eug4ne Marquis, Rennes, France ( J . F . Herry); Servicio de Medicina Nuclear, Centro Ramon y Cajal, Madrid, Spain (A. Crespo Diaz); Servicio de Medicina Nuclear, Hospital Puerta de Hierro, Madrid, Spain (J. Ortiz Berrocal, J. Ramos, J. De Haro); Servicio de Medicina Nuclear, Hospital de Cruces de Barcaldo, Bilbao, Spain (J. Fombellida, J.J. Duque); Servicio de Radiologia, Hospital Reina Sofia, Cardoba, Sapin (A. Mateo, M. Martinez Paredes, M. Torres); Servicio de Medicina Nuclear, Hospital Universitario Virgen del Rocio, Sevilla, Spain (J.R. Rodriguez, D. Garcia Solis); Laboratorio de Isotopos, Instituto Portugfies de Oncologia Francisco Gentil, Lisboa, Portugal (Amalia Nogueira); Department of Nuclear Medicine, University of Bern, Switzerland (H. Roesler, K. Brinner, T. Cerny, M. Rentsch); Department of Nuclear Medicine, I University Clinic for Internal Medicine, Vienna, Austria (R. Dudczak); Department of Nuclear Medicine, University Central Hospital, Helsinki, Finland (K. Liewendahl, A.L. Kairento-Brownell, S.L. Karonen); Department of Nuclear Medicine, National Institute of Oncology, Budapest, Hungary (M. Fiizy); Department of Radiology, Postgraduate Medical School, Budapest, Hungary (U. Szilv~tsi); Department of Nuclear Medicine, Lublin, Poland (B. Siwek).

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