Klin. Pädiatr. 202 (/990)

Radio-immunodetection of Myosarcoma using ll1Indium Antimyosin E. Koscielniae, P. Reuland2 , F. Schilling!, U. Feine and J. Treuner! I Abt. für Onkologie und Hämatologie, Pädiatrisches Zentrum Olgahospital, Stuttgart 'Abt. für Nuklearmedizin, Med. Strahleninstitut der Universität, Tübingen

Summary Radio-Iabelled antimyosin-monoclonal antibodies (AMA) have been introduced to demonstrate myocardial necrosis after cardiac infarction or in cardiac allograft transplants. As rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS) are tumors of myogenic origin, thus often containing myosin, we decided to use the 1llln-Iabelled Fab fragment of AMA (Centocor) in scintigraphic tumor detection. We examined 13 children with histologically-confirmed RMS and LMS, and five other children with other types of soft tissue sarcomas. Conventional techniques were used to determine the extent of the tumor. An uptake of the tracer was observed in all known tumor sites in seven RMS patients. As the scans were negative in three RMS patients who were in complete remission (CR) and in two other patients (fibrosarcoma and haemangiopericytoma) with a measurable tumor mass, we considered them to be "true negative". In the three remaining CR patients (I RMS, 2 LMS) the scans were positive but weak in the primary tumor site in two cases and in a distant site (bone) in the third respectively. We considered them to be "false positive" as no tumor cells were evident in the biopsy specimen. In one case, the antimyosin uptake was presumably the result of damage to the muscles after radiation. Interestingly, in three patients with other malignancies such as rhabdoid and peripheral neuroectodermal tumors there was a noticeably strong uptake of the tracer in the primary tumors and the scans turned negative after complete remission was achieved. The diagnostic AMA scanning showed no side-effects. The reason why antimyosin antibodies permeate the membrane of the tumor cells is yet undetermined. It has been postulated that tumor cell membranes are more permeable than their normal counterparts. At present we are conducting in vitro studies on the mechanism of the AMA transfer through the cell membrane, on the specificity of the binding and on the retention in the cells. The AMA scan could possibly prove to be a valuable diagnostic tool in RMS but, clearly, further investigation is necessary to come to adefinite conclusion.

Klin. Pädial r. 202 (1990) 230-234 1990 F. Enkc Verlag Stllltgart

Radioimmunologischer Nachweis beim Myosarkom mittels 111 Indium Antimyosin Isotopenmarkierte monoclonale Antimyosin-Antikörper (AMA) sind zur Darstellung von Myocardnekrosen nach Herzinfarkt und der Abstoßung von Herztransplantaten eingeführt worden. Da Rhabdo- und Leiomyosarkome Tumoren myogenen Ursprungs sind, findet man in diesen Tumoren häufig Myosin. Aus diesem Grund wurde das mit IlI Indium markierte FabFragment des AMA (Centocor) von uns zur szintigraphischen Tumordarstellung verwendet. Wir untersuchten 13 Kinder, bei denen ein Rhabdomyosarkom (RMS) oder ein Leiomyosarkom (LMS) histologisch gesichert war und 5 Kinder mit anderen Weichteilsarkomen. Die Tumorausdehnung wurde mit konventionellen, bildgebenden Verfahren bestimmt. Bei 7 Patienten mit RMS wurde eine Aufnahme des Tracers in alle bekannten Tumorlokalisationen beobachtet. 3 Patienten mit RMS in kompletter Remission und 2 Patienten mit einem meßbaren Tumor aber anderen histologischen Diagnosen (Fibrosarkom, malignes Hämangioperizytom) zeigten negative Scans. Die restlichen 3 Patienten in kompletter Remission (I x RMS, 2 x LMS) zeigten eine schwache Anreicherung, 2 davon in der ursprünglichen Tumorlokalisation. Da histologisch keine Tumorzellen in den Biopsien gefunden werden konnten, wurden diese Fälle als "falsch positiv" bezeichnet. In einem Fall war wahrscheinlich die durch die Bestrahlung verursachte Muskelschädigung Ursache für die Aufnahme von Antimyosin. Interessanterweise konnte bei drei Patienten mit anderen histologischen Diagnosen (z. B. PNET, Rhabdoidtumor) eine sehr starke Aufnahme des Tracers in die Primärtumoren beobachtet werden. Die Szintigraphie wurde negativ als die Patienten eine komplette Remission erreichten. Der diagnostische Einsatz von Antimyosin-Antikörpern zeigte keine Nebenwirkungen. Eine Ursache für das Durchdringen des AMA durch die Zellmembran in die Tumorzelle ist bis jetzt nicht bekannt. Es wurde vorausgesetzt, daß eine erhöhte Durchlässigkeit der Tumorzellmembran im Vergleich zu normalen Zellen besteht. Zur Zeit führen wir in vitro Studien durch, um die Mechanismen des AMATransfers durch die Zellmembran, die Spezifität der Bindung und die Aufnahme in die Zellen zu untersuchen. Es scheint, daß die AMA-Szintigraphie ein hilfreiches Verfahren in der Diagnostik des Rhabdomyosarkoms darstellt. Bevor eine abschließende Beurteilung möglich ist müssen zunächst noch weitere Erfahrungswerte mit dieser Methode gesammelt werden.

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Radio-immunodetection oj Myosarcoma using lI/Indium Antimyosin

Radio-labelled monoclonal antibodies (Moab) raised against tumor-associated antigens have been used to locate tumors during external body scintigraphy. Another potential application of Moab is in the delivering of chemotherapy, toxins or radionuclides which specifically destroy tumor cells. Recent studies have dealt mostly with solid tumors such as gliomas and ovarian or colon carcinomas (Kalojonos et al. , 1989), adenocarcinomas (Bumol et al. , 1989), melanomas (lrie et al. , 1989) and neuroblastomas (Cheung et al. , 1987). We investigated the '1lIndium labelled antigen-binding Fab fragment of the RIIDIO antimyosin antibody in radio-imaging of myosin-positive tumors such as rhabdo- and leiomyosarcoma. Myosin is a major intracellular protein present in muscle cells and has unusual properties: it is both a globular enzyme (ATPase) as weil as a fibrous structural protein. The antimyosin antibody has already been applied in the detection of myocardial damage in cases of acute infarction, myocarditis and in allograft rejection (Khaw et al., 1979). There is no uptake in normal myocardial cells, but it is likely that tumor cell membranes are more permeable than normal ones (Coxet al. , 1975). Thus, studies in which the amimyosin antibody was used in the scintigraphic detection of myosarcoma were initiated by Hoefnagel (Hoejnagel et al., 1987) and by our group (Reuland et al. , 1989). The pre1iminary findings have been

Tab. 1 No.

very encouraging, and the fact that the other imaging techniques used in myosarcoma have proved to be non-specific makes it all the more essential for this research to be continued. Material and Methods

On 24 occasions we examined 18 children with histologically proven rhabdo- or leiomyosarcoma. In five of these children, the pathological diagnosis was changed after the consulting pathologist of the German Cooperative Soft Tissue Sarcoma Study (CWS) reviewed the cases. The clinical data of the patients we studied are delineated in Table I. In aIl the cases, the extent of the tumor was determined through conventiona1 techniques like ultrasound, whole-body scintigraphy, computed tomography or nuclear magnetic resonance tomography. For the scintigraphic imaging we used the Fab fragment (5 kD) of the antimyosin monoclonal antibody (RIIDlO, IgG2 with Kappa light chains), which was raised by immunizing Balb/c mice with human cardiac myosin (Centocor Europe, NL-Leiden). Each patient received 0.2 mg to 0.3 mg Fab fragment bound to diethylenetriaminepentaacetic acid (DTPA) labelled with an activity of 74 MBq 111 Indium-chloride (2 mCi). Planar scintigraphies as weIl as the ECTstudies were done using a double-head camera (Picker dyna digit) with a high-energy collimator. Total body scanning was carried out at intervals of 4, 24, and 48 hours.

Clinical characteristics 01 studied patients Sex






Antimyosin uptake 1st/2nd


1 2 3 4 5 6 7


5 11 14 2 12 1 4


head extremity extremity head head bladder/prost. head


-/+ + + + +/+ +

8 10 11 12 13


1 17 11 6 8 18

rhabdoid tumour rhabdoid tumour PNET aRMS LMS LMS

abdominal head head head extremity stomach

MT MT/MT MT/Rem Rem Rem Rem

+ +/+ +/+ + +

14 15 16 17 18


6 14 2 5 2

eRMS eRMS eRMS haemangiopericytoma librosarcoma

bladder head buttock head orbita

Rem Rem Rem Rem Rem



measurable tumor Remission embronyal rhabdomyosarcoma alveolar rhabdomyosarcoma leiomyosa rcoma peripheral neuroectodermal tumor

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K/in. Pädiatr. 202 (1990) 231

Klin. Pädiatr. 202 (1990) Result

In seven rhabdomyosarcoma ca es with evidence of measurable tumor found through conventional techniques, the antimyo in scans also showed an uptake in all the known tumor ite . For this reason, we considered them to be "true positive" (Figures 1, 2 and 3). The antimyosin scan proved to be negative in three rhabdomyosarcoma cases and there was also no tumor evident when conventional techniques were used. Two other patients who had macroscopic tumor residues but histopathological diagnosis other then myosarcoma (patient nos. 17 and 18) also showed no uptake of antimyosin. Thus, this group was considered to be "true negative". In patient no. 12, who had leiomyosarcoma, the antimyosin scan carried out one year after cardiotherapy was very weakly positive in the primary tumor area (Figure 4). Similarly, in the magnetic resonance te t, the

Fig. 1 a + b Uptake of I I Iindium antimyosIn In an mtraeranial metaslasls of an embryonal rhabdomyosareoma (Pat. no. 1). The CT is ineluded as a eomparlson

E. Koscielniak et al. T2-weighted images produced an intensified signal in the same area. This was presumably due to the damage caused by radiation in this area. Patient no. ] 3, also a leiomyosarcoma case, was considered to have been in remission and a weak, diffuse antimyosin uptake in the oesophagus was observed. The pathological examination of the biopsy specimen taken from this site, however revealed no tumor cells and there was no tumor relapse at this site during the emire 16-month observation period. Patient no. ] 1, a rhabdomyosarcoma case, showed a positive antimyosin uptake in the banes. Positivity was also observed in the same sites in the bone seintigram although no tumor cells were found in the histopathological examination. These cases must, therefore, be considered as false positive. Two patients (patients no. 8 and 9) with rhabdoid tumor and another with a peripheral neuroec-

Fig. 2 a + b Uptake of 1 l11ndium anllmyosin in an intrathoraeie metastasis of an embryonal rhabdomyosarcoma. Below is the ehest X-ray of the same patient

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Radio-immunodetection 0/ Myosarcoma using li/Indium Antimyosin

Klin. Pädiatr. 202 (1990)


/ '6128/8'




Fig. 4a + b Diffuse uptake of I I Iindium antlmyosin in a primary tumor region after resection. chemotherapy and radiation of a leiomyosarcoma in the nght thigh (Pat non. 12) Below is the magnetic resonance examination, showing an increased signal In the T2 weighted images

Fig. 3a + b Uptake of I I Iindium antimyosIn in an alveolar rhabdomyosarcoma (right thighl. Below is the ultrasound image included as a comparison

todermal tumor (patient no. 10) howed a very strong uptake of antimyosin in all the known tumor sites. There was no change in the uptake even when the examination was repeated. The 13II-meta-iodobenzylguanidine (MIBG) scintigrams were negative in these patients. 0 pathological concentration of 1 J lIndium antimyosin was found after chemotherapy led to a complete regression of the tumor in patient no. 10. The distribution pattern of the antimyosin uptake was a folIows: the kidneys showed the strongest uptake in almost every patient, followed by the liver and bone marrow. A constant uptake in the nasopharyngeal region was found. It was possible to achieve a clear and constant imaging of the tumor as early as three hours after the injection. The best tumor depiction wa achieved after 48 hour ,although there was no marked change in quality between the e images and those of the 24-hour scans. No adverse reactions resulted, even after repeated antimyosin injections.

Discussion The potential diagnostic and therapeutic application of monoclonal antibodie raised against a tumor-associated antigen is currently a subject of great interest. Upto now, studies on the radio-imaging of solid tumors have used antibodies against surface antigens. In contrast, myosin is an intracellular protein. Thus, antimyosin antibody circulating in the bloodstream does not have access to myosin in situ and does not localise in normal muscle tissue. Following irreversible cell damage, due to ischemic injury, for example (as in the case of myocardial infarction), the cell membrane becomes permeable to macromolecules, thus allowing the antimyosin antibody to interact with intracellular myosin.

Cox and van de Pompe (Cox et al., 1975) discovered that, in fact, tumor cell membranes are highly permeable to ionic radio-pharmaceuticals. This observation, and the fact that most rhabdo- and leiomyosarcomas contain a certain amount of myosin, led to investigations on the radio-Iabelled antimyosin antibody in the imaging of particular tumors. The preliminary results of Cox et al. (Cox et al., 1988), Planting et al. (Planting et al. , 1989) and our group (Reuland et al., 1989) provide corroborating evi-

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Klin. Pädiatr. 202 (1990)

E. Koscielniak et al.: Radio-immunodetection 0/ Myosarcoma using

dence. We regarded this method as promising enough for further research. Our current analysis shows that the antimyosin scintigram was true positive in seven out of II patients with rhabdomyosarcoma, and true negative in three cases. A weak antimyosin uptake in the bones showed up only in one alveolar rhabdomyosarcoma patient, although no evidence of tumor was found in the histo-pathological examination. It must be mentioned that, in the latter case, a bone scan also showed a positive image in the same site: it is very difficult to explain this phenomenon. In two patients with leiomyosarcoma, the antimyosin scan was considered to be false positive. In one of these patients, a plausible explantation for the antimyosin uptake could be the damage to the muscles caused by radiation, especially because there was a significant difference between the kinetics of the antimyosin uptake and normal kinetics. Planting et al. also described false antimyosin scan results in leiomyosarcoma patient (Planting et al. , 1989). Their cases, however, were false negative. We believe that the number of our leiomyosarcoma patients is too low for a definitive conclusion to be drawn. Another major problem we faced was the very high uptake of antimyosin in one neuroectodermal and two rhabdoid tumors. Recently, we postulated the neuroectodermal ongm of rhabdoid tumor (Kimmig et al. , 1989), although the definitive origin and immunocytochemical features of these tumors remain a matter of controversy. In our two antimyosin-positive rhabdoid tumors, the immunocytochemical staining for myoglobin was also weakly positive. The high uptake of antimyosin could thus be explained not only by a cross-reaction of antimyosin with other cytoskeletal elements, but also by the extreme heterogeneity of rhabdoid tumors. Further in vitro investigations in respect to this problem are being carried out. We did not observe any side-effects during or after the delivering of the antibody, even when the examination was repeated. This was probably due to the use of the Fab fragment whose antigenicity is lowered by virtue of the fact that it remains in the body for a short length of time. Our data shows that radio-Iabelled antimyosin is practicable in imaging tumor mass in rhabdomyosarcoma patients. Until an even better antibody is found, one could, theoretically, go ahead with using the ra-

dio-Iabelled antimyosin antibody in therapy. In this case however the whole antibody would be more suitable. As far as leiomyosarcomas are concerned, more data is necessary before a definitive conclusion can be drawn. We are currently conducting in vitro and animal-model experiments with the aim of clarifying the specificity of antimyosin binding in myosarcoma and rhabdoid tumors.

References l




, •





Bumol. T. F.• S. V. De Herdt. D. L. Zimmerman. L. D. Apelxren: Monoclonal antibody-oncolytic conjugates for site-directed therapy of human Adenocardinomas. Proceedings of the American Association for Cancer Research. 30 (1989) 647-648 Cheung. N.-K. v.. H. Lazarus. F. D. Miraldi. C. R. Ahramowsky. S. Kallick. U. M. Saarinen. T. Spitzer. S. E. Strandjord. P. F. Coccia. N. A. Berger: Ganglioside G u, Specific Monoclonal Antibody 2F8: A Phase I Study In Patients with Neuroblastoma and Malignant Melanoma. J. Clin. Oncol. 5 (1987) 1430-1440 Cox. P. H.. W. B. Van de Pompe: An ionic model to explain the distribution patterns of tumour see king radiopharmaceutical in normal and pathological tissues. In: Munkner T. (ed). Nuklearmedizin Fadl. Copenhagen 1975 Cox. P. H.• J. Verweij. M. Pillay. G. Stoter. D. Schon/eid: Indium'" antimyosin for the detection of leiomyosarcoma and rhabdomyosarcoma. Eur. J. Nucl. Med. 14 (1988) 50-52 Hoe/nagel. C. A .• P. A. Voute. J. De Kraker. H. Behrendt: Scintigraphie detection of rhabdomyosarcoma. Lancet I (1987) 921 {rie. F. R .• T. Matsuki. D. L. Morton: Human Monoclonal Antibody To Ganglioside GM2 For Melanoma Treatment. Lancet I (1989) 786-787 Kal%nos. H.. A. A. Epenetos: Radioimmunotherapy with lodine 131-1abelled Antibodies in Ovarian, Colonic and Brain Tumours. In: H. G. Beger et al. (Eds.): Cancer Therapy, Springer Berlin-Heidelberg 1989 Khaw. B. A .• J. T. Fallon. G. A. Beller. E. Haher: Specificity of Localization of Myosin-specific Antibody Fragments in Experimental Myocardial Infarction. Circulation 60 (1979) 1527-1534 Kimmig. A .• E. Koscielniak. R. Handgretinger. K. Schi/hach-Stückle. C. O/lenlinger. G. Rudolph. H. Wolhurg. W. Paulus. D. Schmidt. M. Altmannsherger. J. Treuner. D. Niethammer: Experimental evidence for neural origin of extrarenal rhabdoid tumour. Clin. ehern. Enzym. Comms. 2 (1990) 321-327 Planting. A .• J. Verweij. P. Cox. M. Pillay. G. Stoter: Radioimmunodetection and Radioimmunotherapy in Myosarcoma. In: H. G. Beger et al. (Eds.), Cancer Therapy. Springer Berlin-Heidelberg (1989) Reuland. P.• E. Koscielniak. J. Treuner. U. Feine: Monoclonal Antibodies Against Myosin For The Diagnosis Of Myosarcoma. In: H. A. E. Schmidt, G. L. Buraggi (Eds.) Nuclear Medicine, Trends and Possibilities, Suppl. 25 (1989) 572-576

Dr. med. E. Koscielniak Pädiatrisches Zentrum Olgahospital Abt. für Onkologie und Hämatologie Bismarckstrasse 8 D-7000 Stuttgart I

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Radio-immunodetection of myosarcoma using 111indium antimyosin.

Radio-labelled antimyosin-monoclonal antibodies (AMA) have been introduced to demonstrate myocardial necrosis after cardiac infarction or in cardiac a...
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