RADIOLOGY AND ONCOLOGY Established in 1964 as Radiologia Iugoslavica in Ljubljana, Slovenia. Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiology, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical aru! experimental oncology, radiophysics and radiation protection. Editor in chief Tomaž Benulic Ljubljana, Slovenia Associate editors Gregor Serša Ljubljana, Slovenia Viljem Kovac Ljubljana, Slovenia Editorial board Bela Fornet Budapest, Hungary Marija Auersperg Tullio Giraldi Ljubljana, Slovenia Udine, ltaly Andrija Hebrang Mat{ja Bistrovic Zagreb, Croatia Zagreb, Croatia Durda Horvat Haris Boko Zagreb, Croatia Zagreb, Croatia Berta Jereb Malte Clausen Ljubljana, Slovenia !Gel, Germany Vladimir Jevtic Ljubljana, Slovenia Christoph Clemm Miinchen, Germany H. Dieter Kogelnik Salzburg, Austria Mario Corsi Udine, Italy Ivan Lovasic Rijeka, Croatia Christian Dittrich Marijan Lovrencic Vienna, Austria Zagreb, Croatia Ivan Drinkovic Luka Milas Zagreb, Croatia Houston, USA Gillian Duchesne Maja Osmak London, Great Britain Zagreb, Croatia Branko Palcic Vancouver, Canada ]urica Papa Zagreb, Croatia Dušan Pavcnik Ljubljana, Slovenia Stojan Plesnicar Ljubljana, Slovenia Ervin B. Podgoršak Montreal, Canada Miran Porenta Ljubljana, Slovenia Jan C. Roos Amsterdam, The Netherlands Horst Sack Essen, Germany Slavko Šimunic Zagreb, Croatia Lojze Šmid Ljubljana, Slovenia Andrea V ero nesi Gorizia, Italy Publishers Slovenian Medica/ Society -Section of Radiology, Croatian Medica/ Association -Croatian Society of Radiology Affiliated with Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.l.R.M. (ltalian Society of Medica! 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Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EXCERPTA MEDICAIELECTRONIC PUBLISHING DIVISION TABLE OF CONTENTS INTERFERONS -EXPERIMENTAL STUDIES Type I interferons spontaneously expressed during human pregnancy Duc-Goiran P, Robert B, Navarro S, Lopez J, Chavinie J, Ferre F, Chany C, Doly J 265 Changes in the quantity of cathepsin D in irradiated human cells following treatment with hyperthermia and interferon a Ferle-Vidovic A, Kaštelan M, Petrovic D, Škrk J, Vrhovec 1 271 Interactions of interferon and vinblastine on experimental tumor model melanoma B-16 in vivo Jezeršek B, Novakovic S, Seda G, Cemažar M, Auersperg M, Fleischmann WR Jr 275 Combined treatment of murine SA-1 tumors by human leukocyte interferon alpha and electrotherapy Serša G, Miklavcic D 280 Antitumor effect of interferon-a administered by different routes of treatment Novakovic S, Fleischmann WR Jr 286 Anti-tumor effect of interferon alpha in combination with cisplatin animal experiments Štabuc B 293 Serum interleukin -2 levels in malignant melanoma patients Rudolf Z, Novakovic S 298 Natura( porcine interferon gamma (PoIFN gamma) Filipic B, Rozman S, Carlsson K, Cencic A 302 Biological activity of rat fibroblast interferon beta Cencic A, Filipic B 307 INTERFERONS -CLINICAL STUDIES Our experience with alpha-2b interferon in the treatment of chronic active hepatitis B Brinovec V 312 Alpha interferon in the treatment of chronic hepatitis C Palmovic D, Crnjakovic-Palmovic J Treatment of cervical intraepithelial neoplasia associated with human pappilomavirus by interferon vaginalettes Singer Z, Šooš E, Feichter G Natura! IFN-a for non small celi lung cancer with pleural carcinosis Jereb B, Petric-Grabnar G, Tercelj-Zorman M, Us-Krašovec M, Mažuran R, Šooš E, Stare J 321 326 Adjuvant treatment of malignant melanoma with human Ieukocyte interferon after radical surgery: I. general analysis ..z m REPORT Breast imaging at ECR'93 Jancar B 339 BOOK REVIEW Gastrointestinal radiology Hebrang A NOTICES 342 AUTHOR INDEX and SUBJECT INDEX, 1993 1 st The papers were presented on the SI ovene Microbiological Congress with Internati ona! Participation; Section of Interferons; Bled, Slovenia, October 24-27, 1993. Radio! 011col 1993; 27: 265-70. Type I interferons spontaneously expressed during human pregnancy Paulette Duc-Goirnn,1 Brigitte Robert,1 Sebastien Navarro/ Jacqueline Lopez, Jacques Chavinie,3 Fran.oise Ferre,1 Charles Chany, Janine Doly2 1 1NSERM U.361, 2 CNRS UPR-37, Faculte de Medecine, 3Hopita!St-Vincent-de-Paul, Paris, France Our previous studies have shown the presence of Interferons (IFNs) in human fetal annexes, in the ahsence of any apparent incluction. The characterization of the IFN proteins was performecl and their analysis by SDS-PAGE revealed the presence of a ancl f3 IFNs and unusually large TFN components. These results were confirmecl by the demonstration of unusually large IFN-a transcripts, by means of Northern blot analysis. The large IFN-a transcript (4.3 kb) has been characterized ancl correlated with the presence of functionally active IFN protein. It may be specifically expressecl during fetal clevelopment. In human species, the IFN-a proteins are cletected throughout fetal gestation, particularly in the amniotic fluicl. However, early embryos do not produce significant levels of IFN. In contrast, in domestic ruminants, some IFNs are only expressed in early embryos. A relation between the type of placenta/ion ancl the I FN expression is suggested. Key words: pregnancy; placenta-analysis; interferon type 1 Introduction During gestation, development of the uterus and of the fetal-placental unit is specially inten­se. It is mediated and regulated by a number of endogenous hormones, growth factors and cytokines. Cytokines are a heterogeneous fa­mily of proteins. They comprise interferons (IFNs), colony-stimulating factors, and cytoki­nes produced by activated cells in inflammatory situations or in immune responses. Cytokines Correspondencc to: Paulcttc Duc-Goiran, Ph.D., IN­SERM U.361, 123, Bld de Port-Royal, 75014 Paris France. UDC: 618.36-074 are protein mediators of celi-to-celi communica­tion. They have pleiotropic activities, in a va­riety of cellular processes. Their action is usually local, unlike that of hormones, and is restricted to the micro-environment of the pro­ducer cells, specially at the fetal-maternal inter­face. Interferons were the first cytokines identified. Their name was originally derived from their ability to interfere with vira! replication. IFNs are now known to be involved in celi prolifera­tion and tumor growth inhibition. They may alter immune functions and modulate a variety of physiological responses. IFNs are inducible proteins. Their production is usually subject to stringent control. However, a low spontaneous Duc-Goiran et al. expression has been reported in a variety of tissues. 1• 2 In humans, endogenous IFNs have been detected in human fetal annexes, in the absence of any apparent induction. Sponta­neous IFN-a has been detected in the amniotic fluid withdrawn by puncture at seiected periods after the 15th week of pregnancy.3 Some IFNs ( a/B) were also found to be diffusing from the amniotic membrane maintained in a tissue cul­ture medium, in placentae obtained after caesa­ 5 rean section,4• in fetal blood and in decidua, while they cannot be detected in materna! 5 blood.4• In addition, an immuno-reactive IFN­a protein has been localized in the syncytiotrop­hoblast of chorionic villi6 and in spindle shaped 7 cells, which were thought to be macrophages.There is also a low leve! of IFN-a in human embryo culture media. 8 Moreover, specific bin­ding sites for human IFN-a have been described in human placenta! membranes.9 Patients and methods Human placentas were obtained between the 35th and 40th week of pregnancy. Biological preparations, affinity chromatography, NaDod SO4/P AGE, RNA preparations and Northern blot hybridization were described previou­ sly_ 4, 10 Results IFN activity IFN activity was found in 29/37 placentae, 14/29 amniotic membranes and 3/10 umbilical cord blood samples.4 Characterization of IFN proteins In order to characterize the IFNs present in human fetal annexes, we have previously analy­zed a set of twenty placentas, in the third trimester of pregnancy.4 First, we performed affinity chromatography on Concanavalin A-Sepharose. IFN-a did not bind to the immobilized Concanavalin A and was found in the breakthrough fractions. After elution with methyl-a-D-mannoside and with ethylene glycol, IFN-B was recovered.4 We also performed chromatography using as a ligand, specific polyclonal lmmunoglobulins against Interferon-a. Most IFN bound to speci­fic immunoglobulins .was _ eluted with pH 2.2 buffer.4 Analysis by SDS-PolyAcrylamide-Gel-Elec­trophoresis, under denaturing and reducing conditions, revealed the presence of a and B IFNs. Besides these typical IFNs, three IFN components of unusual size and antigenic pro­perties have been detected, reminiscent of those previously found in the human amniotic mem­brane after vira] induction. These three compo­nents are recognized by antibodies against IFN­ a. The 43 kDa component is neutralized by antibodies against IFN-a or IFN-B to about the same extent.4 Type I IFN transcripts We investigated the presence of type I IFN transcripts in placenta! samples by Northern blot analysis and showed the results of four cases.10 IFN-a transcripts have been identified, using an antisense riboprobe, complementary to the human IFN-a C gene. This probe gives a strong signal with the 1.1 kb IFN-a transcript which is usually detected in leukocytes after vira! induction. This species is lacking in all placenta! samples. In contrast, in 3 out of 4 polyadenyla­ted RNA samples, we found transcripts of 4.3, 2.8 and 2.1 kilobases, and a minute signal at 4.3 kb in placenta 3. The intensity of the signals displayed on the autoradiogram was estimated by densitometer scan. The area of the peak corresponding to the 4.3 kb transcript is shown in Figure lA. The hybridization of the placenta! mRNAs to the IFN-a C gene appears to be specific. No artefactual signal with the 28 S ribosomic RNA could be detected with the corresponding non-polyadenylated RNAs. Ho­wever, this hybridization is observed in mode­rate or low stringency conditions and the signals disappear after stringent washes and after Type 1 inte1ferons spontaneously expressed during human pregnancy RNAse A treatment. These results suggest that the mRNAs detected here are only partially homologous to the IFN-a C gene. As IFN-a genes belong to a multigene family clustered on the same chromosome, we have suggested that the mRNA detected in the fetal tissue annexes, belongs to one of the IFN-a family genes, but it is not the IFN-a C gene. The presence of IFN proteins was studied in the same placentas, by immune affinity chroma­ tography for IFN-a. In placentas 1, 2 and 4, IFN-a was bound to the specific IgG and eluted with pH 2.2 buffer (Figure 1 B). The IFN-a-like transcripts correlate with the presence of the corresponding functional IFN-a proteins, purified by immuno-affinity. The le­ vels of both IFN-a-like transcripts and IFN-a Area 100 Transcripts A a: IO 3 4 lfN unils B 1000 100 Placentae Figure l. IFN-a: expression in human placentae. A) IFN-a: transcript signals, measured by densitometer scan (area). B) IFN proteins (International Units) isolated by immune affinity chromatography for IFN-a:. poly (A) + ­ 1,7 kb ­1kb ­ Figure 2. Hybridization of an ovine trophoblast-IFN cDNA to poly (At and poly (Af RNAs from a placenta, analyzed by a Northern blot. proteins differ from one placenta to another; being high in placentas 1 and 4, moderate in placenta 2 and absent or minimal in placenta 3. IFN [3. In placentas 1, 2 and 3, some IFN was excluded from the immuno-absorbent IFN­a column and was found in the flowthrough fractions (Figure 1 B). The antiviral activity appeared to be related to an IFN-. protein and could be purified by a second affinity chroma­tography, using as a ligand, antibodies against IFN-.. However, we could not show the pre­sence of IFN-. transcripts in any placenta! sample. The absence of IFN-. transcripts might be due to either minute amounts or to a rapid turnover of mRNAs. Trophoblast-Interferon. In order to study the transcripts of a human trophoblast-IFN gene in placentas, we used an ovine Trophoblast-IFN cDNA probe, 11 in low stringency conditions of hybridization: Two mRNA species of 1.7 kb and 1 kb were detected. No signal was detected with the non-polyadenylated RNA (Figure 2). These preliminary results suggest the presence of mRNA transcripts possibly related to a trop­hoblast-IFN gene. Duc-Goiran et al. Discussion Type I Interferons form a group of three distinct gene families: a, . and w, based on sequence divergence and antigenic differences. Recently, trophoblast proteins from ovine and bovine species have been identified as type I IFNs and 12· 13 cloned. ll· These trophoblast IFNs appear to be immunologically distinct from IFN-w and IFN-a and form a new class of IFNs, the "trophoblast-lFNs" or "IFN-TAU". These IFNs are poorly virus-inducible. In ruminants, trophoblast IFNs are only ex­pressed in early embryos, secreted by the extra­embryonic trophoblast. Their primary amino­acid structure ranged from 40 to 55 % identity with IFNs-a and 70% with lFNs-cD. These Inter­ferons play a key role in materna! recognition of pregnancy and maintain the corpus luteum by inhibiting the synthesis of the prostaglandin F2a, which is a luteolytic factor by endo­metrium. A type 11 IFN is also expressed by the early porcine trophectoderm. 14 In contrast, in human species, early embryos do not produce significant levels of IFN. IFN-a proteins seem to appear later and are detected materna] vesscl cndothclium matcrnal connc1.:tivc tissuc -. \ ,i UlC. \ . ra( .. ­ „ 0 Jl(& rfi"J throughout gestation, being found in the amnio­tic fluid from the fifteenth week of pregnancy onwards. Thus, Interferons seem to be expressed uni­versally in mammalian species during gestation. However, the nature and amount of the lnter­ferons released differ markedly, according to the specics and placentation. We have observed variations both in the yield and in the nature of the type l Interferon expressed in human placenta. It could be suggcsted that the synthe­sis of these IFNs and the extent of their action could only take place at a specific tiine under certain conditions. The situation seems to be the same in ro­dents: l. Endogenous IFNs, which were first identi­fied in murine placenta, 15 have been found throughout fetal murine gestation but not in significant amounts in early embryos. 16 In hu­mans as in rodents, the initiation of implanta­tion occurs early. In contrast, in ungulates with an epitheliochorial placenta, embryos which express a high leve! of trophoblast IFN, attach to the uterine epithelium later and penetration into this materna! layer does not occur. fctal conncctivc tissuc troph_oblast /; B'."ctal cndothd,um rt _, Jl( . rt J& ., . jJ( & 1 .--,)ft[\.,< 0 /.. O !Oj .. J5 „ e 0 ,. ·.. & .. ,,, )-.:-JI( .. ,3/ ­ j§{;f "0 0 :,,r " JI{.,,, ; 0'!J'." 0 ) rt & "0 :,,r t\ .,e -. ) .[!1.El. -.J ,-r. t\. J .,,,. .. &"0 . 0 „ Jl( 0 M01ernal 8/ood Fetai 8/ood Epilhelio-chorial Placenta Synepilhelio-chorial Placenta Endothelio-chorial Placenta Hemo-chorial Placenta Figure 3. Diagrammatic. rcprcscntation of comparativc placcntation in rclation to invasion of thc utcrinc laycrs. 'f)'pe I i11terf'eron1· spontaneously expressed during human pregnancy 2. Like humans, rodents have a hemochorial placenta, but some differences are observed: the murine trophoblastic epithelium is trilami­nar (ancl not monochorial) ancl is arrangecl in a labyrinthic pattern, without forming villi. Once the implantation of the blastocyst has been initiatecl, the fetal trophoblast invades ali materna! layers, penetrating as far as the mater­na! bloocl, in these placentae (Figure 3). IFNs may be considered as negative growth factors,17 inhibiting celi proliferation by inducing cellular cycle arrest in the G0/G 1 phase and the decrease of c-myc expression. 18 Therefore, we suggest that the early cytotrophoblast, which is highly proliferative and invasive, with a high expres­sion of proteases, is not controlled by a mole­cule which has properties of an IFN. 3. One of the IFNs detectecl in murine fetal 20 annexes seems to be an atypical molecule. 19· In humans, the RNA population of the same placenta! sample might contain different IFN transcripts and, among them, some high mole­cular weight transcripts relatecl to an IFN-a gene. These high molecular weight placenta! IFN-a like transcripts could be those of a new IFN gene involvecl in fetal development. Conclusion Presently, functions of a human IFN-a in preg­nancy are almost speculative. As previously suggested: Human placenta! IFN-a coulcl protect the fetus against vira! infections. -IFN-a coulcl inhibit the lymphocytes proli­ferative response to Interleukin-2 and thus pro­tect the fetus against rejection by materna! cytolytic cells. -IFN could also down-regulate tissue growth previously promoted by growth-factors, and thus limit tissue invasion. -As IFN is a powerful inclucer of class l antigen, it may induce expression of a non polymorphic class I antigen of the Major Histo­compatibility Complex, the HLA-G antigen in cytotrophoblast cells.21 -Moreover, it could enhance production by syncytiotrophoblast of Human Chorionic Gona­dotropin (hCG) which is presumecl the main luteotrophic factor in early human pregnancy. This increase of hCG by IFN-a has been shown in ectopic bladcler tumor cells.22 IFNs are immunoregulatory molecules. They might serve as intercellular communication sig­nals between the immune and reproductive systems, ancl seem to be requirecl for successful pregnancy. The presence of IFNs in human and murine fetal annexes, the diseovery of the trophoblast Interferon in ruminants, the expression of a type II Interferon gene and that of a new type l Interferon gene in porcine trophoblast23 pro­vide a eonsiclerable insight into interactions between the embryo and mother cluring preg­nancy thus opening a new area of Interferon biology. References l. Tovey MG, Streuli M, Gresser I. Gugenheim M. Blanchard B, Guymarho J, Vignaux F ancl Gigou M. Interferon messenger RNA is producecl consti­tulively in the organs of normal inclividuals. l'roc Natl Acad Sci US/\ 1987; 84: 5038-42. 2. Vanden Broecke C and Tovey MG. Expression of the genes of class I Interferons and Interleukin­6 in inclividual cells. J lnterf' Res 199 l; It: 9!-!03. 3. Lebon P, Girard S, Thcpot F and Chany C. The presencc of a Interferon in human amniotic fluicl. J Gen Virol 1982; 59: 393-6. 4. Duc-Goiran P, Robcrt-Galliot B, Lopcz .1 and Chany C. Unusual apparcntly constitutive Interfc­rons and antagonists in human placenta! blood. Proc Natl /\cad Sci USA 1985; 82: 5010-4. 5. Chard T, Craig PH, Menabawey M ancl Lee C. A c;-interferon in human pregnancy. Br J Obst and Gynaecol 1986; 93: 1145-9. 6. Howatson AG, Farquharson M, Mcager A, McNi­col AM, Foulis AK. Localization of a-interfcron in thc human fcto-placental unit. .I Endocr 1988; 119: 531-4. 7. Khan NU-D, Pulford KAF, Farquharson MA, Howatson A, Stewart C, Jackson R, McNicol AM and Foulis AK. The distribution of immunoreac­tive intcrfcron-alpha in normal human tissues. Jmmunology 1989; 66: 201-6. 8. Jones KP, Edwin SS, Warnock SH, Mitchcll MD, Urry RL. Immunosuppressivc activity and alpha interferon conccntrations in human cmbryo cul­ture media as an indcx of potential for succcssful implantation. Fertility a11d Sterility 1992; 57: 637-40. Duc-Goiran et al. 9. Branca AA. High-affinity rcceptors for human interferon in bovine Jung and human placenta. J Inte1f Res 1986; 6: 305-11. 10. Duc-Ooiran P, Chany C, and Doly J. Unusually large Interferon-a-like mRNAs and high expres­sion of Interleukin-6 in human fetal annexes. J. Biol Chem 1989; 264: 16507-11. 11. Charpigny O, Reinaud P, Huet JC, Ouillomot M, Charlier M, Pernollet JC and Marta] J. High homology between a trophoblastic protein (trop­hoblastin) isolated from ovine embryo and a-Inter­ferons. FEBS Letters 1988; 228: 12-6. 12. Imikawa K, Anthony RV, Kasemi M, Marotti KR, Polites HO and Roberts RM. Interferon-like sequcnce of ovine trophoblast protein secreted by embryonic trophectoderm. Nature 1987; 330: 377-9. 13. Imikawa K, Hanscn TE, Malathy PV, Anthony RV, Polites HO, Marotti KR and Roberts RM. Molecular cloning and charactcrization of comple­mentary deoxyribonucleic acids corresponding to bovinc trophoblast protcin-1 : a comparison with ovinc trophoblast protcin-1 and bovinc Interferon­aII. Molec Endocr 1989; 3: 127-39. 14. La Bonnardiere C, Martinat-Bottc F, Tcrqui M, Lefevre F, Zouari K, Marta] J and Bazcr FW. Production of two specics of interferon by Large White and Meishan pig conceptuscs during the pcri-attachmcnt period. J Reprod Feri 1991; 91: 469-78. 15. Fowlcr AK, Rced CD and Oiron DJ. Identifica­tion of an Interferon in murine placentas. Nature 1980; 286: 266--7. 16. Baker DJ and Nieder OL. Interferon activity is not detccted in blastocyst secretions and does not induce dccidualization in mice. J Reprod Fert 1990; 88: 307-13. 17. Sporn MB and Roberts AB. Autocrine growth factors and cancer. Nature 1985; 313: 745-7. 18. Einat M, Resnitzky D and Kimchi A. Close link between reduction of c-myc expression by interfe­ron and Oo/O1 arrest. Nature 1985; 313: 597-8. 19. Weislow OS, Kiser R, Allen PT, Fowler AK. Partial purifieation of a placenta] interferon with atypical characteristics. J Inte,f Res 1983; 3: 291-8. 20. Yamada K, Shimizu Y, Okamura K, Kumagai K and Suzuki M. Study of interferon production during pregnancy in mice and antiviral activity in thc placenta. Am J Obstet Gynecol 1985; 153: 335-41. 21. Kovats S, Main EK, Librach C, Stubblebine M, Fisher SJ and DeMars R. A class I antigen, HLA-O, exprefsed in human trophoblasts. Science 1990; 248: 220--3. 22. Iles RK and Chard T. Enhancement of ectopic .-human chorionic gonadotrophin expression by Intcrferon-a. J Endocr 1989; 123: 501-7. 23. Lefevre F and Boulay V. A novel and atypieal type One interferon gene cxpressed by trophoblast during early pregnancy. J Biol Chem 1993; 268: 19760-8. Radio/ Oncol 1993; 27: 271-4. Changes in the quantity of cathepsin D in irradiated human cells f ollowing treatment with hyperthermia and interferon a Ana Ferle-Vidovic, 1 Marija Kaštelan, 1 Danilo Petro vic, 1 Janez Škrk,2 Ivan Vrhovec2 1 2 Ruder Boškovic Institute, Zagreb Croatia, Institute of Oncology, Ljubljana, Slovenia In the presen! work, the changes in quantity of cathepsin D, an aspartic proteinase, in proliferating human nonmalignant (HEF) and malignant (HEp2) cells after combined treatment by gamma irradiation, hyperthermia and by interferon a-2b (IFNa) were followed. Correlation between the antiproJiferative effect of these combined agents and changes in the concentrations of this cathepsin D were expected. Evidently, the treatment of cells in culture by IFNa, combined with irradiation a,;,d elevated temperature, produces an increased quantity of cathepsin D. In nonmalignant HEF cells these effects are more expressed than in malignant HEp2 cells. Key words: cathepsin D; cell, cultured-radiation effects; hyperthermia, induced; interferon-alfa-2B Introduction Intracelular proteinases participate in the vita! cellular processes such as growth and rnultipli­cation, response to DNA darnage and radiation 1234 -response. • • • Recent studies suggest that the aspartic proteinase cathepsin D; rnay also be implicated in the process of tumor invasion and rnetastasis, 5 Severa! in vitro observations s110­wed that this proteinase rnay facilitate the spread of neoplastic cells through different rnec­hanisms releated to its proteolytic activity, by acting at different levels of the rnetastatic casca­de. Cathepsin D was also shown to be able to degrade in vitro the extracellular rnatrix, and Correspondence to: Ana Ferle Vidovic, Ph. D., Ruder Boškovic Institute, Bijenicka cesta 54, 41000 Zagreb, Croatia UDC 615.281.7:577.156.2 to activate Iatent precursor forrns of other pro­teinases involved in the invasive steps of the rnetastatic process. Interferon a-2b (IFNa) initially recognized for its antiviral effects, has also been shown to have antiproliferative, imrnunoregulatory and antiturnor activities.6 There is some experirnen­tal evidence supporting the concept that rnodest levels of hypertherrnia rnight be beneficial to the action of interferons. This concept is sup­ported by in vivo experirnents showing that rnodest levels of hypertherrnia enhanced the action of interferon.7 In vitro it was found that hyperthermia acted synergistically by enhancing the proliferative effects of IFNa.8 There is definitive experirnental evidence in vivo for synergistic effect of this cornbined treatrnent.9 Sirnilar synergistic effects were also observed, when interferon was applied in combination with irradiation. 10 Ferle-Vidovic A. et al. Such results may have clinical importance, because they suggest that hyperthermia could be used in combination with IFNa. to provide 10 a synergistically enhanced antitumor action.9· Therefore, it is possible that the combination of hyperthermia and IFNa. therapy may have clinical application in cases when technically feasilbe. Such combined treatment can, how­ever, induce an increase in proteolitic enzymes in cells, which might enhance metastasing of thc treated tumor. In this work we determined the changes in the quantity of the aspartic intracellular pro­teinase -cathepsin D in irradiated human non­malignant (HEF) and in malignant (HEp2) cells after combined treatment with hyperther­mia and/or IFNa.. Correlation between the an­tiproliferative effect of these combined agents and the changes in concentrations of this cat­hepsin was found. Materials and methods Cell cultures and experimental procedure Human embrional fibroblasts (HEF) and hu­man laryngeal carcinoma (HEp2) cells, were cultured as monolayers in Eagle's minimal es­sential medium, supplemented with 10 % foetal calf serum. Celi cultures were prepared by plating 105 of 106 cells per Petri dish of 10 cm in diameter (3 dishes per experimental point) and after two days of growth, cells were irradia­ted, treated by IFNa. and by hyperthermia in the following combinations: irradiation only, IFNa. only, irradiation plus IFNa., irradiation plus IFNa. plus hyperthennia. Following the mentioned treatment, celi cul­tures (106 per dish) were kept at 37 ° C and samples taken after one hour were stored at -20 ° C until proteinase quantity assay. The number of proliferating cells (105 per dish) following irradiation and combined treatment were counted 24 and 96 hours after treatmen. Interferon Recombinant interferon a.-2B (INTRON-A, Schering-Plough-Baltimore-USA) was added to the growth medium to reach fina! concentration of l X 104IU/ml. Cells were incubated in the IFNa.-containing medium at 37 ° C for 1 hour and then incubated until additional treatment or harvesting. Hyperthermia Heat treatment was conducted by submerging the Petri dishes in a water bath at 44 ° C for 20 min. HEF and HEp2 cells were exposed simul­taneoulsy to IFNa. and to hyperthermia. Jrradiation For gamma irradiation, a Gamma Celi 220 (Atomic Energy of Canada, a.td) unit was used. The dose rate was 4,13 Gy/min, with the total dose 15 Gy/sample for cathepsin D, or 5 Gy for the growth inhibing effect. Cathepsin D concentration Following treatment, celi cultures were incubat­ed in the growth medi um at 37 ° C. Samples were taken after different tirne intervals, placed on ice and washed three times with cold phos­phate buffered saline. The cells were harvested by a rubber policeman, concentrated by centri­fugation (10 min at 1000 rpm), lysed in distilled water and frozen at -20 ° C until assay. The concentrations of cathepsin D were de­termined using specific enzyme immunometric assay (ELSA-CATH-D kit, CIS Bio Internatio­nal, Solid phase two-site immunoradiometric assay), for the quantitative determination of total cathepsin D in cytosol. Results The combined effects of IFNa., hyperthermia and irradiation on celi proliferation, expressed as the number of growing HEF celi population, are shown in Table l. The antiproliferative effects were expressed as percentage of cel! numbers in control samples. White irradiation or IFNa. alone, moderately inhibited celi growth, the treatments, by IFNa. plus irradia­ lrradiation, lzypertlzermia, inte1feron ,1 and catlz!'psi11 D tion, and in particular, when IFNn was cornbin­ed with irradiation and hypertherrnia, the anti­proliferative effect was rnarkedly enhanced. Table l. Celi numbers (x105 HEF cells) after combined treatment with irracliation ancl/or interferon a and/or hyperthermia. Group of Incubation time Percent of treatment after treatment control 24h 96h 24h 96h Controls 10,9 ± 2,6 20,4±3,1 Table 2. Celi numbers (x 105 HEp2 cells) after combin­ed treatment with irracliation and/or interferon a and/ or hyperthermia. Group of Incubation time Percent of treatment after treatment control 24h 96h 2411 96h Controls 16,4± 1,4 41,0 ± 4,2 lrradiation 12,0±0,9 18,6 ± 1,6 73 45 Interferon a 11,0±2, l 21,8 ± 4,0 67 53 Irradiation + Interferon a 8,0±1,1 l0,0 ±0,7 48 24 Irradiation + Irradiation 5,0 ± 0,9 7,5 ± 1,1 46 Interferon n + 3,6 ± 0,8 7,7 ± 0,7 22 Interferona 6,6 ± 0,2 8,7± 1,7 61 Hyperthermia lrradiaton + Interferona 3,7 ±0,1 4,0±0,4 I rracliation + 20 The changes in the concentrations of catheps­ 1,8±0,1 1,9±0,2 16 9 Interferona+ in D were dependent on the agent used. Hyperthennia Gamma iradiation alone revealed little change in the concentration of cathepsin D in the malignant celi line (1.1), whereas the same changes were more evident in the nonmalignant cells (1.3). Interferon n increased the levels of cathepsin D in both celi strains. These effects are more expressed in malignant cells (1.5), The combined effects of IFNn, hypertherrnia and irradiation on celi proliferation, expressed as the numbers of growing HEp2 celi popula­ tion, are shown in Table 2. The antiproliferative effects were expressed as percentage of celi numbers in control samples. While irradiation than in nonrnalignant cells (1.2). Interferon n or IFNn alone, moderately inhibited HEp2 celi plus irradiation increased the concentration of growth, the treatmens by IFNn plus irradiation, cathepsin D significantly more than in the for­and in particular when IFNn was combined mer two cases, when the agents were applied with irradiation and hyperthermia, the antipro­separately. The increased concentrations of the liferative effect was markedly enhanced. enzyme were similar in rnalignant (1.6) as in Changes in the concentrations of cathepsin nonmalignant (1.8) cells. Combined application D measured in irradiated, proliferating human of ali three agents together was most effective nonmalignant (HEF) and human malignant in increasing the amounts of cathepsin tested. (HEp2) celi lines following combined treatment Most evident effects were achieved in both celi by interferon n and by hyperthermia are shown lines after combined treatment with ali three in Table 3. agents and again, with more expressed effects Table 3. Changes in the quantity of Cathepsin D in irracliatecl HEF ancl HEp2 cells following combinecl treatment. Group of HEFcells HEp2 cells treatment Cathepsin D T/C* Cathepsin D T/C* ng/mg proteins ng/mg proteins Controls l05 ± l1 562 ± 14 Irracliation 138 ±7 1,3 604 ± 12 1,1 Interferon n 127±9 1,2 875 ±22 1,5 1 rracliation + Interferon n 198 ±26 1,8 856 ± 31 1,6 Irracliation + Interferon n + 332 ± 16 3,1 1407 ± 53 2,5 Hyperthermia * T/C Treatecl/Control Ferle-Vidovic A. et a/. on the nomnalignant HEF celi line (3.1), the in the malignant HEp2 cells (2.5). Discussion Cathepsin D, as mentioned before, plays an important role in tumor invasion and metasta­sis. Conelation between elevated levels of the enzyme in tumor cells and their ability to meta­stasise were found.5 On the other hand in our previous experiments we found that agents used in tumor therapy can influence the concentra­tions of various intracellular proteolytic enzy­mes, either by increasing or decreasing their concentrations11 or activities. 12 This raises the question, particularly when cathepsin D is con­cerned, whether a particular tumor treatment could perhaps, apart of its cell killing potential, have some unwanted effects due to possibly elevated levels of cathepsin D. Our results show that such agents (irradia­tion, interferon a and heath), when given in amounts that evidently produce celi growth inhibition, can significantly increase the intra­cellular concentrations of cathepsin D, and the­refore could consequently enhance the potential of the tumor cells to infiltrate the neighbouring tissues or to metastasise. lf this occurs in a tumor bearing organism, this should also be kept in mind, when predicting the outcome of a particular tumor therapy. This may be even more important at combined modality therapy regimens. Acknowledgement We thank Mrs. Ljiljana Krajcar for her excel­lent technical assistance. This project was sup­ported by the Ministry of Science of the Repu­blic of Croatia and the Ministry of Science and Technology of Republic of Slovenia. References l. Holzer H, Heinrich PC. Control of proteolysis. Ann Rev Biochem 1980; 40: 63-91. 2. Korbelik M, Škrk J, Suhar A, Turk V. The role of proteinases, interfcrons and hormones in proli­fcrative activities of nonmalignant and malignant cells. Neoplasma 1988; 35: 555-63. 3. Schcr W, Scher BM, Waxman S. Proteases stimu­late mouse erythroleukemia celi differentiation and multiplication. Biochem Biophys Res Comm 1982; 109: 348-54. 4. Walker CG. Induclible DNA repah-systems. Ann Rev Biochem 1985; 54: 425-57. 5. Leto G, Gebbia N, Ransa L, Tuminello FM. Cathepsin D in the malignant progression of neo­plastic diseases (Review). Anticancer Research 1992; 12: 235-40. 6. Baron S, Tyring SK, Fleischmann WR Jr et al. The interfcrons: mechanisms of action and clinical applications. JAMA 1991; 266: 1375-83. 7. Heron I, Berg K. The actions of interferon are potentiated at elcvated temperatures. Nature 1978; 274: 508-10. 8. Anjum A, Fleischmann WR Jr. Effect of hyper­thermia on the antitumor actions of interfcrons. Journal of Biologica/ Regulators and Homeostatic Agents 1992. 6: 75-86. 9. Park RI, Richtsmeier WJ. Hyperthermia effects on the growth of a laryngeal squamous celi carci­noma celi line treated with recombinant human interferons a. and y. Oto/ Laryngol-Head and Surgery 1989; 101: 542-8. 10. Perez CA, Nussbaum G, Emami B., Vongerichten D. Clinical results of iradiation combined with local hyperthermia. Cancer 1983; 52: 1597. 11. Ferle-Vidovic A, Kaštelan M, Petrovic D, SveticB, Škrk J, Gabrijelcic D, Turk V. Cytotoxicity potentiation of irradiation and cytostatic -mcasur­cd by changes in quantity of intracellular proteina­ses. Proc. of the First Symp. of Croatian Rad. Protect. Ass., Zagreb, Croatia 1992; 68-71. 12. Petrovic D, Fcrle-Vidovic A, Škrk J, Suhar A, Turk V. Effects of irradiation and THP-Adriamy­cin on the proteinase activity profiles in cultures V 79 cells. Radio/ Oncol 1993; 27: 44-8. Radio! Oncol 1993; 27: 275-9. Interactions of interferon and vinblastine on experimental tumor model melanoma B-16 in vivo Barbara Jezeršek,1 Srdjan Novakovic,1 Gregor Serša,1 Maja Cemažar,1 Marija Auersperg,1 W. Robert Fleischmann Jr.2 1 Institute of Oncology, Ljubljana, Slovenia, 2 University of Texas Medica! Branch, Department of Microbiology, Galveston, Texas, USA In the study, we investigated the in vivo interaction of two antitumor agents, that have different sites and different mechanisms of action. Vinblastine (VLB) in combination with human recombinant interferon a AJD (rHulFN-a AJD) and in combination with human leukocyte interferon a (HuLIFN-a) was tested on intraperitoneal (i.p.) melanoma B-16 tumor model. The effect of the combination was determined with follow-up of animals' survival and the interaction defined by means of Spector's formula. Only subadditive enhancement of interferon's (IFN's) antitumor activity was observed when rHulFN-a AJD was combined with VLB and supraadditive, but not synergistic, interaction when HuLIFN-a was combined with VLB. Synergism between VLB and rHulFN-a AJD on B-16 melanoma in vitro, that had been observed in our previous study, did not come true in vivo. Key words: melanoma, experimental-drug therapy; vinblastine; interferon alpha, recombinant; Introduction Chemotherapy and biotherapy are the two sy­stemic modalities available for cancer treat­ment. However, because it is apparent that neither one nor the other are perfect treatments for cancer, the combination of cytotoxic drugs and cytokines offers a new approach to increase the therapeutic index in the treatment of neo­plastic diseases. 1 •2 Interferons (IFNs) are a complex group of cytokines with antiviral, antibacterial, antitu- Correspondence to: Jezeršek Barbara MD, Institute of Oncology, Zaloška 2, 61105 Ljubljana, Slovenia, Tel. + 386 61 323 063 ext. 29 33, Fax + 386 61 131 41 80 mor and immunomodulatory activities.3 .4 They exert antiproliferative effect on tumor cells, while IFNs . and y also have a direct cytotoxic activity.5 Antitumor activity of VLB is a conse­quence of its binding to microtubular proteins of the mitotic spindle, which causes metaphase arrest of cells in mitosis.6• 7 VLB is, in higher concentrations, also directly cytotoxic for inter­phase cells.8 While in vitro studies have demonstrated both direct cytotoxic and cytokinetic effects of IFNs, a more interesting role derives from their ability to sinergistically potentiate the wide va­riety of cytotoxic agents against multiple human and rodent tumors, both in vitro and in animal models.9 The broad spectrum of cytotoxic drugs whose activity can be enhanced by cytokines UDC: 616-006.81-085 argues for multiple levels of drug interaction in Jezer§ek B. el al. vitro: alteration of cellular drug uptake, modu­lation of drug target enzymes, and changes in metabolism or disposition of a drug. In vivo interaction between cytokines and cytotoxic drugs involves an additional layer of complexity because of the effects of cytokines on the host immune system and on drug-metabolising enzy­mes. 2 The ability of IFNs to directly modulate the biochemical effects of cytotoxic agents indepen­dent of immunomediated or host-protective ef­fects has been evaluated in a variety of in vitro systems.9 Since synergistic cytotoxicity has been observed in vitro for IFN-a combination with VLB on BG-1 human ovarian carcinoma line, 10 on RPMI 8226 human myeloma line, on MCF-7 human breast carcinoma line, on WiDr human colon carcinoma line11 and on murine B-16 melanoma line, 12 we wanted to define the inte­raction of VLB with rHuIFN-a AJD or Hu­LIFN-a in vivo on i.p. B-16 melanoma tumor model. Materials and methods Reagent.1· Recombinant HuIFN-a AJD was provided by Hoffmann-LaRoche (Nutley, New Jersey) and HuLIFN-a by Immunological Institute (Zagreb, Croatia). Both were diluted with phosphate buffered saline (PBS). Vinblastine sulfate (Lymphomed, Deerfield, Illinois) was used in combination with rHuIFN­a AJD and Velbe (Lilly, Firenze, Italy) with HuLIFN-a. Both were diluted with PBS. Animals Six to eight weeks old pathogen-free female C57B1J6 mice were purchased from Jackson Laboratories, Bar Harbor, USA. Animals were maintained in a pathogen-free state in animal rooms with alternating cydes of 12 h light and 12 h darkness. Each experimental group consi­sted of 10 to 11 mice. These animals were used for experiments with rHuIFN-a AJD and Vin­blastine sulfate. Femalc C57Bl/6 were purchased from Rudjer Boškovic Institute, Zagreb, Croatia. Animals were maintained at a natura! dayJnight cyde in a standard animal colony. Eight to ten weeks old mice in good condition without any signs of fungal or other infections were used in the experiments. Each experimental group consi­sted of ten mice. These animals were used for experiments with HuLIFN-a and Velbe. Tumor cells Murine B-16 melanoma cells (done Fl, Ameri­can Type Culture Collection, Rockville, Mary­land) were grown in Eagle's Minimal Essential Meditlll1 supplemented with 10% fetal calf se­rum, penicillin (100 unitsJml), streptomycin (100 .tgJml) and gentamycin (11 .tg/ml). These cells were used for experiments with rHuIFN-a AJD and Vinblastine sulfate. Murine B-16 melanoma cells ( done B6, Ru­djer Boškovic [nstitute, Zagreb, Croatia) were grown in RPMI 1640 mcdium supplementcd with 10% fetal calf serum, gentamycin (500 .tgJml) and 7,5% sodium bicarbonate (27 ml/!). Thcse cells were used for experiments with HuLIFN-a and Velbe. Experimental procedure Mice were i.p. injected with 106 B-16 melanoma ( done Fl or B6) cells on day O and randomly divided into four groups. Ali treatment was intraperitoneal and was administered as follows: control group -PBS for five consecutive days, starting day 1 -vinblastine group -Vinblastine sulfate or Velbe (30 µg per animal) on day 4 only -interferon group -rHuIFN-a A/D (1 x 105 I.U. per animal) or HuLIFN-a (5 x 105 I.U. per animal) for five consecutive days, starting day 1 combination group -Vinblastine sulfate or Velbe (30 µg per animal) on day 4 only + rHuIFN-a AJD (1 x I.U. per animal) or 105 HuLIFN-a (5 x 105 I.U. per animal) for five consecutive days, starting day l. Interactions of IFN-a mul VLB in vivo The mice were monitorecl for the clay of cleath ancl the average clay of cleath was deter­minecl. Statistical analysis The Mante! -Cox test (BMDP Statistical Sof­tware, Los Angeles, California) was employecl for comparison of the animals' survival ancl Spector's formula 13 to clefine the interaction of rHuIFN-a AJD or HuLIFN-a with VLB. Results Vinblastine sulfate ancl rHuIFN-a AJD as single agents or in combination were testecl for their effect on survival of animals with i.p. B-16 melanoma (Fl). Intraperitoneal application of 30 .tg of Vinblastine sulfate on clay 4 hacl a moclerate (p 0,054) antitumor effect, while treatment with 1 x I.U. of rHuIFN-a AJD 105 for 5 consecutive clays showecl a more pronoun­cecl statistically significant (p<0,001) effect on 10 20 30 40 50 60 DAYS figure l. Survival of mice with i.p. B-16 melanoma (clone Fl) trcatecl with Yinblastine sulfate (e), rHuIFN-a A/D (.) or combination of both agents (111); control (O). The antitumor effcct of the combi­nation was mercly subaclclitivc in comparison to thc one expccted on the basis of separate activities of YLB or IFN. 100 90 80 70 (/) o:: 60 50 .J (/) 40 a'2 30 20 10 o 10 20 40 50 DAYS Figure 2. Survival of micc with i.p. B-16 melanoma (clone B6) treatecl with Yclbe (e), HuLIFN-a (.) or combination of both agents (1111); control (0). The effect of the combination on survival of thc animals was supraaclclitive in comparison to thc one expcctccl on the basis of separate activities of VLB or IFN. survival of mice with i.p. B-16 melanoma. The combination of both agents hacl a statistically significant (p<0,001) antitumor effect, but there was no significant difference (p 0,497) in survival between the "interferon group" ancl the "combination group" of animals (Figure 1). Accorcling to Spector's formula the interaction of rHuIFN-a AJD with VLB was merely subad­ditive ancl the antitumor effect of the combina­tion was 90% of the one expectecl on the basis of their separate activities. Survival of animals with i.p. B-16 melanoma (B6) treatecl with HuLIFN-a or Velbe alone or in combination is presentecl in Figure 2. Treat­ment with 5 x 105 l.U. of HuLIFN-a for five consecutive clays hacl a moderate (p = 0,059) antitumor effect, while i.p. application of 30 µg of Velbe significantly (p<0,001) prolonged sur­vival of the animals. The combination of both agents had a statistically significant (p<0,001) antitumor effect, but there was no significant difference (p 0,058) in survival between the "vinblastine group" ancl the "combination group" of animals. The interaction of HuLIFN­a with VLB was supraadclitive ancl the antitu­ 278 Jezeršek B. mor effect of the combination was 115% of the one expected on the basis of their separate activities. Discussion The results of in vivo studies testing the combi­nation of IFNs with VLB are controversial. Sidkey et al. report that murine IFN a/. increa­sed survival in mice with P388 leukemia cells after treatment with VLB. 14 Harrison et al. on the other hand found no positive interaction when murine IFN a/. was combined with VLB on s.c. Meth A sarcoma tumor model and when recombinant murine IFN-y was combined with VLB on s.c. Meth A sarcoma and s.c. B-16 melanoma.15 Also Mitchell has pointed out that although type I IFN has been found to poten­tiate chemotherapy in cultured cells, "there is very little substantiation in vivo". 16 Our results clearly demonstrate that in vitro synergism between rHulFN-a A/D and VLB observed on Fl done of B-16 melanoma cells12 did not come trne in vivo. However, there is an interesting difference between the antitumor activity of combination of rHulFN-a A/D with VLB and combination of HuLIFN-a with VLB. Even though HuLIFN-a alone bas only mode­rate antitumor activity, the interaction with VLB was supraadditive, in comparison with rHuIFN-a A/D that has significant antitumor activity, but demonstrated only subadditive in­teraction with VLB. In part this difference could be explained with the fact that different clones of B-16 melanoma were used in the experiments, but Sklarin et al. report that in most of the cases where potentiation was obser­ved, human IFN-a alone had only weak antitu­mor activity; however, IFN-a seemed to be most effective in combination with drugs that alone possessed substantial activity against the specific tumor. 17 The question of the mechanism of interaction between IFNs and VLB stili cannot be resolved and it seems likely that multiple factors may be contributing to success or failure in these prec­linical models. The interactions observed are et al. not solely the consequence of the combined effect of two cytoreductive agents, since the enhanced activity of the drug-interferon combi­nation was observed even in instances where IFNs alone lacked activity, and IFNs also failed to potentiate the activity of other efficacious drugs. 9 There is also a complex relationship between the timing of interferon (IFN) with a cytotoxic agent, the doses used, and the efficacy of the regimen. In combination with cytotoxic drug, sequence and duration of exposure to IFN may play as significant a role as 0.05) (Figure 2, 3). Also, there was no statistical difference between the effectiveness of IFN-a after peritumoral and intraperitoneal application (p = 0.6). In order to test for interaction of IFN-a treatment with electrotherapy, both treatment modalities were combined. Electrotherapy (0.6 mA for 1 hour) as a single treatment statistically significantly delayed tumor growth (P < 0.001) (Figure 2, 3). In combined modality treatment electrotherapy was performed one hour after the first IFN-a application. The interaction was better when electrotherapy was combined with intraperitoneal IFN-a treatment than with peri­tumoral application (Figure 2, 3). Additive an­titumor effect was obtained with peritumoral Anti-tumor effect of IFN-a and electrotherapy treatment and more than additive with intrape­ritoneal treatment. . .!"1,t-! ,oo k. . A/ x i 50 . f /y! 5 . '° ..,A • conLrol II WN-o:. o ET O • ip .6mA . ET&IFN o: 1 p 1 O Days afler LreaLrnenL Figure 3. Antitumor cffcct of IFN-a and clcctrothc­rapy on subcutancous SA -1 tumors. IFN-a (5 x 104 U) was injcctcd intrapcritoncally fivc consccutivc days, starting on day O. Elcctrothcrapy was pcrformcd with 0.6mA one hour aftcr IFN-a trcatmcnt on day O. Expcrimcntal groups comprisccl 8-10 animals. 14 13 • ET field 12 o F:T & IF'N a. 1 . 11 crJ 10 -cJ :>-, crJ 8 (1) -cJ 6 . 5 o 4 .... (/ C) 3 2 --------------_ t _ ------_ ! : P: _ :.-. :-_ ':_ ---. o +--,-.-.--.--,-.,-.-.-.-.-. 0.2 0.4 0.6 0.8 1.0 1.2 Current (mA) Figure 4. Tumor growth clelay after electrotherapy or combinecl modality treatment with elcctrotherapy and IFN-a. Elcctrotherapy was pcrformed with different current lcvels one hour after IFN-a treatment on clay O. The clata are presentecl as arithmetic means andstandard deviations of the mcan. Thc tumor growthdelay after treatmcnt with IFN-a alone is presentecl as an average tumor growth delay. Experimcntal groups consistecl of 10 animals. To determine how IFN-a therapy interacts with escalating electrotherapy doses, combined modality treatment was performed with diffe­rent direct current levels, ranging from 0.2 mA to 1.2 mA. IFN-a treatment schedule remained the same as in the previous experiment. Rela­tionship between the effectiveness of therapy, presented as tumor growth delay, in relation to electrotherapy at different current levels, is presented in Figure 4. The interaction of IFN-a with electrotherapy was additive up to 0.4 mA and more than additive from 0.6 mA on (Table Table l. Tumor growth clclay aftcr elcctrotherapy (ET ) alone or in combination with IFN-a. Growth Delay (clays) ET only ET+ IFN-a1 ET0.2 mA 1.0 ± 0.5 (u 15)2 1.9 ± 0.5 (u = 15) ET0.4mA 1.8 ± 0.4 (u =15) 2.8 ± 0.6 (u = 16) ET0.6mA 2.0 ± 0.6 (u = 18) 3.8 ± 0.6 (u = 18) ET0.8mA 4.9 ± 1.4 (u = 14) 8.0 ± 2.4 (u = 16) ET1 .2 mA 8.3 ± 1.7 (u = 15) 11 .6 ± 1.9 (u= 16) 1 Tumor growth clelay after intraperitoneal IFN-a treatment was l.!± 0.5 (u = 15) 2 Tumor growth dclay in clays (AM ± SD), u degrec of frecclom Discussion The study shows that electrotherapy and IFN-a treatment internet in control of fibrosarcoma SA-1 tumor growth. More than additive antitu­mor effect was obtained when electrotherapy was combined with intraperitoneal IFN-a treat­ment. The interaction between the two treat­ment modalities increased by escalating current levels. Electrotherapy is a new treatment modality used in local control of tumor growth.9• 11• 19 Its antitumor mechanisms are probably multiple: biochemical reactions in the vicinity of the electrodes and influences of electric current 20• 21 directly on tumor cells.17• Among bioche­mical reactions are changes of pH and changes of ion composition in extra cellular matrix which ali exert influence on cell growth and Serša G. and Miklavcic D. survival. 17 Effeetiveness of eleetrotherapy is predominantly dependent on eleetrie eurrent intensity.11· 19 With eurrents 1.8 mA a growth delay of approximately 12 days ean be aehieved on SA-1 tumors, while on B-16 melanoma tumor model even tumor eures ean be indu­eed.19 Nevertheless, after the treatment viable tumor eells remain, whieh again give rise to a tumor. In order to potentiate effeetiveness of eleetrotherapy, and eradieate the remaining tu­mor eells, attempts were made to eombine eleetrotherapy with radiotherapy, 22 ehemothe- . 13 · 15 14 · an d b' 1 I response mo d'f' 1 1ers. rapy. 10 og1ea 16 23 · I n mos t eases a 1t1ve or supra-a dd' . ' dd' . 1t1ve effeets were obtained. Our interest was foeused on eombinations of eleetrotherapy with biologieal response modi­fiers. The studies eombining interleukin-2 (IL­2), 14 tumor neerosis faetor alpha (TNF-a)23 and human leukoeyte interferon alpha (IFN-a)16de­monstrated that stimulation of host's defenee meehanisms eontributes to antitumor effeetive­ness of eleetrotherapy. Depending on the biolo­gieal response modifier used, different arms of the eytokine network are stimulated, but in ali eases the effeetiveness of eleetrotherapy was inereased. In our preliminary study we have already tested the eombined modality treatment of hu­man leukoeyte interferon alpha (IFN-a) with eleetrotherapy on SA-1 tumor model.16 In that study IFN-a treatment protoeol was the same as in the present study, however, eleetrotherapy protoeol was different. Repetitive eleetrothe­rapy treatment was not very effeetive, therefo­re, aeeording to later experienee we applied the "field" eleetrotherapy as a single treatment.17 As demonstrated in the present study, the effeet is dose dependent resulting in a moderate anti­tumor effeet at 0.2 mA eurrent leve!, and a signifieant growth delay at 1.2 mA. Comparison of the IFN-a antitumor effeets aeeording to the route of applieation demonstrated that IFN-a is moderately effeetive at the doses used. No differenee in the antitumor effeetiveness of IFN­a was observed, given either loeally or systemi­eally. But when eombined with eytoreduetive eleetrotherapy, systemie treatment was more effeetive than Ioeal treatment. Although IFN-a was demonstrated to be eytostatie to SA-1 eells in vitro, it is very unlikely to reaeh suffieiently high eoneentrations in the tumor to exert sueh an effeet, when injeeted loeally or systemieally in vivo. Therefore, enhaneement of the antitu­mor meehanisms of the organism must be eon­tributing to the supra-additive effeet of eleetro­therapy eombined with systemie IFN-a treat­ment. The interaetion of IFN-a treatment with elee­trotherapy was dependent on antitumor effeetiveness of eleetrotherapy. With esealating eleetrotherapy doses also eombined modality treatment was more effeetive. This demonstra­tes that adjuvant IFN-a treatment was more effeetive on a smaller tumor burden. The doses used in both treatment modalities were low and no treatment related side effeets were observed. Our study shows that IFN-a and eleetrothe­rapy internet in antitumor effeetiveness on fi­brosareoma in miee. Combined use of IFN-a and eleetrotherapy resulted in effeetive tumor eontrol. Thus, eleetrotherapy ean be used to loeally potentiate systemie IFN-a treatment. Further studies are rcquired for possible imple­mentation of the investigated treatment ap­proaeh in clinieal praetiee. Acknowledgment This study was supported by The Ministry of Seienee and Teehnology of the Republie of Slovenia, eontraet No. P3-5252-302. The au­thors wish to express their appreeiation to Srdan Novakovic M.Se., Maja Cemažar B.Se., Mira Lavric B.Se. and Olga Shrestha, ali Institute of Oneology, for their helpful suggestions and teehnieal assistanee. References l. Kurzrock R, Gutterman JU, Talpaz M. Intcrfc­rons-a, f,, y: Basic principles and preclinical stu­dics. In: DcVita VT Jr, Hcllman S, Roscnbcrg SA cds. Biologic threrapy of cancer. Philadelphia, Lippincot Company, 1991: 247-74. Anti-tumor effecl of' IFN-u and electrotherapy 2. Frieclman RL, Manly SP, MeMahon M, et al. Traneriptional ancl posttranscriptional regulation of interfcron-inducecl gene expression in human cells. Cel! 1984; 38: 745-55. 3. Samuel CE. Mechanisms of the antiviral action of interfcrons. Prog Nucleic Acid Res Mol Biol 1988; 35: 27-72. 4. Murray HW. lnterfcron-gamma, thc activatcd ma­crophagc, and host dcfcnse against microbial chal­lengc. Ann !ntern Med 1988; 108: 595-608. 5. Trown PW, Wills RJ, Kamm JJ. The prcclinieal development of Rofcron-A. Cancer 1986; 57: 1648-56. 6. Miehalewicz R, Reve! M. Intcrfcrons regulatc the in vitro differentiation of multilineage lympho-my­lcoicl stem eells in hairy celi Ieukemia. Proc Natl Acad Sci USA 1987; 84: 2307-11. 7. Trotta PP. Preclinieal biology of alpha interfcrons. Sem Oncol 1986; 13 (suppl 2): 3-12. 8. Moormeier JA, Golomb HM. Interfcrons: Clinical applications. In: DcVita VT Jr, Hcllman S, Ro­scnbcrg SA cds. Biologic threrapy uf cancer. Phi­ladelphia, Lippincot Company, 1991: 275-353. 9. Watson BW. Thc trcatmcnt of tumors with dircct clcctric currcnt. Med Sci Res 1991; 19: 103-5. 10. David SL, Absolom DR, Smith CR, Gams J, Herbert MA. Effcct of low leve! clircct currcnt on in vivo tumor growth in hamstcrs. Cancer Res 1985; 45: 5625-31. 11. Miklavcic D, Scrša G, Vodovnik L, Bobanovic F, Reberšek S, Novakovic S, Golouh R. Loeal treat­ment of murinc tumors by clcctric dircct currcnt. Electro Magnetobiol 1992; 11: 109-25. 12. Norclcnstrom BEW. Elcctrochcmical trcatmcnt of eanccr. 1: Variablc rcsponsc to anodie and eatho­dic ficlcls. Am J Ciin Oncol (CCT) 1989; 12: 530-6. 13. Norclenstrom BEW, Eksborg S, Bcving H. Elec­troehcmieal trcatmcnt of canecr. II: Effeet of clcctrophorctie influence on aclriamyein. Am .1 Ciin Oncol (CC1; 1990; 13: 75-88. 14. Scrša G. Miklavcic D, Batista U, Novakovic S, Bobanovic P, Vodovnik L. Anti-tumor cffcct of clcctrothcrapy alonc or in combination with intcr­lcukin-2 in micc with sareoma ancl melano·ma tumors. Anti-Cancer Drugs 1992; 3: 253-60. 15. Serša G, Novakovic S, Miklavcic D. Potentiation of bleomycin antitumor effectivcness by clcctro­therapy. Cancer Letters 1993; 69: 81-4. 16. Scrša G, Miklavcic D. Inhibition of SA-1 tumor growth in mice by human leukocytc interferon alpha combinecl with low-lcvcl direct eurrent. Mol Biother 1990; 2: 165-8. 17. Miklavcic D, Scrša G, Kryžanowski M, Novakovic S. Bobanovic F, Golouh R, Vodovnik L. Tumor treatmcnt by direet clcctric currcnt-tumor tempe­rature ancl pH, clcctrodc material ancl configura­tion. Bioe/ectruchem Bioener 1993; 30: 209-20. 18. Ikic D, Lukic V. Juzbašic M, ct al. Interferon procluction in FS-4, MRC-5 and Wl-38 human cliploicl eclls. In: Procecclings of thc symposium on thc prcparation, stanclarclization ancl clinical usc of interferon. 11th lntcrnational Immunobio­logy Symposium. Zagreb: Yugoslav Acadcmy of Scicncc and Arts, 1977; 8-9, 59-63. 19. Scrša G, Miklavcic D. Thc fcasibility of low Jevci dircct eurrcnt clcctrothcrapy for regional canccr trcatment. Reg Cancer Treat 1993; 6: 31-5. 20. Lytc M, Gannon JE, O'Clock Jr. GD. Effcct of in vitro clcctrical stimulation on cnhanccmcnt ancl supprcssion of malignant lymphoma celi prolifcra­tion . ./ Natl Cancer lnst 1991; 83: 116-9. 21. Vodovnik L, Miklavcic D, Scrša G. Modifiecl celi prolifcration cluc to clcctrical eurrcnts. Med Biol Eng Comput 1992; 30: CE21-CE8. 22. !to H, Hashimoto S. Expcrimcntal stucly of thc antitumor aetivity of clircct currcnt an cffcetivc adjuvant thcrapy in irracliation. Gan To Kagaku Ryoho 1989; 16: 1405-11. 23. Scrša G, Golouh R, Miklavcic D. Antitumor cffcct of tumor nccrosis faetor combinccl with clcctrothcrapy on mousc sarcoma. Anti-Cancer Drugs, 1994; in prcss. Radio/ Oncol 1993; 27: 286-92. Antitumor effect of interferon-a administered by different routes of treatment Srdjan Novakovic1 and W. Robert Fleischmann Jr.2 2 1 lnstitute of Oncology, Ljubljana, Slovenia, University of Texas Meclical Branch, Department of Microbiology, Galveston, Texas, USA Besides the fact that inte1ferons were identifzed as factors capable of i11hibiting vira! infections, they have proved to be antiproliferative, immunomodulatory and differentiation-inducing factors. On the basis of these activities, they have been employecl clinically far treatment of various tumors. The stucly was performed to determine whether there was dtjferent antitumor effect of recombinant human inte1jeron-a A/D (rHulFN-a A/D) when it was given as a local or systemic therapeutical agent. Two clijferent tumor models, i.e. subcutaneous (s.c.) ancl intraperitoneal (i.p.) B-16 melanoma on C57Bl/6 mice, were employed in these experiments. Experimental mice were treated locally or systemically with different doses of rHuIFN-a AID; the treatment was begun 24 hours afier tumor cel! inoculation and continued through five consecutive days. lntraperitoneal treatment of animals with i.p. tumors resulted in significantly longer survival time in c:omparison with c:ontrol group or with subc:utaneously treated animals (p<0.001). Similarly, the delay of tumor detection and tumor growth in mice with s.c. tumors treated subc:utaneously with rHu!FN-a AID was signific:antly greater than in intraperitoneally treated animals (p<0.01). According to these results we c:an conclude that rHuIFN-a AID is muc:h more patent antitumor agent when it is used locally. However, systemic treatment with higher doses was effec:tive in both tumor model.\' ancl it is stil! more convenient for treatment of some tumor lesions which are not ac:c:essible for local treatment. Key words: melanoma, experimental-drug therapy; interferon-alpha; drug administration routes Introduction Interferons are glycoproteins which were iden­tified as factors capable of inhibiting vira! infec­tions. 1 · 2 Besides, interferons have proved to be Correspondence to: Novakovic Srdjan, MSc., Institute of Oncology, Zaloška 2, 61105 Ljubljana, Slovenia, Tel. + 386 61 323 063 ext. 29 33, Fax + 386 61 131 41 80. UDC: 616-006.81-085 antiproliferative, immunomodulatory and diffe­ 45 rentiation inducing factors.3· · Other putative functions include antioncogene activity and mo­bilisation of energy stores during sickness. 6• 7 Three subtypes of interferons (IFN a, . and y) have been identified, differing in terms of their celi surface receptors, their acid stability, their primary sequence and their chromosomal 8 Iocation and organisation.3· Interferon-a and interferon-. produced by leukocytes and fibro­blasts, respectively, are acid stable and share the same receptor, while interferon-y is produ­ Antitumor e}:fect of inte,feron-a administered by different routes of treatmelll ced by T lymphocytes, is acid labile and has a 8 different receptor.4· The precise mechanisms of action for the antitumor effects of interferons are not fully explained. They involve both direct (antiprolife­rative effects, cytotoxic effects and enhance­ment of celi surface antigen expression on tu­mor cells) and indirect antitumor action (activa­tion of macrophages/monocytes, activation of T cells, activation of NK cells and modulation of 10 antibody production).9• More then 20 subtypes of interferon-a are known, but only few of them are used systemi­cally or locally in the treatment of neoplasms as hairy celi leukemia, AIDS -related Kaposi sarcoma, Hodgkin's disease, 11011 -Hodgkin's lymphomas, oral cancer, malignant melanoma, renal celi carcinoma and bladder cancer. 11-16 In our experiments we investigated the rela­tive capability of local versus systemic treatment with rHuIFN-a A/D as an antitumor agent against B-16 melanoma. To address this que­stion we used two different tumor models: i.p. and s.c. B-16 melanoma tumors. Materials and methods Animals Six to eight weeks old female C57Bl/6 mice were used in the experiments. Mice were pur­chased from Jackson Laboratories (Bar Harbor, USA) and held in a pathogen free animal colony. The adaptation period before use was two to three weeks. At least nine healthy animals with normal body weight were included in each experimental group. Tumor model,1· Subcutaneous (s.c.) and intraperitoneal (i.p.) tumors were employed as tumor models. Subcu­taneous tumors were induced subcutaneously in the left lower abdomen with 106 B-16 melanoma cells in 0.1 ml EMEM (Eagle's minimal essen­tial medium) supplemented with 2 % fetal calf serum (FCS), while mice for i.p. tumors were inoculated with the same number of viable cells intraperitoneally. In the experiments with s.c. tumors the day of tumor detection was monito­red and tumor growth was followed by measu­ring two tumor diameters with a vernier caliper. The tumor burden was calculated by the stan­dard formula for a prolate sphere V= ni 6xd1 xd} (d2 000 ---O--lfl Control -o--SC Control ··O·· !P 3KU lrN 4000 ----t:,-•-· se 3KU llN $ . -83---!P lOKU !FN -·-·•----se 1 OKU ffN 3000 ---$---IP 30KU IFN -_,,_ -se 30KU lrN .i p' f-,ooo ? . l -,./ ! / ,v ,'/ / / / . f 'j/ : 1000 :i,,: / _;i ·" ,' #p. , "v . ,-'{Pi •"'-'i.1(.,:·{"-' ' ,,, )" LS.' .• . Jf·!f' 1 10 ,o .10 .H) Day'.> aftcr l umor Cel! moculat1on Figure 2. Growth kinetics of subcutaneous B-16 mela­noma implanted in the left lower abdomen using 106 viablc tumor cells, and treatecl subcutaneously or intraperitoneally with rHuIFN-a A/D. neously) with different doses of rHulFN-a A/D or PBS/BSA. Treatment schedule was the same as the one described above for s.c. tumors. Mice were monitored for the day of death. Table 2 presents the average results of two identical experiments which gave similar results. It ean be seen that also in i.p. tumors local treatment was more effective than systemic Table 2. Average day of death for i.p. B-16 melanoma bearing mice treated subcutaneously or intraperito­nealy with rHulFN-a A/D. Antitumor effects 011 i.p. tumors Avcrage SD* p-valuc p-valuc clay of (comparing (comparing clcath to the thc same It was important to consider previous data from s.c. tumor model in order to asses whether the control) closes) s.c. control 19.9 1.8 differential responsiveness of the tumors would S.C. 3 KU 19.6 1.7 be observed on i.p. tumor model after different S.C. 10 KU 20.9 l.7 routes of treatment with rHuIFN-a A/D. To S.C. 30 KU 21.5 1.8 address this point, mice were inoculated i.p. with B-16 melanoma tumor cells and randomly control 3KU 19.3 22.6 1.3 2.5 0.0001 0.0001 distributed (as mice with s.c. tumors), into i.p. 10 KU 23.3 l.9 0.0001 0.0004 eight groups. Mice were also treated locally i.p. 30KU 24.8 3.1 0.0001 0.0002 (intraperitoneally) and systemically (subcuta-*SD -Standard deviation Novakovic S. and Fleischmann R. Ir. treatment (Table 2, Figure 3). Percent of in­crease in life span ( % ILS±SE) for locally trea­ted mice .with rHuIFN-a A/D was 17.3±2.99 (3 KU), 20.5±2.27 (10 KU) and 28.7±3.68 (30 KU); for systemically treated mice the %ILS was -1.7±1.96 (3 KU), 5.2El.98 (10 KU) and 100 -----.-0--6. ­ eontrol ..-6* ----0------­ IP 3KU IFN-a i l ! 75 : : ----0----IP l0KU IFN-o ' ' ' ' , : : -----6.----IP 30KU IFN-a 6-6-6* : : 1 .t'. 50 lii,r ::1: o 25 .·1.fr---------------. ; • .·0-Q------. 1 i j ! 0;----,i_J---Q--<)>--.--1.-----. 15 20 25 30 35 Days after tumor celi inoculation 100 ---0-t'.l. ' ' '' ---0---eontrol '-6-­ ; --------.--· se 3KU IFN-a : : <>-------l-<>-o 75 ----o---­ : ! se 1 0KU IFN-a : ----6---­ se .30KU IFN-a 4'*' ' ·5 50 66 !-t'.l. ' ' ' ' 6-1-o ' ' ' ' -lf o-+----.-{\--(:}---.---.---. 15 20 25 30 35 Days after tumor celi inoculation Figure 3. Survival curves for intraperitoneal B-16 melanoma bearing mice (C57BI/6); tumors werc indu­ced intraperitoneally with 106 viablc tumor cclls and 24 hours la ter treatcd intraperitoneally ( upper figure) or subcutaneously (lower figure) with different conccn­trations of rHuIFN-a A/D during five consecutive days. .10 /. .o . \P Treatment 10 "' se Treatment ;f A 10+-----..-------­ 1000 !0000 100000 IFN-u concentration {U/Oay) Figure 4. Increasc in life span (ILS) for intraperitoneal B-16 melanoma bearing micc treated systemically orlocally with rHuIFN-a A/D. 7.9±2.08 (30 KU) (Figure 4). Owing to a higher agressivness of i.p. tumors, systemic (subcutaneous) treatment did not statistically significantly affect the average day of death in comparison with control mice, except when the mice were treated with the highest dose (30 KU) (Table 2, Figure 3). However, statistically significant increase in life span was observed when we compared locally (intraperitoneally) treated animals to the ones treated systemically (subcutaneously) with the same dose of rHuIFN-a A/D; the p-value for mice treated with 3 KU was 0.0001, for mice treated with 10 KU 0.0004 and for mice treated with 30 KU 0.0002. Moreover, the animals that received a threefold lower dose (10 KU) of rHuIFN-a A/D locally (intraperito­neally) survived significantly longer than those treated systemically (subcutaneously) with 30 KU (p = 0.005). Based on the results obtained in both tumor models, it is clear that maximal antitumor acti­vity occurred when rHuIFN-a A/D was given locally. Systemic treatment was moderately ef­fective: more effective in s.c. tumors when rHuIFN-a A/D was administered intraperito­neally than on i.p. tumors when it was admini­stered subcutaneously. Antitumor effect of interferon-a administered by different routes of treatment Discussion Previous experimental findings clemonstrated that IFN-a has reproducible antiproliferative 22 effects in vitro20-and in vivo. 23 On the basis of these findings, IFN-a has been employed clinically for treatment of various tumors. Toclay, IFN-a is approvecl as an antiprolifera­tive agent for the treatment of hairy celi teuke­mia ancl AIDS related Kaposi sarcoma.5• 10 Ne­vertheles, IFN-a as a single agent has been reported to induce clinical remission in many hematological malignancies and solid tu­mors.16·24·25 Moreover, IFN-a has reproclucible activity against malignant melanoma, a tumor for which conventional chemotherapy has poor efficacy. 10•25 The present stucly was unclertaken to assess which route of administration is more suitable · for IFN-a treatment. Therefore, we chose s.c. and i.p. B-16 melanoma tumor models. Mice were treatecl locally and systemically for five consecutive days with different doses of rHuIFN-a AJD. Systemic treatment of s.c. tu­mors was performed in the form of intraperito­neal injection, while locally treated animals were injected subcutaneously. In contrast, in the i.p. tumor model intraperitoneal aclministra­tion was performecl as a local ancl subcutaneous as a systemic treatment. In both cases local treatment proved to be significantly superior to systemic. An interesting observation was that systemic (intraperitoneal) treatment of s.c. tu­mors resultecl in a statistically significant delay in tumor detection at all interferon concentra­tions examinecl, while systemic treatment (sub­cutaneous) of i.p. tumors clicl not significantly increase the life span of treatecl animals (except 30 KU). The fact that developecl tumors in systemically treated animals continued growing at the same rate as tumors in control mice, suggests that systemically administered rHuIFN-a A/D exerts antitumor effect only on a very small tumor burden. In accordance with our observations, rHulFN-a AJD is more effective when given as a local therapeutical agent. Nevertheless, when we have to use rHuIFN-a AJD systemically, it is much more aclvisable to administer it intrape­ritoneally than subcutaneously. This is also in agreement with previous pharmacokinetic fin­clings that intraperitoneal administration of IFN-a has good bioavailability (30 times higher) comparecl to the intravenous route.26 The role of IFN-a in the treatment of malig­nancies has not yet been fully established. Many questions remain to be answered concerning the optimal strategy for incorporating IFN-a into anticancer therapy, ancl one of them is the optimal route of its aclministration. However, the future of IFN-a usage in oncology seems to be in its local ( ancl also systemical) use as acljuvant therapy after the tumor burden has been reduced by other therapeutic modalities. Acknowledgement This work was supportecl by U.S. Public Health Service Grant (National Cancer Institute). References l. Isaacs A, Lindenmann J. Virus interference. I. The interferon. Proc R Soc Lond Ser B 1957; 147: 258-67. 2. Lindenmann J. Induction of chick interferon: pro­ccdurcs of the original expcriments. Methods En­zymo/ 1981; 78: 181-8. 3. Friedman MR, Vogel NS. Intcrferons with spccial cmphasis on immunc systcm. Adv lmmuno/ 1983; 34: 97-[40. 4. Baron S et al. The Interferons. Mechanisms of action and clinical applications . .TAMA 1991; 266: 1375-83. 5. Elsdssr-Beilc U, von Klcist S. Cytokincs as thera­pcutic and diagnostic agents. Tumor Biol 1993; 14: 69-94. 6. Kurzrock R, Talpaz M, Gutterman JU. Intcrfe­rons a, .' y: basic principles and preclinical stu­dies. In: DcVita VT Jr., Hellman S, Roscnberg A eds. Biological therapy of cancer. New York: J.B. Lippincott Co. 1991: 247-75. 7. Patton JS et al. Interferons and tumor necrosis factors have similar catabolic effccts on 3T3Ll cclls. Proc Nat/ Acad Sci USA 1986; 83: 8313-8. 8. ltri ML. Thc intcrferons. Cancer 1992; 70 (Suppl 4): 940-5. Novakovic S. and Fleischmann R. Jr. 9. Fleischmann WR Jr., Fleischmann CM. Mecha­nisms of interferons antitumor action. In: Baron S et al eds. Interferon: Principles and medica! applications. Galveston: UTMB 1992: 299-309. 10. Dorr RT. Interferon-a in malignant and vira! diseases. Drugs 1993; 45(2): 177-21 l. 11. Rao SV, Wadler S. Curent use of interferons. Contemp Onco/ 1992; 3: 44-9. 12. Kuo JY et al. Impaired interferon-a production in whole blood cultures from bladder cancer pa­tients. Uro/ Res 1991; 19: 51-6. 13. Rassiga-Pidot AL, Mclntyre OR. In vitro leuco­cyte interferon production in patients with Hod­gkin's disease. Cancer Res 1974; 34: 2995-3002. 14. Ho AD, Moritz T, Rensch K, Hunstein W, Kirc­hner H. Deficiency in interferon production of peripheral blood leucocytes from patients with non-Hodgkin lymphoma. J lnte1feron Res 1988; 8: 405-13. 15. Jamkar AV, Banerjae AC, Gore MM, Sathe PS, Ghosh SN. Interferon produeing capacity of perip­heral mononuclear cells in oral cancer patients. lndian .! Cancer 1989; 26: 76-84. 16. Wadler S. The role of interferons in the treatment of solid tumors. Cancer 1992; 70 (Suppl 4): 949-58. 17. Fidler IJ. Selection of successive tumor lines for metastasis. Nature New Biol 1973; 242: 148-9. 18. Kramer MJ et al. Celi and virus sensitivity studies with recombinant human alpha interferons. J ln­te1feron Res 1983; 3: 425-35. 19. Rosenthal GJ et a/. Organ-specific hematopoetic changes induced by a recombinant human interfe­ron alpha in mice. Fundam Appl Toxico/ 1990; 14: 666-75. 20. Paucker K, Cantell K, Henle W. Quantitative studies of vira! interference in suspended L cells, III. Effect of interfering viruses and interferon on the growth rate of cells. Virology 1962; 17: 324-8. 21. Pfeffer LM, Murphy JS, Tamm l. Interferon ef­fects on the growth and division of human fibro­blasts. Exp Celi Res 1979; 121: 111-5. 22. Jezeršek B, Novakovic S, Serša G, Auersperg M, Fleischmann WR .lr. Interactions of interferon and vinblastine on experimental tumor model me­lanoma B-16 in vitro. In print (Anti-Cancer Drugs). 23. Jezeršek B, Novakovic S, Serša G, Cemažar M, Auersperg M, Fleischmann WR Jr. Interactions of interferon and vinblastine on experimental tu­mor model melanoma B-16 in vivo. Radio/ Onco/ 1993; 27: 275-9. 24. Wandl UB, Niederle N, Kranzhoff M, Seeber S. Clonogenic assay is not predietive but refleets therapeutic efficacy of interferons in the treatment of chronic myelogenous leukemia. Int J Celi Clo­ning 1992; 10: 292-8. 25. Kirkwood MJ. Studies of interferons in the the­rapy of melanoma. Semin Onco/ 1991; 18 (Suppl 7): 83-90. 26. Schuller J et al. Pharmacokinetic aspects of inter­feron alfa-2b after intrahepatic or intraperitoneal administration. Semin Oncol 1992; 19 (Suppl 3): 98-104. Radio! Oncol 1993; 27: 293-7. Anti-tmnor effect of interferon alpha in combination with cisplatin -animal experiments Borut štabne Department of Medica! Oncology, Institute of Oncology, Ljubljana, Slovenia Anti-tumor effect of human interferon-a and cisplatin was studied on B-16 melanoma bearing C57Bl/6 syngeneic mice. When the tumors reached I mm in diameter, applications of cisplatin were started at a dosage of 0.001 mg/g of animal's body weight, or human inter.feron-a was given at a dosage of 5 X 104 IU. A control group of mice received normal .mline solution. The treatment was applied every next day, altogether 12 times. Tumor volumes were measured every next day, and their mean values calculated; all 12 measurements con.firmed that the mice treated with human interferon-a and cisplatin had smaller mean value than the control group, or the groups receiving either inte,feron or cisplatin alone, re.pectively (p < 0.05). The mean tumor volumes o.f ci.1platin treated mice were lower than those of the controls or interferon-treated animals (p < 0.05). The obtained results indicate that human interferon-a enhances the antitumor ejfect of cisplatin. Key words: melanoma, experimental drug therapy; interferon-alpha; cisplatin Introduction Melanoma represents Iess than 5 % of ali malig­nant diseases, though its incidence in the last few decades has been rapidly increasing. 1• 2• 3 Chemotherapy in melanoma has not been particularly effective. Although severa! drugs have a Iow order of antitumor activity, combi­nation chemotherapy has not produced better results that those observed with the use of single agents such as dacarbazine, nitrosoureas, vinca-alkaloids, and cisplatin (CDDP). More­over, none of these drugs as well as their Correspondence to: Borut Štabuc MD, PhD, Dcpart­ment of Medica! Oncology, Institute of Oncology, Zaloška 2, 61105 Ljubljana, Slovenia. UDC: 616-006.81-085 combinations have been proved to definitely increase the survival of patients with metastatic 5 melanoma.4• A number of pre-clinical and clinical trials studying the effects of inteiieron alpha were consistent in confirming its antitumor activity 7 against melanoma. 6• Even if the mechanism of antitumor effects of interferon are stili insuf­ficiently understood, it has become clear that in the majority of the experimental systems investigated, interferons act in a very different way from chemotherapy. Thus, the idea of using interferons in combination with other agents has interested investigators for a Iong time.8 Different experimental data have shown that combined treatment can improve the response rate and prolong the duration of response due 294 Štabuc B. to different actions of drugs on the tumor, interactions between the drugs and, perhaps, 10 influences on the host immune system. 9• Therefore, the aim of this study was to inve­stigate the possibility of enhancing the cisplatin antitumor effect by the application of human interferon alpha in suboptimal doses. Materials and methods Experimental animals The animals, 8-10 week old C57BI/6 mice were obtained from Rudjer Boškovic Institute, Za­greb. Mice used in the experiment were of the same sex and age. Animal colonies were main­tained in accordance with the recommendations issued by the National Cancer Institute in Be­thesda, USA. B-16 melanoma was used as an experimental tumor model. Tumors were im­planted to the animals by subcutaneous injec­tion of 5 x 105 viable tumor cells given dorsola­terally. Tumor celi suspension was prepared by mechanical decomposition of viable tumor tis­sue. Treatment Mice were divided into four experimental groups as follows: 1) control group, 2) group treated with CDDP, and 3) group receiving human interferon-a (IFN-a) and 4) group recei­ving combined CDDP and IFN-a treatment. Each group consisted of 7 animals. Intraperito­neal applications of the cytotoxic agent and/or IFN-a and normal saline solution were started when the tumors reached 1 mm in diameter, or a volume of 0.5 mm3 . The injections of active substances were administered every next day, and the experiment was completed on the 25th day from the beginning of application. The solution of CDDP (Bristol-Myers Co.) and normal saline was injected at a dose of 0.001 mg/g b.w. or 0.01 ml of the solution per gram of the animal's body weight. IFN-a (human interferon alpha from the In­stitute of Immunology, Zagreb, Croatia)' dissolved in normal saline was injected at a dose of 5 x 104 IU which equalled to 0.25 ml of the solution per application. In one group CDDP injections were followed after one hour by IFN-a application; drug do­sage was the same as in groups receiving either of the agents alone. Animals in the control group had the same quantity of normal saline solution injected intraperitoneally every next day. Tumor measurement and statistical analysis Tumor growth was followed up daily by the evaluation of tumor diameter and thickness. Tumor volume was ca\culated using the follow­ing formula: 0.523 X a X b X c, where a, b, c were tumor diameters. Mean volumes, as well as standard deviation and standard error of the mean values were calculated from the results of measurements performed on the same day. The data were statistically analysed by means of CIA softwa­re.11 Results Mean values of tumor volumes (MTV) express­ed in mm3, measured every next day during the treatment with normal saline solution, IFN-a, CDDP, or combination of both are presented in Table l. In ali measurements MTVs of CDDP-treated animals were found to be lower that those of the control group (p<0.05), and the values obtained after the 5th measurement were also statistically significantly lower that those of the IFN-a treated animals. In ali 12 measurements MTV of the animals receiving combined CDDP and IFN-a treat­ment were statistically significantly lower than the relevant values obtained in ali other expe­rimental groups (p < 0.05). Figure 1 shows MTV values and 95 % confi­dence interval resulting from ali 12 measure­ments performed in ali experimental groups. In the groups receiving combined CDDP and IFN-a treatment or CDDP alone 2 animals died immediately after drug application. The Anti-tumor effect of IFN-a and cisplatin Table l. Bl6 melanoma volume in mm3 in ali 4 groups of C57Bl/6 mice. Controls IFN-a CDDP IFN-a and CDDP Measurement MTV(m3) 95-CI MTV(mm3) 95-CI MTV(mm3) 95-CI MTV(mm3) 95-CI 1 0.5 0.5 0.5 0.5 2 3.9 3.1 2.1 1.3 2.7-4.l 2.7-3.5 1.8-2.4 0.9-l.8 3 13 10.4 9.9 6.2 11-15 9.6-11.2 9.2-10.7 5-7.4 4 57.7 46.9 49.1 35.2 55-61 44-50 44.8-53.3 29-42 5 131 122 119 59.3 127-135 116-128 113-125 48-71 6 181 179 145 91.4 173-189 170-188 137-153 79-104 7 324 326 240 219 316-332 314-338 231-249 205-233 8 444 438 369 299 433-455 428-448 349-389 281-317 9 538 524 417 371 525-551 510-538 295-439 349-393 10 622 613 498 443 608-636 600-626 473-523 421-465 11 696 689 602 523 683-709 676-702 573-631 496-550 12 789 798 711 611 --­ 775-803 784-812 672-750 576-646 900 Discussion 800 700 600 .9. /_// p .// According to the obtained results, IFN-a alone does not exert any direct statistically significant effect on the tumor. The antiproliferative effect of IFN-a depends on the dose applied, as well as on the mode of application, tumor size and the type of metasta­ ses. An indirect, immunomodulatory effect of control interferon can be achieved at doses lower than • T IFN-a cisplatin those required for a direct antitumoral effect. : IFN-a + cisplatin In experimental animals human IFN-a does not 500 ,o E 400 300 200 E .?-l:f 100 o -100 ..-'--..-.--'--. ........ -. ........ -.. o 2 " G a to 12 1,1. t6 1a 20 22 24 Oays after treatment initiation Figure 1. The effect of IFN-a and CDDP on the growth of B-16 melanoma. Mice were treated every next day after the tumors reached 1mm in diameter. IFN-a (5 x 104 JU) and CDDP (0.00lmg/g) were injected intraperitoneally. Eaeh experimental group consistcd of seven mice. The vertical bars represent 95% CI. animals in CDDP-treated group had for 1 g lower body weight on average, whereas the body weight of animals in other three groups did not differ from that of the control animals. influence the immune cells such as T-lymphocy­tes and NK cells. 14 Balkwill has reported on the interactions between human IFN-a and chemotherapeutic agents in human tumours grown in mice. The efficacy of sub-optimal doses of cyclophospha­mide and doxorubicin was greatly increased by interferon in a human breast cancer xenograft · growing in nude mice. Even low doses of inter­feron, which alone had no effect on tumour growth, were able to potentiate the activity of anticancer drugs. 15 Numerous preclinical and clinical trials have shown synergistic or additive effects between 296 Štabuc B. interferon and at least 20 different cytotoxic agents including doxorubicim, vinca alkaloids, 9• 16 5-fluorouracil and CDDP. The most striking synergy was demonstrated when low doses of IFN were used and, and it was associated predominantly with lymphoma celi lines. 17 However, not ali studies have established positive interaction between IFNs and chemo­therapy. Antagonistic effects between some cy­totoxic drugs and IFN have also been report­ed_ 1s Little is known about the mode of IFN inter­action with CDDP and other cytotoxic drugs. IFNs could potentially alter drug metabolism or act independently of the other agent. The cytochrom P-450 system was inhibited, thus significantly influencing the celi leve! of gluta­thione transferase. The decreased levels of celi glutathione result in increased cytotoxic effect of CDDP. IFN slows down the celi cycle by inhibiting the production of nucleic acids in its postmitotic 01 phase. IFN also slows down catabolism and elimination of some cytotoxic agents such as cyclophosphamide and doxorubi­cin, and influences celi membrane fluidity, i.e. 19 the transport system for cytostatics. 9• Combined IFN and chemotherapy has result­ed in· clinical benefit in many patients with solid tumours. 20 In general, however, significantly improved response rates were not observed. In many regimens, IFN are combined with cytotoxic drugs with different rationale: bioche­mical modulation, immunopotentiation, immu­nostimulation or host protection. Each ap­proach is valid; however, the complexity of potential interactions requires close considera­tions. 9· 22 Our study showed that IFN, even at a dose insufficient to influence the growth of B-16 melanoma when given as monotherapy, statisti­cally significantly increased the antitumor effect of CDDP (95 % CI). Additional prospective clinical trials are of paramount importance for further explanation of the interaction mecha­nisms involved. References 1. Balch CM, Soong S-j, Shaw HM. A comparison of worldwidc melanoma 51 II 27.3 2 127 <51 14 27.2 2 -200 - Ali 25 27.3 ± 9.3 2 200 * AM -arithmetic mean ** SE -standard error 300 Figure l. Serum IL-2 levels in healthy donors and melanoma patients. Serum IL-2 Ievels in melanoma patients and healthy donors are presented in Figure l. The difference between mean values of serum IL-2 in melanoma patients and in healthy donors was significant (27±9 U/ml versus 269±66 U/ml, p<0.05). In 5 patients the disease was found to have recurred by the time of sample taking. In these patients with recurrence, the mean value was decreased (6.8 U/ml ). Despite of a Iow number of cases, it seems that the extent of disease ( or disease activity) influenced the serum IL-2 levels. IFN treatment melanoma patients Figure 2. Serum levels in all melanoma patients and 11 patients that were on adjuvant treatment with human Ieukoeyte interferon (2 x 106 units weekly, intramuscular application) after surgical removal of the primary tumor. At the time of sample taking 11 patients were on adjuvant treatment with human leukocyte interferon (2 X 106 units weekly, intramuscular application) after surgical removal of the pri­mary tumor. In these patients the mean value of serum IL-2 was 57U/ml (Figure 2), which was higher when compared with otherwise de­creased Ievels of serum IL-2 in melanoma pa­tients. Although, the difference in serum IL-2 levels between ali patients and patients treated with human leukocyte interferon was not signi­ficant. Also, the serum IL-2 levels were not in the range of the levels of controls employed in this study (i.e. 269 + 66 U/ml). However, according to our findings it can be postulated that the extent of the disease could influence the serum IL-2 levels since a decrease in serum IL-2 was found in the group of patients with recurrence when compared with the mean serum IL-2 value in ali patients (6.8 U/ml versus 27U/ml). Discussion The aim of this study was to assess the potential role of serum IL-2 in the diagnosis and predic­tion of recurrence in malignant melanoma pa­tients. Biological significance of altered Ievels of 11-2 is stili not clear; in particular, it is not yet clear whether the increased levels in the blood reflect possible activation of immune cells, or should be ascribed to immune dysfunction. Although the Iymphoproliferative response to mitogens or antigens is frequently depressed in cancer patients, their ability to produce fL-2 by lymphocyte stimulation with PHA appears relatively normal, as reported in literature. Ho­wever, subgroups with advanced disease did 8 have depressed IL-2 prod uction. 1 • Furthermo­re, in some studies the presence of soluble form of IL-2 receptors was evaluated; increased va­lues were found in patients with small-cell lung carcinoma . Additionally, in breast cancer pa­tients serum IL-2R levels after surgery were significantly higher than those before surgery. 10 The values were found to correlate with the extent of disease.11 Serum interleukin-2 leve/s in malignant melanoma patients 30[ Decreased serum IL-2 levels in our study are consistent with some other reports, and so is also the finding that further decrease in serum IL-2 levels in our melanoma patients was asso­ciated with recurrence of the disease. The in­fluence of human leukocyte interferon could be ascribed to various reasons though it is possible that, with respect to a small number of cases studied, interferon could influence the produc­tion of IL-2 in vivo. The increased serum JL-2 levels in patients treated with human leukocyte interferon could also be ascribed to crude ex­tract of interferon containing minor quantities of IL-2. Acknowledgements Research work was supported by the Ministry of Science and Teclmology of Slovenia, Grant No. C3-0563-302/27-40/B. Refereneces l. Wancbo 1-IJ, Pacc R, I-largctt S, Katz D, Sando J. Production of and rcsponsc to intcrlcukin-2 in pcriphcral blood lymphocytcs of canccr paticnts. Cancer 1986; 57: 656-62. 2. Rudolf Z, Scrša G, Krošl G. In vitro monocytc maturation in paticnts with malignant melanoma ancl colorcctal canccr-clinical significancc. Neo­plasma 1986; 33: 71-8. 3. Djcu JY, Kasahara T, Balow JE, Tsokos GC. Dccrcascd intcrlcukin-2 inhibitor in sera of pa­ticnts with autoimmunc disordcrs. Ciin Exp Jmmu­nol 1986; 65: 279-84. 4. Low TLK, Goldstein AL. Thymosins -isolation, structural studics and biologic activitics. In: Stoll B, cel. Relation of immune testing to prognosis. New York, Plenum Prcss, 1988: 21-35. 5. Farrar JJ, cnjamin WR, I-lilfikcr ML, 1-loward M. Farrar WL Fullcr-Farrar J. Thc biochcmistry, biology, and role of intcrlcukin-2 in thc incluction of cytotoxic T-ccll and antibody-forming B-ccll rcsponscs. Jmmunol Rev 1989; 63: 129-36. 6. Thompson .TA, Lee DJ, Cox WW, Lindgrc CG, Collins C, Ncraas KA, Dcnnin RA, Fcfcr A. Rccombinant intcrlcukin-2 toxicity ,pharmacoki­nctcs, ancl immuno-modulatory cffccts in a phasc 1 tria!. Cancer Res 1987; 47: 4202-7. 7. Cadcras JM. Human intcrlcukin-2: quantitation by a scnsitivc radioimmunoassay . .! lmmwwl 1986; 89: 181-6. 8. Lissoni P, Viviani S, Santoro A, Barni S, Tancini G. Serum lcvcls of intcrlcukin-2 in canccr paticnts­prcliminary consiclcrations. lnt .! Biol Markers 1989; 4: 203-6. 9. Yamaguchi K, Nishimura Y, Kiyokawa T, Matsu­zaki 1-1, Ishii T, Kubota K, Kawahara M. Elcvatcd serum lcvcls of solublc intcrlcukin-2 rcccptors in small ccll lung carcinoma . .! Lab Ciin Med 1990; 116: 457-61. IO. Brivio F, Lissoni P, Mancini D, Tisi E, Tancini G, Barni S, Nociti V. Effcct of antitumor surgcry on solublc intcrlcukin-2 receptor serum lcvcls. Am .! Surg 1991; 161: 466-9. 1 l. Sharma S, Saha K, Shingal RN. Maluk GB. Serum solublc intcrlcukin-2 (IL-2) receptor lcvcls in womcn with brcast carcinoma and its corrcla­tion with IL-2 receptor cxprcssion on bloocl lym­phocytcs and lymphocytic infiltration within thc tumor. Cancer lmmunol fmmunotlzer 1991; 33: 198-202. Radio/ Oncol 1993; 27: 302-6. Natu.ral porcine interferon gamma (PoIFN gamma) Bratko Filipic, 1 Sonja Rozman,2 Katarina Carlsson3 and A vrelija Cencic4 1 Institute for Microbiology, Medica! Faculty, Ljubljana, 2 BIA Ltd., Ljubljana, Slovenia, 3 Centre of Chemistry, University of Lund, Sweden, 4 College of Agriculture, University of Maribor, Slovenia The natura! porcine mitogen induced gamma interferon (Po/FN gamma) was studied and compared with the human interferons gamma (Hu!FN gamma) and alpha (Hu!FN alpha). A comparison was performed by the following criteria : pH 2.0 and 56 ° C stability, molecular weight, dot-blot reactions and cross reactivity (neutralisation index). The biological activity of porcine ancl human interferons in vitro (antiviral and antiproliferative) was correlated on human nontransformed (H EF) ancl transformecl cells (FL). Key words: interferon type II-analysis; cells; cultured-drug effects Introduction [nterferons (IFNs) are defined as proteins/gly­coproteins having an ability to protect the infec­ted cells ( causing the antiviral sta te) or to reduce and/or inhibit the growth of transformed cells in vitro and tumors in vivo. L 2 They are divided into at least two classes (Type I -alpha or beta and Type l[ -gamma) according to their mode of induction (Type I -with viruses or pl:C, Type l[ -with T celi mitogens or hetero­ 4 logous celi s). 3• Because of clinical use, the main attention was focused on human interferon alpha (HuIFN alpha)5 and much later on the human interferon gamma (HulFN gamma).6 For the IFN gamma production it was thought that a pure popula- Correspondence to: Dr. Bratko Filipic, Institute for Microbiology, Medica! Faculty, 61105 Ljubljana, Slo­venia UDC: 615.28l.7.0l5.44 tion of T lymphocytes is necessary. Latter on, it was found that partially purified spleenocytes can be used as well. The highest titers (IFN units/ml) were obtained when a mixture of peripheral blood lymphocytes and spleenocytes was used for the IFN gamma production, with specific T celi mitogens as inducers. A comparison between HulFN alpha and HuIFN gamma shows that they are antigenically completely different: with anti-HuIFN alpha antisera it is impossible to neutralise HuIFN gamma and vice versa. At the beginning of the clinical use of human IFNs in natura! form, the main problem was how to produce enough IFN Concomitantly, the optional solutions arose: to use human-like IFNs. In this respect, many studies were carried out to find the antigenic similarity between human and human-like interferons. Surprisin­gly, the highest degree of homology was found between murine and human IFNs.7 Natura/ porcine inte1fero11 gamma Similar interferon system as in humans can be found in other animal species, such as rat, horse, cattle, pig, monkey, etc. Porcine and bovine IFNs8-11 became of interest because of their relatively high antigenic similarity with human one. Such a similarity between I-Iu and PoIFN is about 78.5 % (at nucleotide leve!)12 in the case of IFN alpha. When a recombinant gamma interferons (gamma IFNs) were compa­red (rPoIFN gamma/rHuIFN gamma) a homo­logy was estimated in 59 % ( at nucleotide le­ve!). 13 Correlation of non-recombinant fonns of porcine alpha and human alpha IFNs shows even higher Ievels of homology (86.7 % at mtc­Ieotide leve!) between them. 14-15 It seems to be unusual that PoIFN gamma is cross reactive with HuIFN alpha and not with PoIFN gamma. Experiments presented herein are aimed to compare the natura! porcine interferon gamma (PoIFN gamma) with the human interferon alpha (HulFN alpha) by the following criteria: pH 2.0 and 56 ° C stability, molecular weight, dot-blot analysis and neutralisation on celi cul­ture in vitro. Material and methods Blood collection, buffy-coat prepara/ion and in­te1feron induction Porcine blood was collected aseptically into sterile flasks containing 3.8 % soditllll citrate to prevent coagulation. The porcine buffy-coat was separated by introducing 30 % sacha ro­se. 16· 18 The sedimented buffy-coat was washed twice with saline and resuspended in modified Eagle's medium19 containing 4 % of porcine plasma and antibiotics (Penicillin 5000U/ml, Streptomycin 5 mg/ml, Gentamycin 10 mg/ml, Neornycin l0 mg/1111). The induction was perfor­med by LCL (Lens culinaris Iectin) at a concen­tration of 25 µg/ml. Celi concentration was adju­sted to 107-108 cells/ml. After three days of cultivation on a spinner (37 ° C/140 RPM) the cells were sedimented by centrifugation (2000 RPM/20 minutes) and the supernatant represen­ting the crude IFN was harvested. Partial purijication of Po!FN gamma Interferon was isolated as follows: to 100 ml of IFN containing supernatant, 3 g of avtoclaved SiO2 (water glass) were added and incubated overnight at + 4 ° C. On the next day the su­spension was centrifuged at 2500 RPM for 30 minutes to sediment the water glass. The sedi­mented water glass was resuspended in the 1/8 of the original mixture vol ume of 50 'Yo ethylene glycol monoethyl ether in 1.4 mM NaCl to elute the interferon. After 2 hours the water glass was sedimented by centrifugation (2500 RPM for 30 minutes). The elution procedure was repeated in 1/16 of the original volume. Both eluates were mixed with 0.1 % of FCS (Fetal calf serum, Flow) and dialysed against distilled water. Measurement of antiviral and antiproliferative activity IFNs were tested for antiviral (A V) activity by 50 % cytopathogenic inhibition assay against HSVl (Herpes simplex virus type 1) as a chal­lenge virus.20 The antiproliferative activity (AP) was determined by 50 % growth inhibition assay on HEF (human embryonal fibroblasts) and FL (human amniotic celi line) cells.21 Natura! inter­ferons alpha (Institute of lmmunology, Zagreb, Croatia; EGIS Budapest, Hungary) at a concen­tration of 1000 A V (antiviral units/ml) were used as the control standards. Stability tests To determine the type of IFNs the samples were exposed to pH 2.0 and heating at 56 ° C for 30 minutes. Antiviral (A V) and antiprolife­rative (AP) units were determined before and after such treatment. Protein content In each sample, the quantity of proteins was determined by the rnodified Lowry method.21 304 Filipic B. Serological analyses To determine the serological similarity and/or differences between porcine and human IFNs (gamma and alpha) the "constant method" ac­cording to LaBonnardiere et al.14 was used. In summary, constant ( twofold) dilutions of anti­IFN antisera werc added to the FL cells in microtiter plates (NUNC). In the next step, three-fold dilutions of IFNs with the virus (HSV l) were added. Simultaneously, IFN titration was performcd without antisera. The neutralisa­tion index (NI) was calculatcd as follows: NI= log3 (antiserum + interferon) -log3 (interfe­ron). Antigenic properties Immunoblot tests were performed according to Pretnar et al. 22 In summary, the antigens HuIFN alpha (Institute for Immunology, Za­greb, Croatia; EGIS, Budapest, Hungary), HuIFN gamma (Sigma, St. Luis, USA), PoIFN gamma (Institute for Microbiology, Medica! Faculty, 61000 Ljubljana, Slovenia) in the vo­lume of 10 µl (the concentration of proteins in the samples was 0.9 mg/ml) were spotted on nylon films (Hybond, Amersham) and air dried. The nylon films with the bound antigens were incubated for 60 minutes with the primary an­tibodies (polyclonal) as follows: Anti-HuAlpha (Boehring, Manheim, FRG; neutralisation titer 10.000U/mg), Anti-HuGamma (Boehringer, Manheim, FRG; neutralisation titer 10.000 U/ mg) and Anti-PoGamma (INRA, Youi-en-Jo­sas, France; neutralisation titer 10.000 U/mg), Anti-HuAlphal (Boehring, Manheim, FRG; neutralisation titer 10.000 U/mg), Anti-HuAl­pha 2 (Institute of Virology, Bratislava, Slova­kia; ncutralisation titer 4000 U/mg) and Anti­Acidolabile (Institute of Virology, Bratislava, Slovakia; neutralisation titer 10.000 U/mg). Af­ter washing with TTBS (10 mM Tris + HCl, pH 8.0, 150mM NaCl + 0.05 % Tween 80), the films were incubated in anti-rabbit IgG (Boe­hring, Manheim, FRG) conjugated with peroxi­dase. Following three washes in TTBS, the reactions were developed using DAB (diamino- et al. benzidinc, Sigma, St.Luis, USA) in the concen­tration of 50 mg/100 ml TBS and 30 fl of hydrogen peroxide. Results Stability tests Table 1 shows the diffcrcnces betwecn porcine (PoIFNs) and human interferons (HuIFNs, al­pha, gamma) as follows: PoIFN gamma is pH 2.0 stable in contrast to HulFN gamma which is not. On the other side, PofFN alpha is pH 2.0 labile, i.e. different from HulFN alpha which is not. Similar data were found when the resistance to he.1ting to 56 ° C were compared. It seems that PolFN gamma is more similar to HuIFN alpha than to its lu1man counterpart. Table l. Stability tcsts for PoIFNs and Hu!FNs. M.W.1 IFN typc pH2.0 56 ° C (Daltons) PolFN gamma Stablc Stablc 21000 Po!FN alpha Labilc Labilc 20000 Hu!FN gamma Labilc Labilc 19000 HulFN alpha Stablc Stablc 19500 1 Molecular wcight was detcrmincd by PAG-SDS clectrop­horcsis Dot-blat analysis Using dot-blot tests, the antigenic properties are described (Table 2) in terms of positive or negative reactions. PoIFN gamma shows a po­sitive reaction with antisera against PoIFN gam­ma, HulFN alpha and HuIFN alpha 2. HuIFN gamma reacts only with the antiserum against HuIFN gamma, and not with PolFN gamma. Table 2. Dot-blot analysis of Po ancl Hu IFNs. IFN typc Rcaction with Anti 1 Po Hu Hu Hu Hu gamma gamma alpha alpha l alpha 2 Po!FN gamma + + + PoIFN alpha -++ Hu!FN gamma ++ - Hu!FN alpha + + + + 1 Dot-blot reaction: "+" = gives positivc rcaction "-" = gives negative reaction Natura! porcine inte1feron gamma In the case of alpha IFNs, PoIFN reacts with the antiserum against HuIFN alpha and HulFN alpha 2. HulFN alpha gave a positive reaction with the antisera against PoIFN gamma, HulFN alpha, HuIFN alpha l and HuIFN alpha 2. Serological test.1· By serological tests in vitro ( on FL cells) (Table 3) the leve! of the neutralisation of IFN's anti­viral activity with anti-IFN antisera was quanti­fied by the Neutralisation index (NI). When PoIFN gamma was tested, the following Nls Table 3. Neutralisation indexes for Po and Hu IFNs. IFN type Neutralisation with Anti1 Po Hu Hu Hu Hu gamma gamma alpha alpha 1 alpha 2 PoIFN gamma -1.15 o PoIFN alpha oo o -0.98 --0.92 o log3 (antiserum + IFN) -log3 (IFN) were found: With anti-PoIFN gamma -1.15, with anti-HulFN alpha -1.94 and with anti­HulFN alpha 2, -0.29. PoIFN alpha shows much higher NI for anti-HuIFN alpha (NI= ­ 2.43) as well as for anti-Hu alpha 2 (---0.53). HulFN alpha shows the following values for Nls: Anti-PoIFN gamma + 0.92, anti-HuIFN alpha -2.85, anti-HulFN alpha 1, -2.00, and anti-HuIFN alpha2, -l.60 Discussion PoIFN alpha and HuIFN alpha were found to be antigenically similar, though the differences between them were found when some of the physico-chemical characteristics were tested (temperature stability, acid-resistance ). 9-11 Contrarily, natura! PolFN gamma is different from its natura! counterpart in humans. lts pH and temperature stability are more similar to that of HuIFN alpha. PoIFN gamma also shows cross reactivity in vitro with HuIFN alpha (com­plete) and its natura! subtype HuIFN alpha 2 When these data were correlated with those obtained by comparison of recombinant IFNs (human, porcine) in the case of gamma interfe­rons, homology was established in 59 % (at nucleotide leve!). It seems possible that in the case of recombinant forms only selected mole­cules, in contrast to the natura! ones (partly purified or purified) when complete molecules composed from different numbers of natura! subtypes, were compared. In this respect it should be mentioned that the comparison by NI (Neutralisation index) in vitro gives a picture of biological activity. Future experiments with pure porcine interfe­rons (alpha and gamma) are expected to dis­close the real leve! of similarity/differences with human interferons (alpha, gamma), and thus enable clinical use of porcine IFNs in veterinary and human medicine. Acknowledgment This work was supported by the grant from the Slovenian Ministry for Research and Techno­logy (URP Molecular Biology, Cl-509/381-93; Research field: Biochemistry with Molecular Biology, PI-5064-0381-93). References 1. Gresser 1, Tovey MG. Antitumour effccts of inter­feron. Biochem Biophys Acta 1978; 516: 231-47. 2. Hubbell RH, Graft AJ, Leibowitz .JP, Gillespie DH. Synergistic antiproliferative effect of recom­binant gamma-interferons. J Biol Resp Mod 1987; 6: 141-53. 3. Bonnem EM, Spiegel RJ. Interferon-alpha: Cur­rcnt status and future promise. J Biol Resp Mod 1984; 3: 580--94. 4. Toth M, Endresz V, Toth S, Beladi l. Human interferon alpha and beta have more potent pri­ming activities than interferon-gamma . .T Gen Vira! 1985; 66: 893-6. 5. Borden EC. Interferons: In pursuit of the promise. In: Mirand EA,Hutchinson WB,Mihrich E. Eds. 13th lnternational Cancer Congress, Part E., Can­cer Management. New York: Alan R. Liss 1983: 287--296. 6. Osborne LC, Georgiades .JA, Johnson HM. Largc scale production and partial purification of mouse immune interferon. Jnfect Immunol 1979; 23: 76-80. Filipic B. et al. 7. Solovicv VD, Pokidyshcva LN, Volodnina TN. On thc antigcnic similarity of some alpha intcrfe­rons. Vapr Virusa/ 1982; 27: 526-8. 8. Richmond .TY. An Interferon-like inhibitor of foot and-mouth discasc virus induccd by phytohacm­maglutininc in swinc lcukocytc culturcs. Arch Ges Virus Fars<;h 1969; 27: 282-9. 9. Solovicv VD, Ogarkov VI, Marchcnko VI, Mona­styrcva LN, Parfenov VV, Bukharova II, Prco­brazhcnskcja NK, Zubanova NA Swinc blood lcukocytcs, a ncw sourcc of production of interfe­ron activc in human cclls. Vapr Virusa/ 1980; 25: 716-20. 10. Solovicv VD, Pokydishcva LN, Volodnina TN.On antigcnic similarity of some alpha-intcrferons. Vapr Virusa/ 1982; 27: 526-8. 11. Piasccki E. Properties of natura! porcinc intcrfe­rons. J IFN Res 1988; 8: 61-73. 12. Lcfcvre F, LaBonnardicrc C. Molccular cloning and sequcncing of a gene cncoding biologically active porcinc-intcrfcron . .l IFN Res 1986; 6: 349­60. 13. Charlcy B, McCullough K, Martinod C. Antiviral and antigenic propcrtics of rccombinant porcine interferon gamma. Vet lmmunal Immunalapathal 1987; 7: 357-68. 14. LaBonnardicrc C, Laudc H, Bcrg K. Biological and antigcnic relathionship betwcen virus-induced porcinc and human interfcrons. Ann Inst Pasteur! Vira/ 1986; 137 E: 171-80. 15. Lavrukhina LA, Gutman NR, Manakhanova LS. Antiviral effect of swinc lcukocyte interferon in experimcntal mice. Vapr Virusal 1981; 20: 414-8. 16. Filipic B, Golob A, Toth S, Mecs I, Bcladi I, Likar M Intcractions bctween human and porcine intcrferons. Acta Vira/ 1991; 35: 19-26. 17. Filipic B, Golob A, Golcc-Wondra M, Vitez Lj, Keše D, Likar M. Preparing and partially purifica­tion of swinc immunc interferon. In: Filipic B. cd. Yugoslav Colloquium on IFN. Ljubljana: Slo­vcnian Microbiological Socicty 1986: 137-9. 18. Filipic B, Golob A, Sinadinovska R, Babic D, Struna A, Pretnar G. Prcparing and purification of porcinc immunc interferon. In: Likar M. cd. Posvetovanje ob 45. obletnici Instituta za Mikro­biologijo, Medicinske Fakultete v Ljubljani. Ljub­ljana: Institute for Microbiology, Medica! Faculty: 1990: 71-80. 19. Filipic B, Carlsson K, Zupan S, Kneževic M, Raspor P. Prcparation and partial purification of porcinc interferon gamma (PoIFN gamma). Acta Pharm 1993; 43: 71-4. 20. Forti LR, Schuffman SS, Davies AH, Mitchcll MW. Objcctivc antiviral assay of thc intcrfcrons by computcr assistcd data collcction and analysis. In: Pcstka S. ed. Methad in Enzimalagy, vol. 119, part C. London, New York: Acadcmic Prcss 1986: 533-40. 21. Filipic B, Carlsson K, Hartman-Pretnar K, Pod­gornik A, Kosclj P. A novci protein adctermina­tion micromcthod. Acta Pharm 1992; 42: 355-60. 22. Pretnar G, Filipic B, Golob A, Škodic A, Toth S, Mccs I, Suhar A. Electroinduction of Interfe­ron-like proteins. Biaelectrachem Biaenerg 1991; 25: 183-92. Racliol Oncol 1993; 27: 307-11. Biological activity of rat fibroblast interferon beta A vrelija Cencic1 and Bratko Filipic2 1 College of Agriculture, University of Maribor, Slovenia, 2 Institute far Microbiology, Medica! Faculty, Ljubljana, Slovenia The biological activity of rat fibroblast beta interferon was studied. Rat embryonal fihroblasts (Wistar strain-WiREF) were used as a source of interferon. Cells were grown in suspension and on the microcarriers (Cytodex 3). In the exponential phase of celi growth the induction was performed. The hiological activity of the obtained interferon was tested on the homologous cells. Interferon 's influence on celi growth, morphology and intracellular leve! of some hydrolases was measured. Key words: fibroblasts-drug effects; interferon-beta; rats Introduction Generally, interferons (IFNs) are very small glycoproteins/proteins produced by eukaryotic cells in response to induction by certain chemi­cals or infection with various viruses. 1 They are divided into two groups: Type I with three distinct families: alpha, beta and omega, and Type II with only one family: gamma.2• 3 Beta IFN can be obtained from various types of mammalian fibroblasts, such as human, bo­ 5 vine and murine4• by vira! or pI:C induction. An interferon molecule has a molecular weight of around 22.000, although some smaller com­ponents with molecular weights between 17.000 and 18.000 can be found. It is less stable than alpha interferon. Schiff's staining revealed that beta interferon is a glycoprotein. Aminosugar Corrcspondcncc to: Asist. Avrclija Cencic, Collcgc of Agriculturc, Univcrsity of Maribor, Vrbanska 30, 62000 Maribor, Slovcnia. UDC: 616-091.8-085 :615.281. 7 .015.44 and aminoacid analyses confirmed its hydropho­bic characteristics. Among other biological activities of beta IFNs, the antiproliferative activity was descri­bed. 6 This activity became important because of possible use of IFN as an antineoplastic agent. Mostly, the attention has been focused on the human interferons. In our previous experiments7• 8 we have found that spontaneously transformed rat embryonal fibroblasts of Wistar strain (WiREF) can pro­duce relatively large amounts of beta interferon. In search of the possible mechanisms of the anticellular activity of IFN the growth inhibition was found to be parallel to the intracellular enzymatic changes in various model systems.9· 10 Different hypotheses were postulated on the anticellular effects of IFN, though little is known about the distinct changes of the normal or transformed cells after a short-term treat­ment. It is generally accepted that at least 18-hour treatment is needed to produce an inhibition of celi growth in vitro. Cencic A. and Filipic B. The experiments presented are aimed to show the distinet ehanges (morphological, growth characteristics, nuclear blebs, alkaline phospha­tase) after the treatment of nontransformed and transformed WiREF cells with IFN for a short tirne (15, 30, 45, 60, 90 and 180 minutes). Materials and methods Cells In these experiments we used rat embryonal fibroblasts (WiREF) in their nontransformed 7 (Phase A) and transformed (Phase E) form.Cells were grown in Eagle's medium supple­mented with 10 % of FCS (foetal calf serum). Interferon To obtain rat fibroblast (beta) interferon, the transformed WiREF cells were grown in up to two liters of suspension (Hf-108 cells/ml). IFN was induced using pI:C (polyionosinic: polyciti­dilic acid) at a concentration of 25 µg/ml. After 8 hours Actinomycin (10 µg/ml) was added. The cells were then sedimented (1200 RPM/20 minutes) and resuspended in fresh medium. The cultivation was carried on for the next 24 hours. Thereafter, the cells were pelleted (1200 RPM/ 20 minutes) and the supernatant was tested for the IFN content (antiviral units/ml). The IFN was further purified using the selective precipitation method.8 The IFN used in the experiments has a specific activity of 108 antivi­ral unit/mg of proteins. Analysis of cel! morphology In order to establish the differences in celi morphology, the cells were cultivated by two methods: (i) on small glass slides for four days, and (ii) on small glass slides in test tubes for 15, 30, 45, 60, 90 and 180 minutes after adsor­btion (control) and treatment with 1000 units of IFN/ml or mock IFN for 15, 30, 45, 60, 90 and 180 minutes. To analyse morphological changes, the cells were fixed with 4 % parafor­maldehyde, washed with PBS (phosphate buffer saline pH 7.2) and stained with Giemsa. In the parallel experiments, the cells were fixed and washed as in the case of staining with Giemsa, but afterwards stained with acetoorcein for two minutes. The following parameters were analy­sed: the diameter of the nucleus (> 1/2 of the celi), presence or absence of phylopodia, pre­sence or absence of binuclear cells. Analysis of nuclear blebs Throughout the experiments the number of nuclear blebs was determined using the method described by Fraccaro et al. II In summary, cells were adsorbed for 45 minutes and treated with 1000 units/ml of IFN (mock in control) for 15, 30, 45, 60, 90 and 180 minutes. The cells were then detached using trypsin (0.25 % ) and resu­spended in 5 ml of 0.83 mM of Ammonium chloride for 45 minutes. The nuclei were sedi­mented by centrifugation for 10 minutes at 1200 RPM, and resuspended in PBS and put onto glass slides, air dried and fixed with methanol. Thereafter, they were stained with Giemsa and analysed by dar k field microscopy. Growth characteristics The following growth characteristics were ana­lysed: growth index (GI), cumulative popula­tion density (CPD) and calcium dependence growth assay.7 Growth index (GI): Cells were seeded into 5 cm Petri dishes for 45 minuts to allow the adsorbtion. After the treatment with 1000 units of IFN/ml (mock in control) for 15, 30, 45, 60, 90 and 180 minutes, the medium was replaced with medium containing 1 % and/or 10 % of FCS. The incubation was continued for four days. Cells were detached using trypsin (0.25 % ) and counted by means of a hematocy­tometer. Cumulative population density (CPD): The values were obtained from the data (number of cells/ml) of growth experiments. Calcium dependence growth assay; To obtain the values for calcium dependence growth in Biological activity o{ rat .fthroblast interferon beta vitro, the eells were seeded into 5 cm Petri clishes for 45 minutes and treatecl with 1000 units of IFN/ml (mock in control) for 15, 30, 45, 60, 90 ancl 180 minutes. Afterwards, the meclium was replacecl with meclium eontaining 10 % FCS, ancl incubatecl for 18 hours. On the next clay, the medium was replaeecl with anot­her one containing 0.01 mM Ca or 1.00 mM Ca with 10 % of clialysecl FCS. The number of cells was cleterminecl after four clays of incubation by means of a hematocytometer. Alkaline phosphatase The enzyme leve! was measurecl according to the methocl describecl by Chou. 12 In summary, l ml of distillecl water was addecl to 50 µl of substrate (p-nitrophenyl phosphate in 0.05 M Tris-HCI, pH 8.6). After 5 minutes of preincu­bation, celi supernatant (100 .ti) was aclclecl. Thereafter, the incubation was continued for 20 rninutes, in the presence of PBS pH 9.0. The extinction was rneasured at 550 nm using a spectrophotometer. Protein c:ontent 1 n each sample the protein content was determi­necl by modified Lowry methocl. 13 Results Cell rnorphology Even though a significant clifferences can be seen between nontransfonned and transformed WiREF cells, we were interestecl to find out whether a short tilne JFN treatment could change the following parameters: diameter of the nuclei, number of binuclear cells and the number of cells without philopodia. The results obtainecl (Figure l) show a relatively fast in­crease in the number of cells with nuclei bigger tlrnn l/2 of the celi. In contrast to this, in transformed cells these changes were much slower. % OF CONTROL 40 ··-­r-::--·· . II. III. 351 I. 30 1/ 25 20 15 10 5 I• Q ..L.-LL .. 1.-L._L-L .L„L._l_ 15 30 45 60 90 160.15 . 45 60 90 160.15 30 45 60 . 160, TIME (Mlnutes) Figure l. Morphological changcs aftcr interferon trcat­mcnt of nontransformcd ( •) and transformcd ( +) WiREF cclls. I. % of big nuclci, II. % of fuscd nuclci, lil. % of cclls without phylopodia. The analysis of binuclear cells showe.d no differences between nontransforrned and tran­sformecl cells at a higher rate, but it can be seen, that the percentage of binuclear cells begins to increase at a higher rate in nontran­sformed than in transformed cells. When the phylopodia were analysed, the following results were obtainecl: in nontransfor­rned cells the increase of their number began after 30 rninutes, whereas in transformed cells this began after 15 rninutes of treatment. Growth charac:teristic:s When the effect of IFN on the growth charac­teristics (Table l, Figure 2) was analysecl, in general the expected data were observed, i.e. a higher sensitivity of transformed versus non­transformed cells. A completly reverse situa­tion, however, was observecl in the case of CPD. The recluction rate was higher in nontran­sformed cells. Alkaline phosphatase In the case of alkaline phosphatase (Figure 3), the kinetics of enzyme levels seemd similar in both nontreated and IFN-trated transformed cells, but quantitative clifferences could be seen. In nontransformed cells, the enzyme leve] de­creased in untreatecl cells, whereas in IFN-trea­ 310 Cencic A. Table l. Growth characteristics of nontransformcd and transformcd rat embryonal fibroblasts (Wistar strain) (WiREF). Cclls Growth index"f>cumulativc2) Ca Dcpcndcncc3> population X2) for al lcast 12 monlhs Anli-HCV positivity Chronic hepatitis with or without cirrhosis on !iver biopsy Exclusion of other ctiologic faetors for ehronic hepa­titis Exc/usion criteria Previous treatmcnt with interferons or steroids Severe eirrhosis (Grade B and C) Drug addietion Alcohol abuse Anti-HIV positivity Malignaney Autoimmune markers or diseasc lmmunosupression (drugs, severe ehronie diseases) Palmovi<' D. (lil(/ Cmjakovic-Palmovic J. Table 2. Charactcristics of patients on two month trcatmcnt with rlFN-a. two month trcatmcnt No. of paticnts Mcan agc (ycars) Sex (M/F ratio) CPH-C 100 45.2 ± 14.0 3.2 35 CAH-C 60 CAH-C and cirrhosis 5 Baseline demographic and histologic features of treated patients are shown in Table 2. We treated 100 patients of both sexes (69 males and 31 females, M/F ratio 2.2). The mean age of ali patients was 45.2± 14 yrs. Liver sections stained by conventional proce­dures revealed features of chronic persistent hepatitis (CPH-C) in 35 cases, chronic active hepatitis (CAH-C) in 60, and chronic active hepatitis with cirrhosis (Grade A) in 5. The characteristics of response to 2-month rIFN-a treatment are presented (Table 3). Af­ter two months of therapy 10 of 35 patients with CPH-C were good responders (28.6 % ), 14 were partial responders ( 40 % ) , and 11 were non-responders (31.4 % ). Among 60 patients with CAH-C, 26 (43.3%) presented with good response, 19 with partial (31.7 % ) and 15 with unfavourable response (25 % ) . None of the five patients with combined CAH-C and cirrhosis was a good responder; only one (20 % ) had partial response to rIFN-a treatment, whereas the remaining four (80 % ) were non responders. In the group with CAH-C a higher percentage of good responders than among patients with CPH-C is evident (43.3 % vs. 28.6 % , p = 0.22). The treatment was performed during 12 month Table 3. Responsc to rIFN-a aftcr two month trcat­mcnt. Good Partial Non- Table 4. Rcsponse to rlFN-a aftcr 12 month treat­ment. Good Partial Non-Tota! rcsponder (%) rcsponder (%) responder (%) lnitial histology CPH-C 6 (46.1 %) 5 (38.5%) 2 (15.47) 13 CAH-C lO (38.5%) 9 (34.6%) 7 (26.9%) 26 CAH-Cand cirrhosis 1 1 Tota!(%) 16 (40) 15 (37.5) 9 (22.5) 40 period in 36 good responders and four partial responders (Table 4). After one year of therapy 6 of 13 patients with CPH-C were goocl responders ( 46.1 % ) , nine were partial responclers (38.5 % ) ancl two were non-responders (15.4 % ). In 10 of 26 patients with CAH-C good response occurred (38.5 % ), nine had partial response (34.6 % ) and sev en were non-responclers (26. 9 % ) . One patient with combined CAH-C ancl cirrhosis, who previously hacl partial response to two month rlFN-a treatment, was non-responcler after full course of 12 months therapy. Al­though the clifference among patients with CAH-C ancl CPH-C is now negligible, it seems surprising that the rate of good responclers among patients with CPH-C is obviously increa­sing (46.1 % vs. 28.6%, p=0.31). Long-term follow-up in the cluration of one year after cessation of therapy was feasible in 11 good responclers (CPH-C : 3, CAH-C : 8) after 12 months of rIFN-a treatment. In three of them (27 % ) ALT values flared up; in one of them CPH-C and in two others CAH-C was found on initial !iver biopsy; ali of them were men. The major side-effects during one-year rlFN­a treatment were flu-like syndrome, especially with initial doses, nervousness, depression, loss rcsponder (%) rcspondcr (%) respondcr (%) of hair, leucopenia and lymphocytosis. Serious sicle effects were not noted. lnitial histology CPH-C 10 (28.6%) 14 (40%) ll (31.4%) CAH-C 26 (43.3%) 19 (31.7%) 15 (25%) 60 Discussion CAH-Cand cirrhosis 1 (20%) 4 (80%) Multiple randomisecl controllecl trials have clo­ Tota!(%) 36 (36) 30 (30) 100 cumented that alpha-interferon suppresses cli­ IFN-n i11 tlze treatment of chrcmic hepatitis C sease activity and induces remissions in a high proportion of patients with chronic hepatitis C. Biochemical response (ALT levels) has been associated with doses of 3-5 million units (MU) in 30-70 % of patients with compensated chronic hepatitis C if ALT levels are at least 1.3 times the upper limit of normal and disease activity is demonstrated by !iver biopsy. A long-terrn, sustained improvement cxprcsscd by normal serum ALT occurred in 10-25 % of pa­ IO. 11. 12 , Although, our study is stili ongoing, a preli­minary evaluation of 12 month treatrnent with 3 MU rlFN-a three tirnes weekly is eligible. Normalisation or improvement in ALT values occurrecl in 70 % of patients after two months of treatment. Although the percentage of goocl responclers is significantly higher arnong pa­tients with CAH-C than among those with CPH-C, interestingly, this clifference climinis­hecl after 12 months of treatment. On the other hancl, regarclless of the small number of patients a significantly lower rate of response was notecl in patients with CAH-C ancl cirrhosis on initial biopsy. By the encl of one-year rIFN-a therapy, the percentage of good responclers among pa­tients with CPH-C significantly increased, whe­reas in patients with CAH-C this rate gradually decreased, but altogether, almost equal ratc of good response in both groups was noted after two rnonths of treatment. Thus, in good respon­ders after two months of treatment with rlFN-a 3 MU three times weekly, regardless of initial histological diagnosis of CPH-C or CAH-C further continuation of IFN therapy seems to be warrantecl. tients. I Although the influence of age ancl sex on our results of one year treatment were not estima­ted separately, it seems that women were better responclers, ancl that in younger persons ( <45 years) the efficacy of treatment was significantly better. In general, the results reportecl by some other authors commonly confirm a positive influence of younger age on the probability of response to IFN therapy. On the other hancl, the significance of sex remains to be elucicla­ s. 9. u , tecl. 1 The percentage of rclapse (27 % ) one year after the ccssation of therapy, which we have noted among good responders, is similar to some previously describecl observa­ 9· 14· tions. 1. 8· 15 Our data suggest that long-term treatment with 3 MU rIFN-cx thrice weekly coulcl be beneficial for about one half of pa­tients with chronic hepatitis C. There are many unresolvecl questions associa­ted with the use of rIFN-a in the treatment of chronic hepatitis C, such as increasing of re­sponse rate, fincling of optimal dose and clura­tion of therapy, treating of patients with severe disease or immunosupression, predicting of re­sponse or relapse etc. Although interferon-al­pha is now consiclered standard therapy for patients with active HCV-inducecl !iver clisease, further research of antiviral therapy is neeclecl in order to provide safe ancl highly effective therapy for ali patients with HCV-induced !iver injury. References l. 1-Ioofnagle JH, Di Bisceglie AM, Shindo M. An­tiviral therapy of hepatitis C -present and future. J Hepatol 1993; 17 (Suppl 3): 130-6. 2. Bonino F, Bruneto MR, Ncgro F, Bakli M, Sa­racco G, Abate ML, Fabiano A, Verme G. Hepa­titis C virus infcction and disease. Diagnostic problems. J Hepatol 1993; 17 (Suppl 3): 78-82. 3. 1-Iayashi N, Higashi 1-1, Kaminaka K. Sugimoto l-1, Esumi M, Komatsu K, 1-Iayashi K, Sugitani M, Suzuki K, Tadao O. Nozaki C, Mizuno K, Shikata T. Molccular cloning and heterogeneity of the human hepatitis C virus (HCV) genome. J Hepatol 1993; 17 (Suppl 3): 94-107. 4. Lau JY, Davis GL, Kniffen J, Quian K-P, Urclea MS, Chan CS, Mizokami M. Neuwald PD, Wilber JC. Significancc of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993; 341: 1501-4. 5. Kotwal GJ. Routine laboratory cliagnosis of hepa­titis C virus infection. J Hepatol 1993; 17 (Suppl 3): 83-9. 6. 1-Iagiwara 1-1, 1-Iayashi N, Mita E, Takehara T, Kasahara A, Fusamoto 1-1, Kamada T. Quantita­tive analysis of hepatitis C virus RNA in serum cluring interferon alfa therapy. Gastroenterology 1993; 104: 877-83. 7. Yatsuhashi 1-1, Jnoue O, Inokushi K, Koga M, Nagataki S, Cha T-A, lrvine 13, Stempien M, Kolberg J, Urdea MS, Yano M. Short and long­ Palmovic D. ancl Crn.jakovic-Palrnovic .l. term effects of interferon on serum markers of hepatitis C virus replication . ./ Gcwroenterol Hepa­ tol 1993; 8: l-6. 8. Alberti A, Chemello L, Bonetti P, Casarin C, Diodati G, Cavaletto L, Cavaletto D, Frezza M, Donada C, Belussi F, Casarin P, Pozzato G, Ruol A, and the TVVH Study Group. Treatment with interferon (s) of community-acquired chronic he­patitis ancl cirrhosis type C. ./ Hepatol 1993; 17 (Suppl 3): 123-6. 9. Carreno V, Marriot E, Quiroga JA. Other ap­proaches to the treatment of chronic vira! hepati­tis . ./ Hepatol 1993; 17 (Suppl 3): 127-9. 10. Castilla A, Camps-Bansell J, Civeira M-P, Prieto J. Lymphoblastoid interferon for chronic hepatitis C: A randomized controlled study. Am ./ Ga­stroenterol 1993; 88: 233-9 . 11. Jansen HLA, Brouwer JT, Nevens F, Sanchez-Ta­pias JM, Craxi A, Hadziyannis S, European con­certed action on vira! hepatitis (Eurohep). Fatal hepatic decompensation associated with interferon alfa. B M./ l993; 306: 107-8. 12. Bosch O, Tapia L, Quiroga JA, Carreno V. An escalating dose regime of recombinant interfcron­alpha 2A in the treatment of chronic hepatitis C. .! Hepato/ 1993; 17: 146-9. 13. Battezzati PM, Podela M, Bruno S, Zuin M, Crosignani A, Camisasca M, Chiesa A, Petroni ML, Russo A, Gallotti P, Borzio M, Borzio F, Busearini L, Fornari F, Sbolli G, Lanzini A, Pigozzi MG, Salmi A, Dastoli G, Carriero PL. Factors predicting early response to treatment with recombinant interferon alpha-2a in chronic non-A, non-B hepatitis. Preliminary report of a long-term tria!. !tal./ Gastroentero/ 1992; 24: 481­4. 14. Varagona G, Brown D, Kibbler H, Scheuer P, Ashrafzadeh P, Sherlock S, Mclntyre N, Dusheiko GM. Response, relapse and re-treatment rates ancl viraemia in chronic hepatitis C treated with 2-b interferon: a phase lll stucly. Eu.r .! Gastroen­terol Hepatol 1992; 4: 707-12. 15. Okacla S-1, Akahane Y, Suzuki H, Okamoto H, Mishiro S. The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interfe­ron therapy in viremic patients. Hepatology 1992; 16: 619-24. Radio/ Onco/ 1993; 27: 321-5. Treatment of cervical intraepithelial neoplasia associated with human pappilomavirus by interferon vaginalettes Zvonimir Singer, 1 Eugen Šooš, 2 George Feichter3 1 University Hospital Merkur, 2 Institute of Immunology, Zagreb, Croatia, 3 University Institute for Pathology, Basel, Switzerland The randomized prospective study of the manifested 176 cases of cervical intraepithelial lesions (CIN) associated with human papillomavirus infections during 24 to 36 months was performed. The control cases were 240 patients which have had the same findings but were not treated with human leukocyte interferon (n-IFN-a). For the diagnosis and follow up, cytology, histology, colposcopy and dot-blat HPV typing were used. Patients were treated with n-IFN-a vaginalettes containing 1 x J(f l. V. of the natura! interferon alpha and Excip. Macrogoli as a daily dose. In the course of two menstrual cycles 42 vaginalettes were given to each patient. Vaginalettes may be given as a monotherapy, or in combination with other remedies, depending on the micro biological findings or persistence. For statistical evaluation Chi-square, Fisher exact probability and Kolmogarov-Smirnow goodness of fit test were used. After 24 to 36 months follow up significant improvement in the treated group was found, and progressions of carcinogenesis was not registered. At the same tirne significant persistence of CIN was found in the control cases as well as twenty five or 42 % controls progressed into higher grade lesions. Better results of treatment were obtained with dot-blot hybridization negatives and with low-grade cases of cervical intraepithelial lesions. Our conclusion is that especially in young women, for the preservation of fertility and in pregnancy, n-IFN-a vaginalettes may be a remedy of choice. Key words: cervix neoplasms-drug therapy; papillomaviruses; natura! interferon-alpha; administra­tion, intravaginal Introduction Human papillomaviruses (HPV) are clearly re­lated to genital squamous precursor lesions and invasive carcinomas. 1·5 Correspondence to: Eugen Šooš, V.M.D., Ph.D., Institute of immunology, 41000 Zagreb, Rockefelle­rova 2, Croatia. UDC: Prospective, randomized, double blind study on the natura! human leukocyte interferon treatment have been perfonned and published previously. 6• 7 Preliminary results of treatment with n-IFN-a vaginalettes resulted in significant improvement in the treated group.8 The aim of our work is to eliminate the cervical intraepithelial neoplasia (CIN) as a potential cancer precursor. Singer Z. et al. Materials and methods Results Out of 250 randomized9 cases 176 were treated with vaginalettes. The remaining 74 cases were without n-IFN-a treatment for different reasons, or were !ost from evidence. Each vaginalette, as 106 a daily dose, contained 1 x I.U. of curde interferon preparation and Excip. Macrogoli. A total dose of 42 vaginalettes was given to each patient. For diagnosis and follow up cyto­logy, 10 colposcopy,11 histology and dot-blot hy­bridization of the HPV deoxyribonucleic acid 9 (DNA)were used. Before treatment, Chlamydia, Gardnerella, fungi or other microbiological causes of inflam­mation were treated. Prospective study was perfonned after three month interval using cy­tology/colposcopy investigations throughout 24 to 36 months. The treated group was compared with randomized cooperative 240 controls. For statistical evaluation Chi square, Fisher exact probability and Kohnogarov-Smirnow goodness of fit test were used. Table 1 shows randomized groups and treat­ment results. Table 2 shows significant improve­ment (P < 0,01) in the treated group. Important differences may be scen in the eradication of the disease in treated group and progression of the disease in the controls. Table 3 presents the HPV DNA analysis; high-risk types 16/18 were found in more than 50 % of treated cases. Table 4 and 5 show that better results have been obtained with dot blot negatives (P < 0,05) and with low-grade cases of cervical intraepithe­lial lesions (P < 0,05). Discussion Double blind study was not performed in this investigation for two reasons. Firstly double blind study was done in our previous investiga­tions. 6• 7 Secondly a double blind is not always feasible because of the medico legal reasons. Table l. Results of treatment with interferon vaginalettes. Results N % Remarks Eradication 41.48 Two six-month interval negative findings 31.25 Regrcssion 55 "Borderlinc" cases Pcrsistency 48 27.27 Permancntly the same findings Tota! treated 176 l00.00 IFN-a in treatment of cervica/ intraepithelial lesions 323 Table 2. Corrclation of trcatccl cascs with controls. Controls Rcsulls N % Rcsults N Eraclication Eraclication o/c) 73 Trcatccl 41.48 Rcgrcssion 77 32.08 Rcgrcssion 31.25 Pcrsistcncy 138 57.50 Pcrsistcncy 27.27 Progrcssion 25 10.42 Progrcssion Tota! trcatccl 240 100.00 Tota! 176 100.00 CONTROLS TREATED PROGRES­ PERSISTENCY REGRESSION SION -, ERADICA­, TION PERSISTENCY REGRESSION Table 3. Rcsults of trcatmcnt with interferon vaginalcttcs rcgarcling vira! typcs. Rcsults N 16/18 31/33/35 6/11 6/11/16/18 6/l 1/31/33/35 16/ 18/31 /33/35 N l Eraclication 73 14 8 1 1 4 28 2 Rcgrcssion 55 10 4 2 1 - 2 19 3 Pcrsistcncy 48 9 ll - - 1 1 22 4 Excluclccl 74 3 7 5 - - 15 Tota! 2 1 7 250 36 30 8 L.ll .. .···.Jl.. CIJ L!) CD (') CD (') The same considerations have been stated also by other authors. 12 Characteristic of our strategy was that inter­feron vaginalettes were used topically, also in pregnancy (10 % of treated group ), and did not interfere with the human reproduction. Surgical treatments were performed ( cold-knife coniza­tion) in persistent cases, over 30 years of age, CIJ (') CD (')L!) . L!) .(') ro-- CD CD CD . if the condition lasted for more than 30 months, and in lower grades of CIN with intent of a fina! eradication in younger women (C02 la­ser). The vaginalettes were mostly used as monotherapy, but also in combination with other modes of treatments, depending on the case. In order to prevent HPV reinfections, the specific treatment of partners should be taken in consideration. .i..) Singer Z. et al. Table 4. Results of treatment with interferon vaginalettes ancl blot hybriclization finclings. Positive Results Negative Tota! N % N % N % 2 3 Eradication Regression Persistency Tota! Treated 28 19 22 69 40.58 27.54 31.88 100.00 45 36 26 107 42.06 33.65 24.99 100.00 73 55 48 176 74 Exluded Tota! randomizecl 15 84 33.12 59 166 66.88 250 70.40 29.60 [00.00 30 ---7 40 1435 5 TOTAL IEXCLUDED 1 25 20 15 1 30 25 1 20 . 10 1 15 1 1 [REATED 5 o 1 1 2 3 2 3 Table S. Rcsults of treatment with interferon vaginalcttcs and Pap test in Squamous lntracpithelilal Lcsions Low-gradc High-graclc Tota! %176 %176 Rcsults 2 l Eraclication N % N N 27.84 31 [7.61 13.64 27 15.34 16 80 51 Rcgrcssion 24 3 Pcrsistcncy 29 16.48 Tota! 9.09 28.98 25.57 102 57.96 42.04 176 [00.00 ---.-1 50 45 40 -35 30 -25 20 35 TOTAL 30 25 20 15 2 5 o o 2 3 2 3 Morphogenetic grades of CIN and HPV as well as by follow up of the lesions, were DNA types appear to be of value as prognostic analyzed in both groups, but from the patient's indices. 13 The low or high-grade of CIN and point of view it is essential that clinical termino­data obtained by colposcopy and HPV typing, logy does not result in over treatment. The !FN-u in treatment oj' cervica/ intraepithelial lesions rcsults of interferon treatment wcre also found to be in close correlation with CIN and Pap grade classification. 15 In our opinion, patients carrying high-risk HPV typcs should have regu­lar checks at shorter intervals. 16 Some findings indicatc long (up to 10 years) persistence of HPV infection in the natura! course of progres­sion to the carcinoma of the uterine cervix. 17 Thcrefore, follow up and further investigations are under way. In conclusion, we want to point out that especially in young women, for preservation of fertility and in pregnancy, the natura! crude interferon alpha vaginalettes may be the treat­ment of choice. References 1. Crum CP, N uovo GJ. Genital Papillomavirus ancl related neoplasms. New York: Raven Press 1991: 65-79. 2. Wernes BA, Mungcr K, Howley PM. Role of the human papillomavirus oncoproteins in transforma­tion ancl earcinogenic progression. ln:Philadcl­phia: Important Advanccs in Oncology 1991: 3-18. 3. Richart RM, Wright TC. Human papillomavirus. Curr Opin Obstetr Gynecol 1992; 4: 662-9. 4. !tri LM. The interferons. Cciizcer 1992; 70: 940-5. 5. Sheets EE, Crum CP. Current status and future clinical potencial of human papillomavirus infce­tion ancl intraepithclial neoplasia. Curr Opin Ob­stetr Gyneco/ 1993; 5: 63-6. 6. Singer Z, lkic D, Beck M, Šooš E, Šips ' Jušic D. Interferon trcatmcnt of utcrine precancerosis. 12th lnternational Immunobiological Syrnposium. Symposium on interferon Yug Acd Sei Arts, Za­1'1'"1) 1979: 127-35. 7. lkic D., Singer Z, Bcek M, Šooš E, Šips D, Jušic D. Interferon treatment of ute rine preeancerosis. .! Cancer Res Ciin Oncol 1981; 62: 625-9. 8. Singer Z, Beek M, Jušic D, Šooš E. Interferon vaginalettes influence on cervical intraepithelial lesion . .fugosl Gine/wl Perinatol 1990; 30: 27-9. 9. Singer Z, Djordjievski E, Feichtcr G, Vrabec B. Investigation of dcoxyribonucleic acicl of the hu­man papillomavirus types in Zagreb population. Gynecol Perinatol 1992; l(Suppl 1): accepted for publication. 10. National Cancer Institute Workshop: The 1988 Bethesda: System for reporting cervical/vaginal cytological diagnoses. JAMA 1989; 262: 931-4. 11. Wilbanks GD. An intcrnational colposcopic termi­nology: lnternational Federation of Cervical Pat­hology/Colposeopy. Europ .! Gynecol Onco/ 1991; 7: 321-2. 12. Sehneider A, Papendick U, Gissmann L, Villiers EM. Interferon treatment of human genital papil­lomavirus infections: lmportance of vira! type. Int .! Cancer 1987; 40: 610-4. 13. Hcllberg D, Nilsson S, Grad A, Hongxiu J, Fuju C, Syrjanen S. Behavior of cervical intracpithelial neoplasia (CIN) associated with various human papillomavirus (HPV) types. Are/z Gynecol Obstet 1993; 252: 119-28. 14. Syrjanen K, Kataja V, Yliskoski M, Chang F, Syrjanen S, Saarikoski S. Natura! history of cervi­cal human papillomavirus lesions does not substan­tiate the biological relcvance of the Bethesda System. Obstet Gyneco/ 1992; 79: 675-82. 15. Manavi M, Czerwenka F, Enzelsberger H, Kno­gler W, Seifert M, Raimann H, Reinolcl E, Kubi­sta E. Humane Papillomavirus (HPV) DNA Infck­tionen an cler Cervix uteri. Geburtslz u Freuenheilk 1992; 52: 283-6. 16. Feiehter G, Heinzl S, Uchlinger U, Torhorst J, Vrabec B, Dalquen P. Vergleichende DNS-Hybri­disicrung, Zytologie und Histologie an konclylo­malosen und prakanzerosen Lesionen der Cervix uleri. Geburtsh u Frauenheilk 1992; 52: 758-63. 17. Konno R, Sato S, Yajima A. Progression of squamous celi carcinoma of the uterine cervix from cervical intraepithelial neoplasia infcctecl with human papillomavirus: A retrospective study by in situ and polymcrase chain reaction. fnt .! Gynecol Pat!zol 1992; 11: 105-[2 . Radio! Oncol 1993; 27: 326-31. Natural IFN-a for non-small-cell Jung cancer with pleural carcinosis Berta Jereb,1 Gabrijela Petric-Grabnar,1 Marjet. Tercelj-Zorman/ Marija Us-Krašovec,1 Renata Mažuran,3 Evgen Sooš. Janez Stare4 1 Institute of Oncology, Ljubljana, Slovenia, 2 Institute for Lung Diseases, Golnik, Slovenia, 3 Institute of Immunology, Zagreb, Croatia, 4 Institute for Biomedical Informations, Medica! Faculty, Ljubljana The survival of patients with pleural effusion from bronchial carcinoma is short. Ten patients with ipsilateral pleural effusion from non-small cel! cancer of the lung, localized to the thoracic cavity, were treated with intrapleural application of IFN-a and radiation therapy. All had malignant pleural effusion confirmed by cytology. Radiation therapy was given to the hemithorax with boost to the area of local tumor and mediastinal lymph node metastases. The dose to the hemithorax was 20-25 Gy, whereas the total dose to the tumor bed and mediastinum was 45 Gy. IFN-a 2 X Ia6 IV diluted in 20 ml of distilled water was injected intrapleurally once weekly. The treatment, as a rule, is suitable for palliation only. The effect of IFN-a was evaluated according to the cellular morphology of the pleural fluid and the patients' survival. The median survival of IFN-a treated patients was 17 months. The median survival of the matched control pts treatecl only for palliation with pleuroclesis or racliation therapy was 7 months. The patients in the experimental group had a slightly better chance of prolonged survival. A ranclomized clinical trial seems to be indicatecl. Key words: carcinoma, non-small cell lung-drug therapy; interferon-alpha Introduction Non small cell lung cancer (NSCLC) represents 75 % of lung cancer, the most common cancer in males. The diagnosis is late in the great majority of cases (70-75 % ) and the overall survival of NSCLC patients is poor. The 5-year survival of operable (Stage I and Stage II) Correspondence to: Berta Jereb, M.D. Institute of Oncology, Ljubljana, Zaloška 2, Tel. No. (386 61) 323-063 (37-17), Fax: 1314-180, Slovenia patients treated by surgery is 30-40 % . The survival of patients with Stage III is less than 5 % whereas the survival of those with malig­nant pleural effusion and those with Stage IV 2 is practically nil. 1• Neither chemotherapy nor radiation have contributed much to the survival of these patients. Surgery has been attempted 8 for cure in patients with stage III,3-but was not succesfull, especially not in those with pleu­ral effusion. To palliate symptoms and prolong the survival was the aim of severa! trials; recen­tly, there have been reports with encouraging UDC:616.24-006.6-085 results of treatment with intrapleural applica­ Natura/ IFN-a for no11-small-cell lung cancer witlz pleural carcinosis tions of chemotherapy and biologic response modifiers.9·t2 IFN-a has proved to be locally effective in severa! solid malignant tumors, 13 and systemi­cally effective in severa! haematological malig­nancies.14· 15 As a single therapeutic agent for adenocarcinoma and other solid tumors it has shown only modest results. 16 Enhancement of chemo-and radiation therapy with IFN-a has been shown in vivot 7-20 and in vitro studies.21 Also, some enhancing effect of IFN-a on che­motherapy and radiation has been established.22 Earlier, we have reported on 14 patients with NSCLC and pleural effusion treated with intra­pleural application of IFN-a; it was founcl that IFN-a could clear effusion from cancer cells and haemorrhagic admixture with minimal sicle effects. The treatment also prolonged the survi­val of patients.23 In the presented series, 10 patients with NSCLC and pleural effusion, without distant metastases (Stage Illb ), were treatecl by radia­tion and intrapleural applications of IFN-a, with the aim of permanent local control and prolonged survival. Materials and methods Experimental group Ten patients admitted to the Institute of Onco­logy between december 1988 through november 1991, were treated for pulmonary cancer and pleural carcinosis. They had malignant cells in the pleural effusion proved by cytology; the primary tumor was confirmecl to be adenocarci­noma by bronchoscopy and biopsy in all cases. The extent of the disease was further clefined on plain chest radiograms, by CT of the chest and the brain, abdominal echogram, 99mTc bone scan in acldition to the conventional bioc­hemical and haematological laboratory tests. Their clinical data are presented in Table 1. The clisease was confined to the chest in ali patients, those with metastases outsicle of the chest were not included. Ali the patients had radiation therapy 20-24 Gy to the whole hemithorax with a boost to the primary tumor ancl mediastinal metastases to a total dose of 40-45 Gy (Table 1). IFN-a was given by intrapleural application weekly as long as pleural effusion was present. After that it was given intramuscularly, twice weekly. In one patient (No. 4) it was only given i.m. because of a high risk for bleeding. 2 X 106 units of natura! IFN-a were diluted in distilled water and after thoracocenthesis, with removal of as much fluid as possible, injected into the pleural cavity. Control group During the same period the great majority of patients with Jung cancer and pleural carcinosis were treated for paliation by other methods both, at the Institute of Oncology as well as at the Institute for Lung Diseases, Golnik, the choice being made by the referring physician. Among these, 10 were chosen who matched the patients in the experimental group in tenns of age, sex, and extent of disease. There were, however, 3 patients with squamous celi carci­noma in the control group. None of the patients had radical surgery performed previously, two had pleurodesis with Achromycin, 3 received palliative radiation and 5 analgetics only. The mean age of this group of patients was 56 years as compared with the mean age of 54 in the experimental group. The leve! of IFN-a was measured in the pleural fluid and in the blood serum before and after IFN-a treatment in 2 patients, one from the experimental group and one from the con­ trol group. In our experiments we used WISH ( epithelial cells; European Co!lection for Animal Cell Cul­ tures, ECACC, England) and MDBKK (bovine kidney, epithelial; American Tissue Type Col­ lection, ATCC). As celi-virus combination using MDBK + VSV is not suitable for the detection of IFN-a, recently we have neglected MDBK cells, esspecially after we have got enough monoclonal antiboclies to identify the type of IFN-ain samples of unknown IFN con­ stitution.24 w Table l. Clinical data of patients in the experimental group. N 3600 Pat. Site Other RT IFN-a tre a t men t Survival No. Age Sex (lobe) Chemotherapy dose/volume Spread metastases (mos) dose effect 106 IU effusion complications 1 60 M RLL mediastinum R thorax 2100 8 X i.p. fibrosis brain, L Jung, !iver, 18 - tumor bed 1500 peritoneum DOD 2 26 M LLL 5-Fu, VP 16 } 5x L thorax 1500 tumor bed 3000 4500 5 X i.p. -fever brain lymphadenopathy DOD 3 51 F LLL L thorax 2000 3 X i.p. brain 9 tumor bed 1750 12 X i.m. DOD 3750 - '--, 4 50 F LLL subclavian ven. L thorax 1500 8xi.m. residual coagulopathy 1 thrombosis tumor bed 2800 1 X i.p. tumor on autopsy (S 4300 not proven i):, 5+ 38 M RLL R thorax 4000 15 X i.m. fcver L Jung, R thoracic 46 Q wall AWD f2.. 6 56 M RLL bil. lymph-R thorax 1500 9 X i.p. residual -lymphangio-5 angiocarcinosis tumor bed 2000 carcinosis DOD 3500 7 72 F LLL L thorax1500 9 X i.p. residual lymphangio-18 tumor bed 2000 carcinosis DOD 3500 8 72 F LLL mediastinum L thorax 1650 9 X i.p. residual -8 tumor bed 1800 DOD 3450 9 52 M LUL mediastinurn Thiotepa L thorax 3000 13 X i.m. residual fcvcr !iver, peritoneum 23 i. p. lX DOD 10 66 F RUL mecliastinum R thorax2500 2X i.p. Llung 30 - tumor bed 2000 11 X i. m. DOD 4500 DOD = dead of disease R =right U = upper i. p. = intrapleural A WD = alive with disease L =left L = lower i. m. = intramuscular + = pleuropneumectomy Natura/ IFN-cx for 11011-small-ce// lwzg cancer with pleural carcinosis Ali patients in the experimental group have been regularly followed by clinical examination, laboratory and blood tests, chest X-ray and CT of the brain. The follow up of the patients in the control group was by the referring physi­cian, who has treated thern syrnptomatically. Therefore, only the date of death is reported for these patients and no details about the The survival was calculated by the Kaplan­Meier rnethod frorn the date of diagnosis until death or the date of the last follow up.25 The difference in the survival of the two groups was calculated with the log-rank test. Results At the ene! of the study in July 1993, 3 patients were stili alive one patient from the experirnen­tal group more than 4 years, and 2 from the control group 17 and 15 rnonths respectively (both had squamous celi carcinoma), ali with rcsidual cliscase. The survival is shown in Figure 1. The median survival of the patients in the experimcntal group was 17 months as comparecl to 7 rnonths in control patients. Malignant cells have disappeared frorn the pleural fluid after treatment with IFN-a in ali patients, in the rnajority the fluicl was stili present. Cytology was possible in 9 out of 10 patients, in 2 of thern without the influence of radiation thcrapy. In ali patients it showed essentially the same findings as in a previous26 study of IFN-a in pleural effusions from breast cancer, i.e.: a) increase in the number of transportecl lyrnphocytes and histiocytes in the sediment of the exudate, b) markcd clecrease in the nlllnber of rnalig­nant cells, and c) markecl degenerative changes in the rernai­ning malignant cells. The lcvels of IFN-a in the pleural fluicl ancl serum are presented in Figure 2 for patient No. 1 of the experimental group, ancl in Figure 3 for a patient in the control group. Only a rninirnal rise was observed in the serum in either of the two patients. proportion o---control surving group 1.0 0.8 0.6 0.2 o.o Figure l. Survival of patients with 11011-small-cell lung cancer and pleural carcinosis. Discussion In a previous study of patients with pulmonary cancer ancl pleural effusion it was observed that IFN-a treatrnent may clear the effusion of can­cer cells ancl haernorrhagic admixture and arrest fluicl accumulation with minimal sicle effects.23 -<>-serum 121 pleural fluid 16 17 20 21 22 23/0)/89 Figure 2. Pharmacokinetics of serum IFN during the­rapy. Bars represent IFN in pleural fluid. IFN appliea­tion is indicated by arrows. Curve(s) are daily serum IFN levels. 330 Jereb B. et a/. paticnt S. D. 100 --serum 0 plcural fluicl rapy. Bars reprcscnt IFN in plcural fluicl. IFN applica­tion is inclicatccl by arrows. Curvc(s) are claily serum IFN lcvcls. Improvement in the survival was also noted. Even in this series of patients treated with radiation therapy and JFN-cx, the treatment was tolerated well; an increased temperature and Iocal pain within the 24 hours after application and reactive lymphadenopathy (Pat.No. 2) were the only complications. Coagulopathy (Pat. No. 4) was more likely a complication of the treat­ment for thrombosis than of IFN-cx application. The leve! of IFN-cx in 2 patients under inve­stigation showed only a minimal rise of the serum levels, an observation different from the one in a recent series of patients with pleural effusion due to breast cancer treated in a similar way. 24 As this observation is based only on 2 patients, a larger group studied, though not included in this series, will be part of a separate report. There are stili many uncertainties regarding the treatment of Jung cancer with IFN-cx, the dosage and the timing of treatment being the most obvious. While in our series a tendency to better survival is shown for the patients treated with IFN-cx, the difference in survival is not statistically significant (possibly due to the small series), and on the other hand, it could be due to radiation therapy alone. Radia­tion therapy has not been shown to affect the survival of patients with inoperable Jung cancer. It has, however, not been tried in patients with pleural carcinosis.27 Because of a trend towards improved survival in patients treated with IFN­cx and the good tolerance for combined treat­ment with radiation and IFN-cx we have started a randomized tria!. The patients with Jung cancer and pleural carcinosis will be treated either with radiation alone or radiation and IFN-cx. We will also continue to study the serum levels after intra­pleural applications of IFN-cx. Acknowledgement The study was supparted by the Ministry of Science and Technology of the Republic Slove­nia. Grant No. P3-5249-0302. References l. lhclc DC, Minna JD. Non-small celi Jung canccr. II. Trcatmcnt. Curr Probl Cancer 1991; 15: l05­54. 2. lhclc DC: Chcmothcrapy of Jung canccr. New Engl J Med 1992; 327: 1434-41. 3. Morton RF, Jctt JR, McGinnis WL ct al. Thoracic racliation thcrapy alonc comparccl with combinecl chcmoracliothcrapy for locally unrcscctablc non­small celi lung canccr: a ranclomizecl, phase II tria!. Ann Int Med 1991; 115: 681-6. 4. Wcick JK, Crowlcy J, Natalc RB ct al. A ranclo­mizecl tria! of five cisplatin-containing trcatmcnts in patients with metastatic non-small-cell lung canccr: a Southwcst Oncology Group stucly. J Ciin Onco/ 1991; 9: 1157-62. 5. Lynch TJ, Clark JR, Kalish LA ct al. Continuous­infusion cisplatin, 5-fluorouracil, ancl bolus mctho­trexatc in thc trcatmcnt of aclvancccl non-small celi Jung canccr. Cancer 1992; 70: 1880-5. 6. Schaakc-Koning C, Bogaert van eten W, Dalcsio O et al. Effccts of concomitant cisplatin ancl racliotherapy on inoperablc non-small-cell Jung canccr. New Engl J Med 1992; 326: 524-30. 7. Gatzemeicr U, Heckmayr M, Hossfclcl DK, Kau­kcl E, Koschcl G, Ncuhauss R. A ranclomizccl tria! with mitomycin-C/ifosfamiclc versus mitomy­cin-C/vinclesinc vcrsus cisplatin/ctoposicle in aclvancccl non-small-cell Jung canccr. Am J Ciin Oncol 1991; 14: 405-11. 8. Gurncy H, de Campos ES, Doclwcll D, Kamthan A, Thatcher N. Ifosfamicle ancl mitomycin in combination for thc trcatment of paticnts with Nmural IFN-a far non-small-ce/1 lung cancer with pleural carcinosis progrcssivc advanccd non-small celi Jung canccr. Eur J Cancer 1991; 27: 565-8. 9. Ruckdcschcl JC. Managcmcnt of malignant plcu­ral cffusion: an ovcrvicw. Semin Oncol 1988; 15: 3(Suppl 3): 24-8. 10. Luh K-T, Yang P-C, Kuo S-H, Chang D-B, Yu C-J, Lee L-N. Comparison of OK-432 and mito­mycin C plcurodcsis for malignant plcural cffusion causcd by lung canccr. Cancer 1992; 69: 674-9. 11. Kodama K, Doi O, Tatsuta M, Kuriyama, Tatcishi R. Dcvclopment of postoperativc intrathoracic chcmothcrapy for Jung canccr with objcctivc of improving local curc. Cancer 1989; 64: 1422-8. 12. Masuno T, Kishimoto S, Ogura T, Honma T, Niitani H, Fukuoka M, Ogawa N. A comparativc tria! of LC9018 plus doxorubicin and doxorubicin alonc for thc trcatmcnt of malignant plcural effu­sion sccondary to lung canccr. Cancer 199 l; 68: 1495-500. 13. lkic D, Nola P, Maricic Z ct al. Application of human lcucocytc interferon in paticnts with uri­nary bladdcr papillomatosis, brcast canccr, and melanoma. Lancet 1981; 1: 1022-30. 14. Dianzani F. The inte,feron system. London: Hcalth Scicnces Prcss, 1993: 87-101. 15. Talpaz M, Kantarjian HM, McCrcdic KB, Keating MJ, Trujillo .1, Guttcrman .1. Clinical invcstigation of human alpha interferon in chronic myclogcnous lcukcmia. Blood 1987; 69: 1280-5. 16. Bcngtsson N-O, Lcnncr P, Sjodin M ct al. Mcta­static rcnal celi carcinoma trcatcd with purificd lcukocytc interferon. Acta Oncol [991; 30: 713-7. 17. Pazdur R, Ajani JA, Patt YZ ct al. Phasc II study of fluorouracil and rccombinant interferon alfa-2a in prcviously untrcatcd advanccd colorcctal carci­noma. J Ciin Oncol 1990; 8: 2027-31. 18. Mcadows LM, Walthcr P, Ozcr H. Alpha-intcrfe­ron and 5-fluorouracil: possiblc mcchanisms of antitumor action. Semin Onco/ 1991; 18: 5(Suppl 7): 71-6. 19. Diaz-Rubio E. Trcatmcnt of advanccd colorcctal canccr with rccombinant alpha interferon and 5-fluorouracil: a review. In: The role of alpha inte,feron in solid tumors. New Jersey: Schering­Plough International, 1991: 7-9. 20. Meadows L, Walther P, Lindley C, Bernard S, Misra R, Ozer H. Phannacologic and biochemical modulation of 5-fluorouracil (5-Fu) by alpha inter­feron. In: The role of alpha inte,feron in solid tumors. New Jersey: Schering-Plough Internatio­nal, 1991: 10. 21. Suzuki N, Oiwa Y, Sugano I et al. Dipyridamole enhances an anti-proliferative effect of interferon in various types of human tumor cells. lnt J Cancer 1992; 51: 627-33. 22. Holsti LR, Mattson K, Niiranen A et al. Enhan­cement of radiation effects by alpha interferon in the treatment of small celi carcinoma of the Jung. Jnt J Radiat Onco/ Biol Phys 1987; 13: 1161-6. 23. Tercelj-Zorman M, Mermolja M, Jereb M et al. Human leukocyte interferon alpha (HLI-alpha) for treatment of pleural efffussion caused by non small celi Jung cancer: a pilot study. Acta Oncol 1991; 30: 963-5. 24. Mažuran R, lkic-Sutlic M, Jereb Bet al. Intrapleu­ral application of natura! IFN alpha in breast cancer patients with pleural carcinomatosis. Moni­toring of immunotherapy by assaying serum inter­feron levels. J Biol Regul Homeostat Agents 1992; 6: 46-52. 25. Kaplan EL, Meier P. Non-parametric estimation for incomplete observation . .T Am Stati.1· Assoc 1958; 53: 457-81. 26. Jereb B, Štabuc B, Us-Krašovec M, Cerar O, Starc J. Intrapleural application of human leuko­cyte interferon (IFN-alpha) in breast cancer pa­tients with pleural carcinosis. Adv Radio! Oncol 1992; 175-80. 27. LeChevalier T, Arnagada R, Quoix E et al. Radiotherapy alone versus combined chemothe­rapy and radiotherapy in non-resectable non­small-cell lung cancer: first analysis of a randomi­zed tria] in 353 patients . .T Natl Cancer lnst 1991; 83: 417-23. Radio/ Oncol 1993; 27: 332-8. Adjuvant treatment of malignant melanoma with human leukocyte interferon after radical surgery: I. general analysis Zvonimir Rudolf Institute of Oncology, Ljubljana, Slovenia In our randomized JJrO.JJective study, palienls with malignant melanoma were lreated with human leukocyte interferon (HLJ) afier surgical removal of primary tumor (Clark leve! of invasion IV, V and/or thickness exceeding 1.5 mm). They were randomized in two groups: (!) those treated with HLI and (2) a con/rol group with no immediale lreatment. HLI was applied through 30 weeks in cummulative dose 6 x U ,and 2 X Jr/' U weekly. Both arms of the study included allogether 321 l07 patients. The resulls of 5-year analysis showed significanl dijf'erences in disease-ji-ee interval as well as in survival between both groups in favour of HLI treated palienls (p < 0.005). in the treated group the rate of NED patients was significantly higher than in the con/rol group. According to the stratification by sex, the difference was significant also between jemale as well as male patien/s of both groups (p < 0.005). In a majority c.l patien/s HLJ application caused a jlu-like syndrome, whereas adverse effects on blood count and chemistry could 1101 be established. The treatment (given in the reported dose) was not toxic and could be applied on an out-patients basis. Key words: melanoma-therapy; surgery, operative; interferons Introduction In the world, patients with malignant melanoma of the skin represent approximately 1 % of ali cancer patients. The incidence of melanoma has been rapidly increasing, reaching its double value every 6-10 years, and Iikewise, also mela­noma-related mortality has been exhibiting a trend of constant increase. Also in Slovenia, the yearly incidence of cutaneous melanoma by Correspondencc to: Prof. Zvonimir Rudolf, MD,PhD, Institute of Oncology, Zaloška 2, 611()5 Ljubljana, Slovenia, Tel. + 386 61 1314225, Fax + 386 61 1314180. UDC: 616-006.81-08:615.28!.7.015.45 sex shows tcndency of increase. 1 In thc survival analysis study2 of malignant melanoma in Slovc­nia, overall 5-year survival was 57 .5 % , and median survival 108 months; 5-year survival by sex was 66.4 % for females and 38.5 % for males. Using univariate analysis of the sex and other clinical and pathohistological variables on the survival a statistical significant difference was established so for sex as well as for the extent of the invasion by Clark levels. lrrespec­tive of the sex, a statistically significant better survival was found in the group of patients with thinner melanoma. Considering the high mortality ratcs obscrved in patients with malignant melanoma (with deep leve! of invasion) as well as ineffective treat­ i\djuvant treatment of malignailt melanoma 1vith !FN-a :i:n ment of advanced disease, many studics have bcen invcstigating the potcntial of various treat­mcnt modalitics. Sincc thcsc rcsults of malignant melanoma trcatmcnt are stili unsatisfactory, cspccially in advanced stagcs of discase, an cffort should bc dirccted to carlicr trcatmcnt. U nfortunatcly, the rcsults of adjuvant trcatmcnt in thc carly 3 stage of the diseasc with chemothcrapyhavc also not confirmed thc effcctivcness of treat­mcnt so far. During thc last dccade a number of clinical studics havc bccn pcrformcd to invcstigatc thc thcrapeutic potcntial of interferons in thc treat­mcnt of various malignant discascs.4 Although partial and occasional complcte rcgrcssions havc been observecl in some canccr paticnts5 thc ovcrall results of singlc-agent interferon trcatment point out the need for furthcr clinical and laboratory research in orclcr to cstablish the role of interferon in canccr trcatmcnt, par­ticularly in solid tumors. Besides cxcrting a dircct antiproliferative effcct on marnmalian cells, interferons have provccl to bc patent activators of natura! killer cells and macropha­ges. 6 These cclls have also bccn involvecl as cffcctors in host resistance to tumor devclop­ 8 mcnt ancl in tumor control proccsscs.7· In view of thc previously mentioncd facts, wc dccidcd to cstablished thc role of interferon as an adjunct to surgical trcatment of primary malignant melanoma. A prospcctivc randomiz­ed trial9 was commcnced in 1988 in paticnts with malignant melanoma stagc IIA ancl B according to thc AJCC classification. lO Patients and methods Three-hundred and twenty-onc patient with ma­lignant melanoma entcred thc study. In thc protocol only paticnts with histologically praven primary tumor aftcr radical surgery were includ­ed. As mcntioned prcviously, ali the patients wcre in Stage IIA and IIB of thc discasc which means tl1at thc primary tumors werc classifiecl as Clark IV,V leve! of invasion and/or tumor thickness cxcccding l.5 mm. Thc paticnts were randomizcd in two protocol anns -a group trcatcd with human lcukocyte interferon (HHLI) and control group with no immcdiatc trcatmcnt after radical surgcry (HCON) as shown in the protocol summary (Figure 1). Ali paticnts in both groups wcrc on rcgular clinical follow-up. Complete blood counts, blood chcmistry, rcnal and !iver function tests wcre takcn cach check; thcse wcre performccl monthly in thc first 2 years, and latcr on in 2 month intcrvals. Complctc cvaluation of pa­ticnts was done before and after the trcatmcnt. Paticnts with rclapsc (in both groups) wcre further trcatcd as nccessary (with surgery, ra­diotherapy, chemothcrapy) and wcrc aftcrward also on regular follow-up. Treatment Trcatment consisted of i/m application of crudc human lcukocytc interferon (Imunološki zavod, Zagreb, Croatia) and started within the first month aftcr surgical cxcision. Interferon was appliecl for 30 weeks in curnulative dosc of 6 x 107 units. Each paticnt receivcd 2 x 106 units of interferon weekly. Sincc at thc start of thc study only human leukocytc interferon was available, this analysis refers only to thc appli­cation of this agent, while later in the study thc aclditional group of paticnts treatecl with recom­binant interferon alpha was introduced and thc rcsults will bc publishcd separatcly. HLI group A total of 160 patients, 70 malcs ancl 90 females, havc bccn entercd in thc HHLI group. Thc mcan agc of patients was 48 ycars ( 48 ± 14 years, range 20 -78 years). Patients wcre distributecl according to tthe primary tumor sitc as follows: head and ncck rcgion (HN) -15 ; trunk (T) -79; limbs (L) -66 . Primary tumors werc dctcrmined as supcrficial-sprcading typc (SSM) in 31 cases, nodular typc (NM) in 127 cases and lentigo maligna type (LMM) in two cases. The leve! of invasion was Clark IV in 109 cases, and Clark V in 12 cascs. In 39 cascs t hc leve! of invasion was Clark III, but tumor thickness cxccecled 1.5 mm, which was in accor­clancc with protocol criteria. Rudolf Z. CON group The control group comprised 161 randomly selected patients (71 males and 90 females) in the mean age of 52 years (52 ± 13 years, range 21-84 years). As to the primary tumor site, lesions were located in head and neck region in 16 cases, on the limbs in 70 and on the trunk in 75 cases. In 92 patients tumors were assessed as SSM type, in 3 patients as LMM and in 66 patients as NM type. The leve! of invasion was Clark III in 23 cases (but thickness more than 1.5 mm), Clark IV in 101 cases, ancl Clark V in 12 cases. Patient distribution by various potential prog­nostic factors is presented in Table l. Our analysis showed that both protocol groups, i.e. HHLI and HCON, were similar as to their sex and age distribution. Also, there was no major difference in site and type of primary tumor, and neither in its leve! of invasion. Statistical analysis The statistical analysis was done using the Ka­plan-Meier product-limit method1 l . 12 which is a 11011-parametrical methocl to estimate the pro­bability of an event occuring during a given time-interval. Statistical significance of graphed survival curves was tested using logrank pro­gram which performs a chi-square like analy­ sis.13, 14. 1s Table 1. Comparison of HLI ancl CON gro.p accorcling to sex ancl age clistribution, type ancl localization of primary tumor ancl leve! of invasion. Data HLI Group CON Group No. Percentage No. Percentage 56% Sex: M 70 F 44% 56% Age: <53 l00 63% 81 50% 60 37% 80 50% 127 Type: NM 79% 66 41% SSM 31 20% 92 57% LM2 1% 3 2% Local.: Trunk 47% HNeck 15 9% 16 l0% Limbs 68 41% 70 43% Clark III 24% 38 23% IV l09 68% 101 62% v 12 8% 12 7% TOTAL 160 100% 161 l00% Results Survival analysis Survival curves of patients in HHLI and HCON group are presentecl in Figure 2. Survival of patients treated with human leukocyte interfe­ron (HHLI.dbf) is significantly higher than in the control (HCON.clbf) group (p < 0.005). Survival curves of patients according to sex distribution in control group (HCON) are pre­sented in Figure 3. Control female patients hacl significantly higher survival (HCONF.dbf cur­ve) when compared with male controls (HCONM.dbf curve), which is consistent with previous observations about influence of sex on the prognosis. The difference between both groups is significant (p < 0.001). Similar is the situation in HHLI group (Figure 4), though the difference between female (HHLIF.dbf curve) and male (HHLIM.dbf curve) patients is not significant (p = 0.07). Primary tumor site in­fluenced the survival of patients in the control group. The difference between group of pa­tients with tumors on limbs (HCONL.dbf cur­ve) and patients with tumors in trunk region (HCONT.dbf curve) is significant (p < 0.05) in favour of limbs site, which is presented in Figure 5. In HLI group the similar difference was not significant (HHLIL.dbf curve versus Adjuvant treatment of malignant melanoma with /FN-a RADICAL EXCISION Clark IV,V and/or Breslow >1.5 mm randomization HLI group CON group HulFN 30 weeks 2 M units/week ".te r 0 .------• ! .5 r * HCON. DBF o HHLI. DDF 0.0+-------------,-----, ,--­ 0 50 100 150 200 Figure 2. Survival analysis of melanoma paticnts in both protocol groups. (HCON.dbf-paticnts in control group, HHLI.dbf -paticnts in trcatcd group; thc cliffcrcncc is significant, p < 0.001 ). HHLIT.dbf curve, p = 0.5). The type of pri­mary tumor did not significantly influence the survival in both protocol groups (Figure 6), and also the impact of age of patients could not be established, as illustrated in Figure 7. The difference between treated and control patients is significant also by sex stratification (Figure 8). Females in the treated group had better survival than temale controls; likewise, male patients treated with interferon survived longer than male patients in the control group (HHLIF.dbf curve vs. HCONF.dbf curve, and HHLIM.dbf curve vs. HCONM.dbf curve; p < 0.005). Interferon treatment was well tolerated by majority of patients and no patient declined it because of toxic side effects. In ali patients the ­ FOLLOW-UP treatment as necessary • in cases of tumor recurrence """.!__ ·.··'l,.M Survival Figure I. Protocol summary. r 0.s r .--1_ 1.0..,..,...,.------------- ---- -. -M tlCONl-'. DBF o HCOl'IM. DDF 0,0 1 1 0 H 100 1H Figure 3. Survival comparison bctwccn male ancl fc­malc paticnts in control group. (HCONM.dbf -malcs, HCONF.clbf -fcmalcs; thc cliffcrcncc is significant, p<0.001). s r 0 .5 1 * HHLIF.DBF o HHLIH.DDF 0.0+-------.------.--------l 0 50 100 150 Figure 4. Survival comparison bctwccn male ancl fc­malc paticnts in trcatcd group. (HHLIM.clbf -malcs, HHLIF.clbf -fcmalcs; thc cliffcrcncc is not significant, p = 0.07). 336 Rudolf Z. 1.0 1.0 . l 0.5 f s u " . 0.5 f ' 1 """1..-.. 0.0+----------­. * HCONL. DDF o HCOHT. DDF x HHLIL.DBF HHLIT. DBF - ---,----­-----; 0.0+-­ ---------. * 7.CAGEl. DBF o 2CAGE2, DBF x ZHAGE.1. DBF ~ ZHAGE2, DBF ---­-,----­-----; 0 50 100 200 50 100 150 lligure 5. Survival comparison in bolh protocol groups according to thc primary tumor sitc. (HCONL.dbf ­control paticnts, primary tumor on limbs, HCONT.dbf -control patients, primary tumor in trunk, HHLIL.dbf created patients, primary tumor on limbs, HHLIT.dbf treated patients, primary tumor in trunk; HCONL.dbf vs. HCONT.dbf signifieant, p < 0.05; HHLIL.dbf vs HHLIT.dbf not significant, p = 0.5). application of interferon was followed by mild up to moderate fever (Iess than 39 ° C) which was transient. The patients experienced also 1.0 'f 0.5 v t * TCNM. DBF o TCSSH. DBF >< THNH, DBF THSSH.DBF 0.0-l--------===;:=====---j -------, -­ 50 100 150 200 lligure 6. Survival comparison in bolh protocol groups accorcling to the primary tumor type. (TCNM.dbf ­control patients, nodular melanoma, TCSSM.clbf ­control patients, superfieial spreading melanoma, THNM.clbf treatecl patients, noclular melanoma, THSSM.dbf -treated patients, superfieial spreading melanoma). lligure 7. Survival comparison in both protoeol groups aeeording to the "iŠe of melanoma patients. (ZCA­GE 1.clbf control palients, age <53 years. ZCA­GE2.clbf -control patients, age >53 years. ZHA­GE l.dbf treatecl patients, age <53 years, ZI-IA­GE2.dbf -treated patients, agc >53 ycars). 1.0 s u " ! 05 . l * HCONF .DBF o HCONM, DBF x HHLIF.DBF HHLIM, DBF 0.0+----------.-----,----­ ----1 0 50 100 150 200 lligure 8. Comparison of survival between bolh proto­col groups by scx stratifieation. (1-11-ILIF.dbf treatecl fcmales, 1-IHLIM.clbf -treatecl males, HCONF.dbf fcmalc controls, HCONM.clbf -male controls; 1-IHLl.clbf vs. HCONF.dbf and HHLIM.dbf vs. 1-!CONM.dbf significant, p < 0.005). flu-like syndrom, which was antecipated. The application of interferon in performed dosage exerted no effect on blood counts and chemi­stry. In one case, as reported previously,16 Adjuvant treatment o( malignant melanoma with !FN-u moderate allergie reaction manifested with urti­caria followed the second course of the treat­ment. Since the fever and flu-Iike syndrom were transient, after pilot study it was decided that the regimen should be applied on out-pa­tients basis. Discussion The increasing incidence of melanoma and its tendency to affect younger adults, as well as relative ineffectiveness of treatrnent in advanc­ed stages, point out the neecl for an effective adjuvant therapy. In our study interferon treatment was founcl to have influencecl the survival of patients, since the difference between both protocol groups (HLI and CON) was significant. In acldition, the difference between male as well as female patients of both groups was also significant. Accorcling to these results, it can be postulated that the interferon treatment pro­longecl the survival of patients in treated group. Also, the treatment was not associatecl with significant toxic side effects. The reason for allergic reaction in one case is most probably in crucle extract of human leukocyte interferon containing various potential allergens. Possible mcchanisms of interferon action have not been fully explained yct. Theoretical­ly, interferons could exert their antitumor ef­fects in three ways: (1) via the host immunc system; (2) by altering some non-imrnune host/ tumor celi interactions; or (3) by dircct effects on thc tumor cells. It is known that turnors sensitive to interferon alpha tend to be slow growing and rnoderately well clifferentiated. Moreover, in contrast to other types of cancer therapy, responses to alpha interferons are typi­cally slow, with haematological ancl bone mar­row irnprovernent often taking severa! rnonths in leukernia cases. Data frorn laboratory ani­mals suggest that interferon act best when tu­mor load is low, such as was the situation in 9 our study.4· Interferons are also an irnportant part of lyrnphokinc cascade. It is reasonable to conclude, that interferon could act as biological response rnodifier through rnany yet uknown mechanisms including lymphokine cascadc. According to our results, the interferon adju­vant treatrnent can be advised in cases with prognostically unfavourable melanoma, i.e. pri­mary melanoma turnors with leve! of invasion Clark IV,V and/or thickness more than 1.5 mm. Acknowledgement The financial support by grant No.C3-0563-302/ 27-40/B of the Ministry of Science and Techno­logy of Slovenia is gratefully acknowlcdged. Refercnces l. Cancer incidence in Slovcnia, 1980, 1981, 1982, 1983, 1984, 1985, 1986. Ljubljana: Institute of Oncology-Cancer Registry of Slovenia, 1984, 1985, 1986, 1987, 1988, 1990. 2. Rudolf Z, Roš-Opaškar T. Survival and disease­free interval of malignant melanoma patients in relation to the prognostic faetors. Radio! Oncol 1992; 26: 45-55. 3. Koh HK, Sober AJ, Harmon DC, Lew RA, Carey RW. Adjuvant therapy of cutaneous malig­nant melanoma -a critical review. Medica! and Pediatric Oncol 1989; 13: 244-60. 4. Baron S, Tyrring SK, Fleischmann R, Coppenha­ver OH, Niesel DW, Klimpel GR, Stanton JG, Hughes TK. The interfcrons -mechanisms of action and elinical applications. JAMA 1991; 266(10): 1375-83. 5. Kirchner H. Update on interfcrons. Progress in Oncology 1988; 7: 5-62. 6. Rudolf Z, Serša G, Krošl G. In vitro monocyte maturation in patients with malignant melanoma and colorectal cancer clinical significance. Neo­plasma 1986; 1: 274-9. 7. Beverly P, Knight D. Killing comes naturally. Nature 1979; 278: 119-20. 8. Haberman RB, Ortaldo JR, Bonnarcl GO. Aug­mentation by interferon of human natura! ancl antibody-clependent cell-mecliated cytotoxicity. Nature 1979; 277: 221-3. 9. Rudolf Z, Furlan L. Acljuvant treatment of malig­nant melanoma with human leukocyte, interferon. Period Biol 1990; 92(1): 141-2. lO. American Joint Committee on Cancer: Ma11ual far staging of cancer, 3rd cel. Philadelphia: JB Lippincott, 1987. 338 Rudolf Z. l l. Kaplan EL, Meier P. Nonparametric estimation fr om incompletc observations. J American Statisti­cal Association 1958; 53: 457-81. 12. Matthews DE, Farewell VT. Vsing and understan­ding medica/ statistics. Karger, 1988, 67-78. 13. Peto R, Pike MC. Design and analysis of rando­mized clinical trials requiring prolonged observa­tion of each patient: I.Introduction and design. Br J Cancer 1976; 34: 585-612. 14. Peto R, Pike MC. Design and analysis of rando­mized clinical trials requiring prolonged observa­ tion of each patient: II. Analysis and examples. Br J Cancer 1977; 35: 1-39. 15. Anderson S, Auquier A, Hauck WW. Statistical methods for comparative studies. 1 Wiley, 1980, 199-234. 16. Rudolf Z. Treatment of malignant melanoma with human leukocyte interferon -preliminary results of randomized tria!. Filipic B(ed): Yugoslave col­loquium on interferon. Ljubljana, Slovenian Mi­crobiological Society 1986, 129-33. Radio! Onco/ 1993; 27: 339-40. Breast imaging at ECR'93 Vienna, September 12-17, 1993 The 8th European Congress of Radiology held in Vienna in early autumn this year was attend­ed by about ten thousand professionals. Tech­nical, educational and scientific aspects of nu­merous topics including breast imaging were discussed. Breast imaging, now firmly rooted in early detection and management of breast cancer together with recent advances and rele­vant views about this rapidly evolving discipline were dealt with in a "Categorical course" offer­ed by the organiser. Mammography (MG) won the central role in the diagnosis of breast disease and particularly in the very sensitive field of mass-screening for asymptomatic breast cancer which, in develop­ed countries, will soon afflict up to one third of women. Early objections against extensive use of MG because of the risk of radiation-in­duced cancer, have proven unfounded. The exhibition accompanying the congress showed that the industry had fully embraced the mo­dem concepts of dedicated apparatus which limits the radiation burden of a typical investi­gation to mere 0.5 mGv and simultaneously enables imaging with high contrast and fine detail. This is being achieved with special anti­scatter grids, an automatic exposure-control, a focus of 0.6 mm or less, a voltage range from 25 to 35 kV and high-resolution film-screens (Friedrich M). It was stressed, however, that in order to insure adequate functioning of this equipment, regular daily, weekly, monthly and annual quality-control program should be rigo­rously followed and only accepted phantoms used (Thomas BA). Equal emphasis was laid on the professional skill of image-readers, particularly by German and Dutch authors with wide experience in mass-screening programs (Hessler C; Hendriks JHCL). Intensive, repeated training courses for medica! and technical personnel should replace self-education wherever possible. It was even suggested that the accreditation for MG should be time-limited and attendance in regular re­freshing courses a sine qua 11011 (Friedrich M). The performance quality of the diagnostic temn should be periodically assessed by reviewing the fina! results including the reevaluation of false reports and interval cancer cases (10). In MG, it was said, mediocrity can hardly be tolerated (Hendriks JHCL). MG features of breast cancer are well known and, in most cases, a skilled radiologist can deliver a correct diagnosis. For various reasons, though, false-negatives cannot be avoided, par­ticularly in screening as proved by the incidence of interval cancers. In clinical work, false-nega­tives destroy the clinician's confidence and their rate should not exceed 5 %-10 % of histologi­cally proven cases. The overall incidence of false-positive reports is hard to estimate but they seem to result from overinterpretation of microcalcifications. Microcalcifications themselves are a rather sensitive sign of malignancy though highly un­specific. Efforts to improve the accuracy of MG images by analysing their semiology in great detail were not successful. Therefored, simpler, commonly identifiable and easily describable morphological criteria (according to "Le Gal") Report were advoeatecl, therefore, partieularly to faci­litate reporting, communication ancl teaching (Lamarque JL). High quality of the image is essential ancl magnification techniques may be helpful (Del Turco MR). The racliologist's task of cletecting locally recurrent cancer is becoming ever so frequent since more patients are being new treated con­servatively. Swelling, scarring and haematomas after surgery ancl/or irracliation may utterly change the racliological appearance of the breast tissue and obscure early malignant regrowth. Nevertheless, a recurrence can be recognised during regular MG follow-up after initial treat­ment since its density and size increases, while post-treatment changes tend to clear up with tiine (Schreer I). Benign breast lesions are very frequent, very variable and difficult to diagnose correctly. In ali doubtful cases, additional diagnostic techni­ques should be used liberally including clucto­graphy, pneumocystography, ultrasound, clini­cal examination and stereotaxically guided thin­needle biopsy before surgical biopsy is resortecl to. Unlike malignant lesions where a mere suspicion may suffice for further cliagnostic work-up, benign diseases call for an accurate diagnosis. In orcler to recluce the number of unnecessary biopsies, the skill of the radiologist, again, cannot be overemphasized (di Maggio C). Ultrasound imaging is safe ancl generally ac­ceptecl. In the examination of the breast, howe­ver, its role remains limited in spite of the experience gathered in the last ten years with this diagnostic moclality. In the screening of unsymptomatic women it has no value at ali but has retainecl a few firm indications in clinical diagnostic work particularly in fluid detection in palpable lumps, examination of young wo­men and visual guiclance of the needle during biopsy (van Kaick G). Magnetic resonance imaging (MRI) is a rela­tively new diagnostic moclality. Its main advan­tage is: absence of any radiation risk. It enables a much better clistinction between fat, cellular tissue and fluicl which together with the possibi­Iity of visualisation in any desirecl plane, greatly facilitates recognition of the disease. The clis­aclvantage of MRI is: high cost ancl very limited availability of the procedure (Heywang-Koe­brunner SH). ECR'93 was a great professional event in every respect including the splendid exhibition of racliological equipment. It is a pity that so few Slovene radiologists attendecl the very ins­tructive categorical course on breast imaging. Breda Jancar, MD The Institute of Oncology Radio/ Oncol 1993; 27: 341. . ...... . review Gastrointestinal Radiology: concise Text. By Branko M. Pfavsic, Anin E. Robinson and Brooke Jeffrey New York: McGraw Hm, 1992 (557 pp) The book, absolutely necessary at the time of modem image technique, shortly summarizes ali knowleclge from the conventional gastrointe­stinal racliology. During the past severa! years many clescriptions of the CT, MR ancl ultra­souncl have been founcl in the literature. The place of the conventional barium stuclies of the gastrointestinal tract remains the important base in the racliological diagnostic proceclures. The book presented here fills that important space in the current literature ancl proves the complete overwiev over the methods, teclrni­ques ancl radiological characteristics of the ga­strointestinal racliological procedures. The mo­dem image technique are only sparsely presen­teci to complete the diagnostic approach to the various diagnostic problems of the alimentary canal. The book is clivided logical into 12 chapters. Ali of them are illustratecl with a number of excellent figures. Accorcling to the intention of the authors to give the complete presentation of the problerns, the first three chapters cover normal anatomy of the alirnentary canal, ga­strointestinal physiology and radiological aspects of gastrointestinal pathology. Ali basic data which are of importance for the unclerstancling of the gastrointestinal examina­tions are presentecl. In the fourth chaptcr the authors give very concise clescription of various teclrniques of examining of the gut. The precise presentation of the clouble contrast examination is given, inclucling the basic clata about contrast meclia ancl phannacoracliography. To complete the racliological presentation of the gastrointe­stinal tract, the authors clescribe ali other te­chniques like CT, MR, ultrasouncl, arterio­graphy ancl scintigraphy. Very useful instruc­tions for the evaluation of radiological rnethocls ancl making the racliological reports are given. Also the role of the intervention racliology is shortly presentecl. The main parts of the book are the next eight chapters which clescribe the various pathological changes in the alimentary tract. In the logical rows the radiological pathology of the pharynks, esophagus, stomach, cluoclenum, small bowel ancl colon is describecl an illustratecl with a number of illustrations. Ali illustrations are of excellent qu.ality. Using more than seventy figu­res in each chapter, the authors illustratecl almost each pathological entity. The special attention was given to the cliseases which affect the both small ancl large bowel, such as intesti­nal ischemia, infections ancl infestations, ulcera­tive colitis ancl Chron's clisease. To present the differential cliagnosis between similar racliologi­cal symptoms the authors use very simple ancl instructive tables ancl diagrams. The diseases of very important localization like ileocecal area and rectum are presentecl in the separate chap­ter. That fact indicates that except general knowledge of various problems the authors inclucle in the book their own experience. Because of very simple ancl precise presenta­tion of the problems, the concise ancl illustrative clescriptions ancl excellent functional approach to the gastrointestinal pathology, the book ser­ves an important textbook for residents the source of various clata for the racliologists. At the same tiine it is unavoiclable for gastroente­rologists, surgeons, pecliatricians ancl medica) stuclents. Prof. dr. Andrija Hebrang Clinical Department of Racliology Clinical Hospital "Merkur", Zajceva 19 Zagreb Radio[ Oncol 1993; 27: 342-3. Notices Notices submitted for publication should contain a mailing address, phone andlor fax number of a contact person or department. Lymphomas The ESO seminar will be offered April 21-22, 1994. Contact Miss Gollubics, ESO-Vienna-Officc, Arzte­kammer flir Wien, Fortbildungsrefcrat Weihburggasse 10-12, A-1010 Vienna, Austria; or call + 43 1 51501 293. Fax: + 43 1 51501 240. Oncology The 11th research workshop (Journees Grenobloises de Cancerologie) "Chemoresistance: from bench to clinical trials", will take place in Grenoble, France, March 24-25, 1994. Contact Pr. C. Vrousos, Hopital Albert Michallon. B. P. 217, 38043 Grenoble Cedex 9, France. Nuclear medicine The British Nuclear Medicine Society annual meeting will be held in London, United Kingdom, March 28-30, 1994. Contact Mrs S. Hatchard, The BNMS Confcrence Secretary, 157 Auckland Road, London SE 19 2RH. Magnetic Resonance The 1st Nottingham symposium on Magnetic Reso­nance in Medicine will be held in Nottingham, United Kingdom, April 6-8, 1994. Contact Julie Mills, University Hospital, Nottin­gham NG & 2UH, United Kingdom; or call + 44 602 709 951. Fax: + 44 602 424 994. Brachyradiotherapy ESTRO teaching course "Modem Brachytherapy Techniques" will take place in Ttibingen, Germany, April 10-14, 1994. Contact the ESTRO Secretariat, Radiotherapy De­partment, University Hospital St Rafael, B-3000 Leu­ven, Belgium; or call + 32 16 33-64-13. Fax: + 32 16 33 64 28. Lung cancer -biology and clinical aspects The 2nd central European confcrence on Jung cancer will be offered in Ljubljana, Slovenia, lune 13-16, 1994. Contact The Conference Secretariat, Department of Thoracic Surgery, Medica! Center, Zaloška 7, 61105 Ljubljana, Slovenia; or call + 386 61 317 562. Fax: + 386 61 1316 006. Magnetic Resonance The "llth Annual Scientific Meeting and Exhibition of the European Society for Magnetic Resonance in Medicine and Biology'' (ESMRMB) will be held in Vienna, Austria, April 20-24, 1994. Contact Vienna Academy of Postgraduate Medica] Education and Research, Alser Strasse 4, 1090 Vien­na, Austria; or call + 43 1 42 138 313. Fax: + 43 1 42 138 323. Brachyradiotherapy The 76th annual meeting American Radium Society will take placc in Southampton Princess Hotel, Bermu­da, April 22-26, 1994. Contact Office of the Secretariat, American Radi um Society, 1101 Market Street, 14th Floor, Philadelphia, PA 19107, USA; or call + 1 215 574 3179. Fax: + 1 215 928 0153. Radiotherapy ESTRO teaching course "Principles and Technical Aspects of Clinical Radiotherapy" will be offered in Bari, ltaly, April 24-28, 1994. Contact the ESTRO Secretariat, Radiotherapy De­partment, University Hospital St Rafael, B-3000 Leu­ven, Belgium; or call + 32 16 33-64-13. Fax: + 32 16 33 64 28. Neuroblastoma The international meeting "Ten years of experience Notices 343 in neuroblastoma. Quo vadis MIBG?" will be offered in Kiel, Germany, April 25-26, 1994. Contact H. Bihl MD PhD, Dept. of Nuclear Medi­cine, Katharinenhospital, Kriegsbergstr. 60, 70174 Stuttgart, Germany; or call + 49 711 278 4300. Fax: + 49 711 278 4309. Skin cancer and melanoma The ESO teaching course will be offered May 4-8, 1994. Contact Miss Gollubics, ESO-Vienna-Office, Arzte­kammer ftir Wien, Fortbildungsreferat Weihburggasse 10-12, A-1010 Vienna, Austria; or call + 43 1 51501 293. Fax: + 43 1 51501 240. Brachytherapy The 11th annual braehytherapy meeting GEC-ESTRO will be held in Linz, Austria, May 9-11, 1994. Contaet the ESTRO Seeretariat, Radiotherapy De­partment, University Hospital St Rafael, B-3000 Leu­ven, Belgium; or call + 32 16 33-64-13. Fax: + 32 16 33 64 28. Radiotherapy The refresher course in radiation oncology will be offered in Toronto, Ontario, Canada, May 11-13, 1994. Contact Continuing Education, Faculty of Medicine, University of Toronto, Medica! Seiences Bldg., Toron­ to, M5S 1A8, Canada; or eall + 1 416 978 2719. Fax: + 1 416 971 2200. Cancer research The "25th Annual Meeting American Association for Cancer Rcscarch" will take placc in Dallas, Texas, USA, May 11-14, 1994. Contact Amer. Assoc. Cancer Res., Meetings Mail­ing List AACR, Public Ledger Building, 6th and Chestnut Streets, Suite 816, Philadelphia, Pa 19106, USA; or call + 1 215 440 9300. Fax: + 1 215 440 9313. Radiotherapy The 4th annual meeting of the American College of Radiation Oncology will be offered in Washington, DC, USA, May 14-16, 1994. Contact ACRO Officc, P.O. Box 12920, Philadel­phia, PA 19108, USA; or call + l 215 762 4993. Fax: + l 215 762 8523. Pediatric neurooncology The international symposium will be held in M. D. Anderson Cancer Center, Houston, Texas, USA, May 18-21, 1994. Contact MDACC, Univ. of Texas, Conf. Services, Box 131, 1515 Holcombe Blvd., Houston, TX 77030­4095, USA; or call + 1 713 792 2222. Gynaecology International gynaecological symposium will be offer­ed in Bruges, Belgium, May 26-28, 1994. Contact Hilde Deckers-Van Overmeiren, Vlaamse Vereniging voor Obstretrie en Gynaecologie, Azalea­laan 10, Sint Niklaas, Belgium; or call + 32 (0)3 776 03 64. Fax: + 32 (0)3 766 07 56. Gynecological endocrinology The 4th world congress of gyneeological endocrinology will be held in Antalya, Turkey, May 28 -lune 3, 1994. Contact Medica! Congress and Research S. A., Via della pace 5 (Palazzo Centro), CH-6601 Locarno, Switzerland; or call + 41 93 322 932. Fax: + 41 93 319 836. Surgical oncology The tripartite meeting of European Society of Surgical Oncology, British Association of Surgical Oncology and Chirurgishe Arbeitsgemeinschaft flir Onkologie, subtitled "Frontiers and Perspectives of Surgical Onco­ logy" will take place in Heidelberg, Germany, lune 8-11, 1994. Contact PD Dr. Thomas Lehnert, Department of Surgery, University of Heidelberg, Im Neuenheimer Felci 110, d-69120 Heidelberg, FRG; or call + 49 6221 566 207. Fax: + 49 6221 565 450. Update in urological oncology The ESO teaching course will be offered lune 9-11, 1994. Contact Miss Gollubics, ESO-Vienna-Office, Arzte­kammer flir Wien, Fortbildungsreferat Weihburggasse 10-12, A-1010 Vienna, Austria; or call + 43 1 51501 293. Fax: + 43 1 51501 240. Radio! Oncol 1993; 27: 344-5. Author index 1993 Auersperg M: Supl 7/79-84, Suppl 6/88-93, Suppl 6/100-4, Suppl 6/110-4, Suppl 6/115-9, Suppl 6/187-91, Suppl 6/ 192-7, Suppl 6/198-203, Suppl 6/204-9, Suppl 6/210-6, Suppl 6/217-23, 4/275-9 Avcin J: Suppl 6/46-50, Suppl 6/164-72 Babic M: 2/89-94 Barta M: 2/95-8 Bergant D: Suppl 6/115-9, Suppl 6/187-91, Suppl 6/ 210-6 Bešic N: Suppl 6/110-4, Suppl 6/187-91, Suppl 6/ 204-9 Bizjak-Schwarzbartl M: Suppl 6/74-8, Suppl 6/ 120-130, Suppl 6/217-23 Bobinac D: 1/5-15 Bracko M: Suppl 6/88-93, Suppl 6/105-9 Bricelj V: 3/175-9 Brinovec V: 4/312-5 Brkljacic B: 1/21-6 Brumen V: 2/125-31 Bubic-Filipi Lj: 2/105-10 Budihna N: Suppl 6/46-50 Burnet NG: 3/205-13 Cambj-Sapunar L: 3/186-90 Car M: 2/111-4; 3/223-7 Carlsson K: 4/302-6 Cencic A: 4/302-6, 4/307-11 Cerar A: 2/120-4 Cerovic R: 3/223-7 Cijan A: 3/175-9 Chany C: 4/265-70 Chavinic J: 4/265-70 Cor A: Suppl 6/137-42 Crnjakovic-Palmovic J: 4/316-20 Cemažar M: 4/275-9 Debevec M: 1/36-8 Dimec D: 1/27-30 Dimitrovski L: 2/85-8 Dodig D: 1/31-5; 2/105-10 Dolenc-Stražar Z: Suppl 6/66-9, Suppl 6/ 70-3 Doly J: 4/265-70 Drinkovic I: 1/21-6 Duc-Goiran P: 4/265-70 Duchesne GM: 3/205-13 Dujmovic M: 1/5-15; 2/85-8 Fajgelj A: 3/200-4 Feichter G: 4/321-5 Ferentzi J: 2/95-8 Ferle-Vidovic A: 1/44-8; 4/271-4 Ferre F: 4/265-70 Fettich J: 3/191-9 Fidler-Jenko M: Suppl 6/217-23 Filipic B: 4/302-6, 4/307-11 Fleischmann WR Jr: 4/275-9, 4/286-92 Frkovic M: 1/16-20 Fuckar Ž: 1/27-30; 1/99-104 Golouh R: Suppl 6/85-7, Suppl 6/88-93, Suppl 6/94-9, Suppl 6/105-9, Suppl 6/110-4, Suppl 6/115-9, Suppl 6/204-9 Griffiths MH: 3/205-13 Grošev D: l/31-5; 2/105-10 Hebrang A: 1/21-6; 4/341 Hocevar-Boltežar I: Suppl 6/110-4 Hocevar M: Suppl 6/187-91, Suppl 6/192-7, Suppl 6/ 198-202 Hojker S: Suppl 6/39-45, Suppl 6/46-50 Huic D: 1/31-5; 2/105-10 Ihan A: 1/39-43 Ivancevic D: 1/31-5; 2/105-10 Jancar B: 3/232-5, 4/339-40 Jancar J: Suppl 6/120-30, Suppl 6/217-23 Jereb B: 4/326-31 Jerman J: Suppl 6/178-86 Jezeršek B: Suppl 6/192-7; 4/275-9 Juretic M: 2/111-4; 3/223-7 Kališnik M: Suppl 6/9-14, Suppl 6/26-31 Kamaric Lj: Suppl 6/32-8 Kaštelan M: 4/271-4 Klobovec-Prevodnik V: Suppl 6/115-9 K6nya A: 3/180-5, 3/214-22 Kotnik V: 1/39-43; Suppl 6/149-54 Kozic S: 2/89-94 Krašovec F: Suppl 6/51-7 Kuhelj J: 3/236-7 Lamovec J: Suppl 6/131-6 Lopez J: 4/265-70 Lovasic I: 2/85-8 Luštica I: 3/223-7 Author indcx 1993 Mandic A: 1/16-20 Maricic A: 1/27-30 Maškovic J: 3/186-90 Matovinovic D: 2/89-94 Mažuran R: 4/326-31 Medvedec M: 2/105-10 Miklavcic D: 4/280-5 Miletic D: 1/27-30; 2/99-104 Mimica Ž: 3/186-90 Moravec-Berger D: Suppl 6/39-45 Mozetic V: 1/27-30; 2/99-104 Munkacsi G: 2/95-8 Navarro S: 4/265-70 Novak-Antolic Ž: Suppl 6/173-7 Novak B: Suppl 6/187-91 Novak J: 3/200-4 Novakovic S: 4/275-9, 4/28(>--92, 4/298-301 Oblak-Ruparcic L.: Suppl 6/110-4 Odak D: 1/21-6 Orel J: Suppl 6/178-86 Osmak M: 2/143-6, 2/147-9 Pajer Z: Suppl 6/137-42 Palrnovic D: 4/3ff>--20 Pavcnik D: 3/175-9 Petric-Grabnar G: Suppl 6/120-130, Suppl 6/204-9, Suppl 6/ 217-23; 4/326-31 Petrovce M: Suppl 6/143-8 Petrovic D: 1/44-8; 4/271--4 Pogacnik A: Suppl 6/100-104, Suppl 6/187-91, Suppl 6/192-7, Suppl 6/204-9 Poljak M: Suppl 6/143-8 Pompe F: Suppl 6/187-91 Pompe-Kirn V: Suppl 6/58-65 Porenta M: Suppl 6/46-50, Suppl 6/51-7 Poropat M: 1/31-5; 2/105-10 Primic-Žakelj M: 2/132-42 Puskas T: 2/95-8 Racz P: 2/95-8 Rah6ty P: 3/214-22 Rakuljic I: 3/223-7 Robert B: 4/265-70 Rode M: 1/39-43 Rok B: 1/39-43 Rozman S: 4/302-6 Rudolf Z: 4/298-301, 4/332-8 Sadler GM: 3/205-13 Safran A: 2/95-8 Schnurrer T: 1/5-15 Serša G: 2/143-6, 4/275-9, 4/280-5 Singer Z: 4/321-5 Snoj-Cvetko E: Suppl 6/20-25 Snoj M: l/49-51 Soldo D: 1/21-6 Sotošek B: 3/228-31 Stanic K: Suppl 6/192-7 Starc J: Suppl 6/204-9 Stržinar V: 2/115-9 Suhar A: 1/44-8 Szepe I: 2/95-8 Škrk J: 1/44-8; 4/271-4 Šmid L: 1/39-43 Šooš E: 4/321-5, 4/326-31 Štabuc B: 4/293--7 Štalckar H: 3/223-7 Šustic A: 2/99-104, 2/111-4 Tercelj-Zorman M: 4/326-31 Tomljanovic Z: 3/223-7 Turk V: 1/44-8 Uravic M: 2/85-8 Us-Krašovec M: Suppl 6/79-84, Suppl 6/100-4, Suppl 6/110-4, Suppl 6/115-9, Suppl 6/187-91, Suppl 6/192-7, Suppl 6/204-9; 4/326-31 Vidjak V: 1/21-6 Vigvary Z: 3/180-5, 3/214-22 Vodnik-Cerar A: Suppl 6/217-23 Vovk M: Suppl 6/217-23 Vraspir-Porenta O: Suppl 6/15-9 Vrhovec I: 4/271-4 Vuckovic R: 1/16-20 Wraber B: Suppl 6/155-63 Zidar A: Suppl 6/94-9 Zupanc A: Suppl 6/74-8 Žargi M: 1/39-41 Žgaljardic Z: 2/111-4; 3/223-7 Župevc A: 1/39-41 Radio! Oncol 1993; 27: 346-8. Subject index 1993 adenocarcinoma: 2/115-9 administration, intravaginal: 4/321-5 Ag-NOR: Suppl 6/137-42 anaplastic carcinoma: Suppl 6/110-4 angioplasty, balloon: 3/175-9 angiosarcoma: Suppl 6/131-6 animal melanoma B-16 tumor model systemic treatment, local treatment: 4/275-9 annual meeting: 2/147-9 antineoplastic agents-toxicity: 2/125-31 aspiration biopsy: Suppl 6/100-4, Suppl 6/110-4, Suppl 6/ 115-9 autoimmune hyperthyrosis: Suppl 6/155-63 autoimmune thyroid disease: Suppl 6/39-45 autoreactive T celi clones: Suppl 6/155-63 behaviour: Suppl 6/94-9 biennial meeting: 2/143-6 bile ducts, interventional procedure, bile ducts neoplasms-surgery, stents, endoprosthesis -transhepatic removal of stents, biliary endoprostheris: 3/180-5 biosynthesis: Suppl 6/32-8 blood flow velocity: 1/31-5; 2/105-10 bone diseases -radionuclide imaging: 1/31-5 breast neoplasms, breast cancer: 3/232-5 Brussels: 2/143-6 cancer, neoplasms -etiology: 2/132-42 carcinoma, non-small celi lung: 1/36-8 -carcinoma, non-small celi lung -drug therapy: 4/326-31 cells, cultured cathepsin D -radiation effects: 4/271---4 cells, cultured -drug effects: 4/302-6 cellular interdependence: Suppl 6/26-31 cervix neoplasms -drug therapy: 4/321-5 cervix neoplasms -therapy: 2/115-9 chemotherapy: Suppl 6/192-7, Suppl 6/198-203, Suppl 6/ 204-9, Suppl 6/217-23 chromosome aberrations: 2/125-31 cisplatin: 4/293-7 classification: Suppl 6/85-7 clear celi: Suppl 6/105-9 clinical pattern: Suppl 6/210-6 clinico-pathological correlation: Suppl 6/120-30 clonal anergy: Suppl 6/155-63 complications: Suppl 6/178-86 -complications, prosthesis failure: 3/180-5 Crohn disease: 1/16-20 cytology: Suppl 6/192-7 cytomorphology: Suppl 6/187-91 cytopathologic diagnosis: Suppl 6/115-9 dexorubicin: 1/44-8 diagnosis: Suppl 6/110-4; Suppl 6/149-54 -diagnosis value of cytological and histological examination: Suppl 6/120-30 differential diagnosis: Suppl 6/79-84 DNA flow cytophotometry: Suppl 6/192-7 DNA measurements: Suppl 6/187-91 drainage: 3/186-90 duodenum -surgery: 2/85-8 EACR: 2/143-6 ectopic gland: Suppl 6/15-9 effects: Suppl 6/32-8 efficience: Suppl 6/137-42 endemic goitre: Suppl 6/39-45 epidemiology: Suppl 6/46-50 -epidemiology of benign diseases of the thyroid gland: Suppl 6/39-45 etiology: Suppl 6/149-54 European Society of Radiation Biology: 2/147-9 facial neoplasms -surgery: 2/111-4 femur head necrosis -ultrasonography: 2/89-94 fetal thyroid: Suppl 6/173-7 fibroblasts: 1/44-8 -fibroblasts-drug effects: 4/307-11 fibrosarcoma-therapy: 4/280-5 fine needle aspiration biopsy: Suppl 6/74-8, Suppl 6/ 79-84 fistula-surgery: 3/223-7 follicular cells: Suppl 6/26-31 frozen section: Suppl 6/88-93 functional diagnostics: Suppl 6/164-72 function of the thyroid: Suppl 6/173-7 gastrointestinal diseases diagnosis: 1/16-20 glomerulonephritis: 3/205-13 goiter: Suppl 6/70-3 goitrogenesis: Suppl 6/51-7 gross anatomy: Suppl 6/20-5 growth factors: Suppl 6/46-50 heart valve diseases-therapy: 3/175-9 hepatitis B -drug therapy: 4/312-5 hepatitis C -drug therapy: 4/316-20 hepatitis, chronic active: 4/312-5 Subject index 1993 histophysiology: Suppl 6/15-9 history: Suppl 6/164-72 human recombinant interferon -alpha A/D: 4/275-9 Htirtle celi tumors: Suppl 6/94-9, Suppl 6/100-4 hyperthermia, induced: 4/ ... -... hyperthyroidism: Suppl 6/173-7, Suppl 6/178-86 hypothyroidism: Suppl 6/173-7 immunohistology: Suppl 6/120-30 immunostimulation: 2/120-4 individual chemotherapy: Suppl 6/187-91 individual planning: Suppl 6/192-7 inflammation: Suppl 6/66-9 infusions, intra-arterial, intraarterial chemotherapy: 3/ 214-22 in Slovenia: 3/232-5 interferons: 4/332-8 -interferon alpha: 4/275-9, 4/280-5, 4/286-92, 4/293-7, 4/316-20, 4/321-5 interferon alpha -2B: 4/271-4 interferon alpha, recombinant: 4/275-9 -interferon -beta: 4/307-11 -interferon type I: 4/265-70 -interferon type II -analysis: 4/302-6 interleukin 2: Suppl 6/155-63 interleukin production, malignant melanoma: 4/298-301 intrathyroid mast cells: Suppl 6/26-31 iodine prophylaxis: Suppl 6/39-45, Suppl 6/46-50 irradiation: Suppl 6/198-203, Suppl 6/204-9 isotope production: 3/200-4 kidney anatomy and histology: 1/5-15 kidney transplantation: 2/105-10 -kidney transplantation -adverse effects: 1/27-30 killer cells, natura!: 1/39-43 laryngeal neoplasms: 1/39-43 laryngeal neoplasms -surgery: 3/223-7 leiomyosarcoma: 3/228-31 limb salvage surgery: 3/214-22 !iver abscess: 3/186-90 lung neoplasms: 1/36-8 lymphocele-therapy: 1/27-30 MALT-omas: Suppl 6/120-30 maxillary neoplasms: 3/228-31 -maxillary neoplasms -surgery: 2/111-4 medial and laterni primordia: Suppl 6/15-9 medullary carcinoma: Suppl 6/15-9 medullary thyroid cancer: Suppl 6/210-6 melanoma, experimental -drug therapy: 4/275-9 melanoma -therapy: 4/332-8 metabolism: Suppl 6/32-8 mice: 4/280-5 molybdenum, molybdenum oxide: 3/200-4 morphological examination: Suppl 6/164-72 morphology: Suppl 6/74-8 multivariate analysis: Suppl 6/204-9 neoplasms: Suppl 6/105-9 -neoplasms, cancer: 3/205-13 neoplasms, second primary: 1/36-8 nodular goitre: Suppl 6/178-86 non-Hodgkin lymphoma: Suppl 6/217-23 nuclear reactors: 3/200-4 nucleator: Suppl 6/137-42 objections and arguments: 3/232-5 orbita! diseases: 2/95-8 papillomaviruses: 4/321-5 parafollicular cells: Suppl 6/26-31 pathogenesis: Suppl 6/149-54 pathology: Suppl 6/88-93, Suppl 6/94-9 peptide peptidohydrolases: 1/44-8 percutaneous balloon valvuloplasty: 3/175-9 pharyngeal neoplasms: 1/39-43 -pharyngeal neoplasms -surgery: 3/223-7 pharynx: 3/223-7 phenols: Suppl 6/51-7 phthalates: Suppl 6/51-7 physical examination: Suppl 6/164-72 placenta -analysis: 4/265-70 pneumoperitoneum, radiology: 2/85-8 polychlorinated biphenyls: Suppl 6/51-7 postirradiation intraabdominal adhesions: 1/49-51 postoperative complications: 3/223-7 pregnancy: Suppl 6/173-7; 4/265-70 primary prevention: 2/132-42 pulmonary, mitra! and aortic valve: 3/175-9 pyridines: Suppl 6/51-7 radiation effects: 1/44-8 radiology, interventional: 3/214-22 radiotherapy: Suppl 6/217-23 RAS onkogenes: Suppl 6/143-8 rats: 4/307-11 renal arthery diameter: 1/5-15 renal artery radiography: 1/5-15 renal artery-ultrasonography: 1/5-15 renal circulation: 1/31-5; 2/1-5-10 renal position: 1/5-15 renal size: 1/5-15 report: 3/236-237 348 Suhject index 1993 review, update: 3/232-5 risk factors: Suppl 6/58-65 screening for congenital hypothyroidism: Suppl 6/39-45 screening, mass screening: 3/232-5 secretion of hormones: Suppl 6/26-31 sister chromatid exchange: 2/125-31 Slovenia: Suppl 6/58-65 soft tissue neoplasms drug therapy, neadjuvant treatment: 3/214-22 soft tissuc sarcomas: 3/214-22 solitary nodus: Suppl 6/178-86 solublc interlcukin-2 receptor: Suppl 6/155-63 solvent extraction: 3/200-4 splccn -ultrasonography: 2/99-104 squamous cell carcinoma: 2/115-9 Stockholm: 2/115-9 stomach-surgery: 2/85-8 substernal goitre: Suppl 6/178-86 surgcry: Suppl 6/204-9, Suppl 6/217-23 -surgery-operativc: 4/332-8 survival: Suppl 6/58-65 -survival rate: 2/115-9 technetium 99m: 3/200-4 tcchnctium-isolation and purification: 3/200-4 thcrapy: Suppl 6/46-50 thiocyanatcs: Suppl 6/51-7 thyroglobulin: Suppl 6/198-203 thyroid canccr: Suppl 6/58-65 thyroid carcinoma: Suppl 6/198-203 thyroid follicular tumour: Suppl 6/137-42 thyroid gland: Suppl 6/20-5, Suppl 6/66-9, Suppl 6/70-3, Suppl 6/74-8, Suppl 6/79-84, Suppl 6/85-7, Suppl 6/88-93, Suppl 6/100-4, Suppl 6/105-9, Suppl 6/110-4, Suppl 6/115-9, Suppl 6/ 131-6, Suppl 6/164-72, Suppl 6/217-23 thyroid hormones: Suppl 6/32-8 thyroid ncoplasms: Suppl 6/187-91, Suppl 6/192-7, Suppl 6/ 204-9 thyroid tumors: Suppl 6/143-8 tomography: 2/95-8; 3/186-90 topography: Suppl 6/20-5 trcatmcnt: Suppl 6/149-54 trend in sex-and age-specific incidence: Suppl 6/58-65 tumors: Suppl 6/85-7 tumor inhibition: 2/120--4 ultrasonography: 2/95-8 ureteral obstruction: 1/21-6 urogcnital discases -radionuclide imaging: 3/191-9 validity: Suppl 6/137-42 vascular cndothelial hyperplasia: Suppl 6/131-6 vinblastine: 4/275-9 viruscs: 2/120-4 wounds, nonpenetrating: Suppl 6/99-104 x-ray computcd: 2/95-8; 3/186-90 Reviewers in 1993 Boko H, Zagreb Brencic E, Ljubljana -Brinovec V, Ljubljana -Brovet-Zupancic Irena, Ljubljana -Budihna N, Ljubljana -Erjavec M, Ljubljana -Fcttich J, Ljubljana -Filipic B, Ljubljana -Franceschi S, Milano Fras P, Ljubljana -Golouh R, Ljubljana -Jancar B, Ljubljana -Jereb B, Ljubljana -Jevtic V, Ljubljana -Kališnik M, Ljubljana -Klancar J, Ljubljana -Korman T, Ljubljana -Kovac V, Ljubljana -Koren S, Ljubljana -Kranjec 1, Ljubljana -Lamovec J, Ljubljana -Lukic F, Ljubljana -Markovic S, Ljubljana Mihelcic Z, Zagreb -Osmak M, Zagreb Pavcnik D, Ljubljana -Perovic-Višnar A, Ljubljana -Pogacnik A, Ljubljana -Pompe-Kirn V, Ljubljana Porenta M, Ljubljana Rakar S, Ljubljana -Rubinic M, Rijeka ­Rudolf Z, Ljubljana -Serša G, Ljubljana Snoj M, Ljubljana -Sotošek B, Ljubljana -Šimunic S, Zagreb -Škrk J, Ljubljana -Šmid L, Ljubljana -Štabuc B, Ljubljana Šuštaršic J, Ljubljana Umek B, Ljubljana -Vegelj-Pirc M, Ljubljana -Vidmar-Bracika D, Ljubljana -Vlaisavljevic V, Maribor Žakelj B, Ljubljana Žmner-Pregelj M, Ljubljana. Editors greatly appreciate the work of thc reviewers which significantly contributed to the improved quality of our journal. LUNG CANCER BIOLOGY ANO CLINICAL ASPECTS Under the Auspices of foternatiomd Association for the of Lm1g Cancer 13--16 april 1994 lljubl_jana -Slovenia Second a11nou11cement Call for abstracts & registra/ion Important dates Deadline for Early Registration at reduced rate Deaclline for Cancellation Requests Deadline for Guaranteed Hotel Accommodation Date of the Conference Opening Ceremony Organizing committee Chairrnan: J. Orel Secretary: M. Bitenc Treasurer: M. Sok Members: B. Hrabar, V. Kovac, T. Rott, F. Šifrer, S. Vidmar International scientific committee (in alphabetical order) A. Debeljak (Slovenia) M. Debevc (Slovenia) B. Corrin (United Kingdom) D. Ferluga (Slovenia) P. Goldstraw (United Kingdom) H. H. Hansen (Denmark) K. Havemann (Germany) F. R. Hirsch (Denmark) K. Karrer (Austria) K. Kolaric (Croatia) L. K. Lacquet (The Netherlands) T. Lewinski (Poland) K. Mattson (Finland) M. Mermolja (Slovenia) K. Moghissi (United Kingdom) V. Pastorino (Italy) M. l. Perelman (Russia) V. Pompe-Kirn (Slovenia) P. Rocmans (Belgium) J. B. St/Jrensen (Denmark) J. Šorli (Slovenia) J. Tobias (United Kingdom) J. Viale (Italy) l. Vogt-Moykopf (Germany) N. van Zandwijk (The Netherlands) Invited speakers and lectnres V. Pompe-Kirn (Slovenia) Epidermiological features of Jung cancer in Slovenia B. Corrin (V. K.) Premalignant lesions G. Viale (Italy) Oncogenes and tumor suppressor genes in Jung tumours (With special emphasis on neu­roendocrine tumours) J. Šorli (Slovenia) Methodology and results of bronchopulmo­nary cancer detection in Slovenia 1970-1992 D. Ferluga (Slovenia) Significance of immunohistochemistry for classification of lung cancer M. Mermolja (Slovenia) Possibilities and limitations of cytology in the diagnosis of lung tumours A. Debeljak (Slovenia) Transbronchial aspiration needle biopsy with flexible and rigid needle in the diagnostic and evaluation of regional spread of Jung cancer P. Rocmans (Belgium) Limits of surgery for non-small celi Jung cancer M. l. Perelman (Russia) Reconstructive surgery for tracheobronchial tumours L. K. Lacquet (The Netherlands) Complete resection for unsuspected N2 non­small celi Jung cancer (Stage III A) P. Rocmans (Belgium) Patterns of failure after "complete resection" of Jung cancer J. B. S@rensen (Denmark) The role of chemotherapy in non-small celi lung cancer K. Mattson (Filand) New drugs for non-small celi Jung cancer J. B. St/Jrensen (Denmark) Prognostic factors in non-small celi Jung can­cer N. van Zandwijk (The Netherlands) EORTC studies in non-small celi lung cancer T. Lewinski (Poland) Staging of small celi lung cancer and its scope according toclinical implications K. Karrer (Austria) The importance of surgery in the multimoda­lity treatment of small celi Jung cancer K. Havemann (Germany) Receptors of the steroid hormone superfami­ly: a new approachfor the treatment of small celi Jung cancer F. R. Hirsch (Denmark) Treatment of small celi Jung cancer -the Copenhagenexperience J. Tobias (V. K.) Ten years of studies in small celi lung cancer from the LondonLung Cancer group K. Kolaric (Croatia) Farmorubicin a new active compound in the treatment of metastatic small celi lung cancer N. van Zandwijk (The Netherlands) EORTC Studies in small celi Jung cancer L. K. Lacquet (The Netherlands) SurgicaJ treatment of multipJe Jung cancer P. Goldstraw (U. K.) Post pneumonectomy empyema U. Pastorino ( ltaly) Second primary lung cancer: clinical manage­ment and chemoprevention K. Moghissi (UK.) Laser in Jung cancer -The present and the future M. Debevec (Slovenia) The role of radiotherapy in the treatment of Jung cancer l. Vogt-Moykopf (Germany) ResuJts in surgery on puJmonary metastases Call for abstract Free papers Ali participants are invited to submit abstracts for Frce Papers session. The papers will be arranged and seheduled according to the topics of the Confcrence. Posters Posters will be displayed in the Congress Centre and discussedat poster session. Participants must submit abstraets also for poster presentation. The Scientific Committee will decide whether a submitted paper will be selected for oral or poster presentation. Video sessions Video sessions are seheduled to take place during the Confcrence. Authors of videos are requested to submit the abstract of their video on the same form as for other presentations. Technical equipment Double slide projection and overhead projeetion will be madeavailable in ali sessions. Video projection in video sessions and in other sessions on request. Slides Speakers are kindly requested to hand their slides and check them through at least one hour before the beginning of their session, at the slide center. Abstracts Abstracts must be mailed together with 3 photocopies to the Confcrence Seeretariat. Department of Thoracie Surgery, Medica] Center, Zaloška 7, 61 105 Ljubljana, Slovenia. Tel.: + 38661 3175 82 Fax: + 386611316006 Couference information DATE The Confcrence will be held on 13-16 April 1994, Ljubljana, Slovenia Venue CANKARJEV DOM Cultural and Congress Centre, Prešernova 10, 61000 Ljubljana, Slovenia. Tel.: + 38661 210956 Fax: + 38661 217431 Conference organizers CANKARJEV DOM Congress Department, Prešernova 10, 61000 Ljubljana, Slovenia. Tel.: + 386 61 210 956 Fax: + 38661 217431 Conference secretariat Department of Thoracic Surgery University Medica! Centre, Zaloška 7, 61I05 Ljubljana , Slovenia. Tel.: + 38661 3175 82 Fax: + 38661 1316006 Conference Ianguage The language of the Confcrenee is English. There will be no simultaneous translation facilities. .o·.'uAni,.. HEPATQBILIARY S fHBci c, L)::t .'Y,v, o"'j? LJUBLJANA, SLOVENIA A·S"I: JUNE 27 . JULY l, 1994 ndPOSTGRADUATE COURSE ON HEPATOLOGY 2POSTGRADUATE COURSE ON HEPATOBILIARY SURGERY Two courses, surgical and medica!, will be held simultaneously with a number of sessions assembled. The aim of the courses is to expand practical methological and rational diagnostic knowledge as well as therapeutic approach to the most common diseases of the !iver and biliary tract. Teaching will include lectures, seminars, case presentations and workshops. INVITED LECTURERS Bengmark S., Boeckl O., Coggi G., Czygan P., Ferenzi P., Krejs G., Mazziotti M., Paquet K.-J., Schaffner F., Scheele J., Schmid R., Tiribelli C., Wiechel K. L., and others SURGICAL COURSE Liver resections Liver trauma Hydatid disease of the !iver SURGICAL WORKSHOP MAILING ADDRESS E.GADŽIJEV Medica! Center Dept. of Gastroenterologic Surgery Zaloška 7, 61000 Ljubljana, Slovcnia te! 386 61 322 282, fax 386 61316 096 ASSEMBLED SESSIONS Extra hepatic biliary obstructions Primary !iver tumors Liver transplantation VENUE University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia FEES HEPATOLOGY COURSE WWWWWWIWWW Vira! hepatitis Liver cirrhosis Portal hypertension Cholestasis MAILING ADDRESS S. Institute ofOncology, Zaloška 2, 61000 Ljubljana, Slovenia tel, fax: 386 61 302 828 - DEADLINE 650 USD up to March 31 850 USD up to May 31 950 USD at registration desk NUMBER OF FOR REGISTRATION LANGUAGE PARTICIPANTS May31, 1994 English Limited­30 for surgery, 50 for medicine! ndPOSTGRADUATE COURSE ON HEPATOLOGY POSTGRADUATE COURSE ON HEPATOBILIARY SURGERY NAME ADDRES TEL FAX . MEDICINE OSURGERY POSTER OYES ONO HOTEL ACCOMMODATION OYES ONO Croatian Medical Association CROATIAN SOCIETY OF RADIOLOGY Zagreb -Croatia and Rijeka University School of Medicine CLINICAL INSTITUTE OF RADIOLOGY Rijeka -Croatia THE FIRST CONGRESS OF THE CROATIAN SOCIETY OF RADIOLOGY rand Hotel "ADRIATIC" Opatija -Croatia October 11-15, 1994 SICENTIFIC PROGRAMME To pi c s: -Diagnostic Radiology -lnterventional Radiology -Workshops -Posters TEHNICAL EXHIBITION Organizing Committee Croatian Society of Radiology President President (Prof. Ivo Lovasic MD, PhD) (Prof. Slavko Šimunic MD, PhD) INFORMATION Prof. Ivo Lovasic MD, PhD, Clinical Institute of Radiology -Clinical Hospital Center, 51000 Rijeka, Tome Strižica 3, Croatia, Phone + 38/51/4418 99, Fax + 38/51/375 36 SIEMENS Vaš partner v ultrazvocni diagnostiki: SIEMENS * SONOLINE SL-1 * Možnost prikljucka sektorskega, linearnega, endo-p in endo-v aplikatorja * Izredno ugodna cena (možnost kredita ali leasing-a) * Servis v Sloveniji z zagotovljenimi rezervnimi deli in garancijo * Izobraževanje za uporabnike SIEMENS O.O.O. Dunajska 47, Ljubljana Tel. 324-670 Fax. 132-4281 !:SANOLABOR Ztij«di.. Pri nas dobite vse za rentgeni O O O O KODAK AGFA GEVAERT POLAROID 3M PHILIPS 0 O O O GENERAL ELECTRIC SIEMENS TOSHIBA HITACHI 0 NICHOLAS O BYK GULDEN ° MAVIG ° CAWO • rentgenski filmi in kemikalije • kontrastno sredstvo • rentgensko zašcitno sredstvo • rentgenski aparati, aparati za ultrazvocno diagnostiko, stroji za avtomatsko razvijanje in drugo opremo za rentgen !:SANOLABOR Cigaletovo 9, UUBUANA ii' 061 133 -231 FAX: 061 325 -395 A Bristol-Myers Squibb Cornpany Vodilni svetovni proizvajalec pripomockov za nego stome VALENCIA STOMA -MEDICAL d.o.o. Županciceva 10, 61000 Ljubljana, Slovenija Tel. 061 214-959 Posvetovalnica za stomiste deluje v naših prostorih vsak delavnik med 9. in 16. uro. Ab a kt a ampoules 400 mg (pefloxacin) A new potent drug against infections • May be given orally and parenterally • Effecive in life-threatening infections caused by nosocomial strains resistant to many drugs • May be given to patients hypersensitive to penicillins and cephalosporins • lts favourable pharmacokinetic properties allow twice-a-day dosage • 1s very well tolerated Contraindications Pefloxacin is contraindicated in patients with known hypersensitivity to quinolones, in preg­ nant women, nursing mothers, children under 15 years of age, and patients with inborn glucose-6-phosphate dehydrogenase deficiency. Precautions During pefloxacin therapy exposure to strong sunlight should be avoided because of the risk of photosensitivity reactions. In patients with a severe liver disorder dosage of pefloxa­cin should be adjusted. Side ettects Gastro-intestinal disturbances, muscle and/or connective tissue pains, photosensitivity reactions, neurologic disturbances (headache, insomnia), and thrombocytopenia (at doses of 1600 mg daily) may occur. Dosage and administration The average daily dosage for adults and children over 15 years of age is 800 mg. Oral: 1 tablet twice daily after meals. Parenteral: the content of 1 ampoule 400 mg diluted in 250 ml of 5 % glucose as a slow 1-hour infusion twice daily. The maximum daily dosage is 8 mg of pefloxacin per kg body­weight. In severe hepatic insufficiency pefloxacin is administered only once daily (jaundice), once every 36 hours (ascites), and once every 48 hours (jaundice and ascites). @ lek Pharmaceutical and Chemical Company d.d. Ljubljana Klimicin ® Klimicin is an effective (Clindamycin) It stimulates the action bactericidal or bacteriostatic as evidenced by the MIC/MBC ratio. of polymorphonuclear leukocytes (PMN) principal factors in host immune system. PMN leukocyte. ,, I' .. Anaerobes Aerobes Bacteroides spp. (including Bacteroides fragilis) Streptococcus spp. (including Fusobacterium spp. Streptococcus pyogenes), except Propionibacterium Enterococcus Eubacterium Actinomyces spp. Pneumococcus spp. Peptococcus spp. Staphylococcus spp. (including Peptostreptococcus spp. B-lactamase producing strains) Clostridium perfringens Contraindications: In patients hypersensitive to lincomycin and clindamycin. Precautlons: Klimicin should be prescribed with caution to elderly patients and to individuals with a history of gastrointestinal disease, particularly colitis. Side Effects: Gastrointestinal disturbances (abdominal pain, nausea, vomiting, diarrhea). When significant diarrhea occurs, the drug should be discontinued or continued only with close observation of the patient. The posibility of pseudo­membranous colitis must be ruled out. @ lek Pharmaceutical and Chemical Company d.d. Ljubljana lopami.rQ 150 -200 -300 -370 mgl/ml FOR ALL RADIOLOGICAL EXAMINATIONS MYELOG RAPHY ANGIOGRAPHY UROGRAPHY C.T. D.S.A. THE. FIRSl WATER s·ol!.l!JBLE1 READY TO· l!JSE NON-IONIC CONT:RAST MEDll!JM Manufacturer: Distributor: Bracco s.p.a. Agorest s.r.l. Via E. Folli. 50 Via S, Michele, 334 20134 -Milan -(1) Fax: (02) 26410678 Telex: 311185 Bracco 1 Phone: (02) 21771 e 34170 -Gorizia -(1) Fax: (0481) 20719 Telex: 460690 AF-GO 1 Phone: (0481) 21711 © Eastman Kodak Company, 1990 Kodak systems provide dependable performance for advanced diagnostic imaging. Our quality components are made to work together from exposure to viewbox. Kodak X-Omat processors are the most respected in the field. Kodak X-Omatic cassettes are known the world over for unexcelled screen-film contact and dura­bility. Kodak multiloaders have earned an enviable reputation for reliability. The Kodak Ektascan laser printer is changing the look of digital imaging. The list goes on. There are quality Kodak products throughout the imaging chain. Equally important, they are made to work together to achieve remarkable performance and diagnostic quality. Contact your Kodak representative for more information. 1 1 1 NI . 1 PREPRECEVANJE Ena sama intravenska injekcija po 4mg pri uvajanju v anestezijo POOPERATIVNA SLABOST IN BRUHANJE NELAGODJE IN STISKA OGROŽATA USPEH OPERATIVNEGA POSEGA e g .­ M!;Q .. c •-.. 1 .. . . ;...' .WJ .. ..-.;•ti: . -... ,,,...:11i· .. : b, -.:.., . ZDRAVLJENJE Ena sama intravenska injekcija po 4mg ondansetron Glaxo Podrobnejše informacije dobite pri: Glaxo Export Limited Predstavništvo Ljubljana, Tržaška cesta 132, 61111 Ljubljana tel. (061) 272-570; fax (061) 272-569 Your Partner in Diagnostic Ima.ng. 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ZAUPAJO NAM NAŠI KUPCI IN DOBAVITELJI, ZNANI PROIZVAJALCI IZ TUJINE PA SO NAM ZAUPALI TUDI ZASTOPSTVA IN KONSIGNACIJE: ZASTOPSTVA IN KONSIGNACIJE: KONSIGNACIJE: -BAXTER EXPORT CORPORATION -HOECHST AG -BOEHRINGER INGELHEIM -HOFFMANN LA ROCHE -NOVO NORDISK -SANDOZ -ORTHO DIAGNOSTIC SYSTEMS -SCHERING & PLOUGH ­ ESSEX CHEMIE SALUS LJUBLJANA d. d. -61000 LJUBLJANA, MAŠERA SPASICEVA 10, TELEFON: N.C. (061) 168-11-44, TELEFAX: (061) 168-10-22 'llT.eLlira/5 11111 Trocar drainage catheter sets A simple, sate technique tor percutaneous nephrostomy, abscess and other fluid collection drainage. A full range of drainage products; • Available from 4 upto 14 french • Variaty of materials .2 standard lengths, 20 and 30 cm. eAvailable with string safity lock ial POLVSTAN Address De Stei"ger 172 Phone 036-5312844 P.O. 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LJ CJ VJ1//--.·. · .A· = INTERNATIONAL Y our Partner in Radiology ACTIV A International Sri Tel. 040-212856 Via di Prosecco, 2 Telefax 040-213493 34016 Opicina-Trieste Telex 460250 I ltaly BV 29 INTEGRIS C 2000 DIAGNOST 76 + DIAGNOST93 NOV CLAN DRUŽINE PHILIPSOVIH TELEDIRIGIRANIH RENTGENSKIH STATIVOV, KI JIH ODLIKUJE KVALITETNA MODULARNA IZDELAVA, VISOKA ZANESLJIVOST, ENOSTAVNA UPORABA IN IZREDNA KVALITETA APARAT JE MOGOCE OPREMITI S SISTEMOM ZA DIGITALNO RADIOGRAFIJO PREKO SVETLOSTNEGA OJACEVALNIKA (OSI), KI OLAJŠA DELO UPORABNIKOM, MANJŠA DOZE, NIŽA STROŠKE IN KRAJŠA PREISKAVE. ZA VSE DODATNE INFORMACIJE SE OBRNITE NA ZASTOPNIKA PHILIPS MEDICAL SYSTEMS V SLOVENIJI: skupina avtotehna d.d. AVTOTEHNA d.d. Ljubljana, Slovenska 54, tel.: (061) 320 767, faks: (061) 322 377 E F R IJ Lekarne, bolnišnice, zdravstveni domovi ,n veterinarske ustanove vecino svojih nakupov opravijo pri nas. Uspeh našega poslovanja temelji na kakovostni ponudbi, ki pokriva vsa podrocja humane medicine in veterine, pa tudi na hitrem in natancnem odzivu na zahteve naših kupcev. KEMOFARMACIJA -VAŠ ZANESLJIVI DOBAVITELJ! 1(: KEMOFARMACIJA Veletrgovino zo oskrbo zdravstvo, p. o. / 61001 ljubijo no, Cesto no Brdo 100 Telefon: 061 268-145 / Telex: 31334 KEMrAR / Telefox 271-362 The publication of the ,journal is subsidized by the Ministry of Science and Technology of the Republic Slovenia. CONTRIBUTIONS OF INSTITUTIONS: Inštitut za diagnosticno in intervencijsko radiologijo, KC Ljubljana Klinika za otorinolaringologijo in maksilofacialno kirurgijo, KC Ljubljana Klinicki zavod za dijagnosticku i interventno radiologijo, KBC Rebro, Zagreb Onkološki inštitut, Ljubljana DONATORS ANO ADVERTISERS: ACTIVA INTERNATIONAL S.r.I Trieste, ltaly AGOREST s.r.I. Gorizia, ltaly A VTOTEHNA d.d. LjubUana, Slovenia GE MEDICAL Systems Zagreb, Croatia GLAXO EXPORT Ltd. Ljubljana, Slovenia KEMOFARMACIJA p.o. Ljubljana, Slovenia KRKA p.o. Novo mesto, Slovenia LEK d.d. Ljubljana, Slovenia MEDITRADE-KODAK Ljubljana, Slovenia POL YSTAN-BENELUX Almere Haven, Tite Netherlands SALUS d.d. Ljub.jana, Slovenia SANDOZ PHARMA SERVICES AG Basel Representative Office Ljubljana, Slovenia SANOLABOR p.o. Ljubljana, Slovenia SIEMENS O.O.O. Ljubljana, Slovenia MEVI d.o.o. TOSIDBA MEDICAL SYSTEMS EUROPE Miklavž, Slovenia VALENCIA STOMA-MEDICAL d.o.o. Ljubljana, Slovenia Radio[ Oncol 1993; 27: 370n. lnstructions Thc journal Radiology ancl Oncology publishcs ori­ginal scicntific papcrs, profcssional papcrs, rcvicw ar­ticlcs, casc rcports and varia (rcvicws, short communi­cations, profcssional information, cct.) pcrtincnt to diagnostic and intcrvcntional racliology, computcriscd tomography, magnctic resonance, nuclcar mcclicinc, racliothcrapy, clinical and cxpcrimcntal oncology, ra­cliobiology, racliophysics and radiation protcction. 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Better control of acute and delayed chemotherapy-induced emesis 2. Safer and better tolerated 3. Simplicity of a once-a-day standard dose Recommended dosage schedule * 5 mg ampoule to be administered shortly before chemotherapy as intravenous infusion or slow injection ** 5 mg capsule to be taken in the morning one hour before breakfast Navoban® Always once a day. Always 5 mg. f3\. SANDO%