ADIOLOGY 1,.11 NCOLOGY 1998 ISSN 1318-2099 UDC 616-006 CODEN: RONCEM RADIOLOGY AND ONCOLOGY Radiologij and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiology, conzputerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncology, radiobiology, radiophysics and radiation protection. Editor-in-Chief Gregor Serša Ljubljana, Slovenia Executive Editor Viljem Kovac Ljubljana, Slovenia Editor-in-Chief Emeritus Tomaž Benulic Ljubljana, Slovenia Editorial board Marija Auersperg Be1aFornet Maja Osmak Ljubljana, Slovenia Budapest, Hungary Zagreb, Croatia Nada Bešenski Tullio Giraldi Branico Palcic Zagreb, Croatia Trieste, Italy Vancouver, Canada Karl H. Bohuslavizld Andrija Hebrang ]urica Papa Hamburg, Germany Zagreb, Croatia Zagreb, Croatia Haris Boka Laszl6 Horvath Dušan Pavcnik Zagreb, Croatia Pecs, Hungary Portland, USA Nataša V. Budihna Berta Jereb Stojan Plesnicar Ljubljana, Slovenia Ljubljana, Slovenia Ljubljana, Slovenia Marjan Budihna Vladimir Jevtic Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada Malte Clausen H. Dieter Kogelnik Jan C. Roos Hamburg, Germany Salzburg, Austria Amsterdam, Netherlands Christoph Clemm Jurij Lindtner Slavko Šimunic Miinchen, Germany Ljubljana, Slovenia Zagreb, Croatia Mario Corsi Ivan Lovasic Lojze Šmid Udine, Italy Rijeka, Croatia Ljubljana,Slovenia Christian Dittrich Marijan Lovrencic Borut Štabuc Vienna, Austria Zagreb, Croatia Ljubljana, Slovenia Ivan Drinkovic LukaMilas Andrea Veronesi Zagreb, Croatia Houston, USA Aviano, Italy Gillian Duchesne Metka Milcinski Živa Zupancic Melbourne, Australia Ljubljana, Slovenia Ljubljana, Slovenia Publishers Slovenian Medical Association -Slovenian Association of Radiologij, Nuclear Medicine Society, Slovenian Society for Radiotherapy and Oncologij, and Slovenian Cancer Society Croatian Medical Association -Croatian Society of Radiologij Affiliated with Societas Radiologorum Hungarorum Friuli-Venezia Giulia regional groups of S.I.R.M. (Italian Society of Medica/ Radiology) Correspondence address Radiologij and Oncology Institute of Oncology Vrazov trg 4 SI-1000 Ljubljana Slovenia Tei: +386 61 132 00 68 Tel/Fax: +386 61 133 74 10 Reader far English Olga Shrestha Design Monika Fink-Serša Key words Eva Klemencic Secretaries Milica Harisch Betka Savski Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 750 copies Banic account number 50101 679-901608 Foreign currency account number 50100-620-133-27620-5130/6 NLB -Ljubljanska banica d.d. -Ljubljana Subscription fee far institutions 100 $ (16000 SIT), individuals 50 $ (5000 SIT) The publication of this journal is subsidized by the Ministry oj Science and Technology oj the Republic oj Slovenia. According to the opinion of the Government of the Republic of Slovenia, Public Relation and Media Office, the journal Radiology and Oncology is a publication oj informative value, and as such subject to taxation by 5% sales tax. Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica This journal is printed on acid-free paper Radiology and OncologiJ is now available on the internet at: http://www.onlco-i.si/radiolog/rno.htm CONTENTS ULTRASOUND High resolution ultrasound and power-Doppler -advances in pre-invasive diagnosis of solid breast lesions: our one-year experience Brnic Z, Drinkovic I, Hebrang A 353 Hypertrophic pyloric stenosis: Ultrasound diagnosis Raic G, Cop S, Posaric V, Ercegovic S 363 NUCLEAR MEDICINE Palliation of painful osseous metastases in patients with prostate cancer using Re-186-HEDP Klutmann S, Bohuslavizki KH, SchulteU, Kroger S, Bleckmann Ch, Mester J, Clausen M EXPERIMENTAL ONCOLOGY p53 -the paradigm of tumor-suppressor genes? Jezeršek B, Novakovic S 373 Micronuclei in cytokinesis-blocked lymphocytes as an index of occupational exposure to antineoplastic drugs Garaj-Vrhovac V, Kopjar N 385 The role of thyroxin in thyroid radiation carcinogenesis in rats Koritnik K, Car A 393 CLINICAL ONCOLOGY Systemic Lupus Erythematosus diagnosed with extreme skin reaction during radiation therapy: a case report Lee RJ, Proulx CM, Donaldson CW, Orner JB 401 Phase II study of fluorouracil, leucovorin and interferon alpha-2a in patients with advanced colon cancer Štabuc B, Markovic A, Brecelj E, Bešlija S, Cizej TE 407 RADOIOPHYSICS Thyroid doses due to stereotactic radiosurgery of the brain Sibata CD, Almeida CE, Shin K 417 TELEMEDICINE The assessment of telemedical procedures in countries of transition Haris B 423 SLOVENIAN ABSTRACTS 428 NOTICES REVIEWERS IN 1998 AUTHOR INDEX 1998 SUBJECT INDEX 1998 Radio/ Onco/ 1998; 32(4): 353-61. High-resolution ultrasound and power-Doppler -advances in pre-invasive diagnosis of solid breast lesions: our one-year experience Zoran Brnic, Ivan Drinkovic, Andrija Hebrang Department oj Radiology, University Hospital "Merkur", Medica/ Schoo/ oj Zagreb University, Zagreb, Croatia The purpose oj the study was to evaluate high-resolution ultrasound (HRUS) and power-Doppler (CDE) in the differentiation of malignant from benign so/id breast /esions. Patients and methods: HRUS and CDE examinations of solid breast masses were carried out in 25 women. Gray-sca/e criteria of malignancy and benignity were considered. The vessels of the /esion were classified as penetrant, peripheral and non-detectable with CDE. Fina/ diagnose was obtained cytologically and/or with biopsy. Results: HRUS detected more jrequently irregularity of contours, heterogeneity and posterior attenuation than "classic" ultrasound. Lateral shadows in carcinmnas were seen in a considerable number oj cases, but this did not have any impact on the accuracy oj diagnosis. HRUS facilitated the visualization of small carci­nomas intraducta/ calcifications and papillomas. CDE detected flow in 15/25 lesions, of which 8 were malignant. Penetrant vessels were observed in 6/8 carcinomas and only in 2/17 benign changes;6/11 fibroadenomas were avascular, and 4/11 with peripheral vesse/s. In 3/6 other benign lesions, the flow was shown with CDE. Conclusion: HRUS and CDE can successfully help in differentiation malignant from benign solid breast mass, and are a good adjunct to mammography and physical examination in the pre-invasive phase of diag­nostic process. Key words: breast neoplasms; ultrasonography, mammary; ultrasonography, Doppler; high-resolution ultrasound Correspondence to: Zoran Brnic, M.D., B.Sc., Depart­ment of Radiology, University Hospital "Merkur", Zagreb, Medica! School of Zagreb University, Zajceva 19, 10000 Zagreb, Croatia. Phone/Fax: +385 1 233 14 40. Introduction Breast sonography performed until few years ago with 5-7.5 MHz transducers was mainly confined to the differentiation between cystic and solid nature of the lesion and, to some extent, to characterizing solid mammary nod­ules.1 High-frequency transducers (up to 13 354 Brnic Zet ni. MHz) constructed in the last decade allow perfect spatial and contrast resolution, with the former being well lower than a millime­ter. Therefore, non-palpable lesions of a few millimeters can be detected, whereas palpa­ble ones more precisely characterized. This "high-resolution ultrasound" (high definition ultrasound, HRUS) was "bom" approximately at the same tirne as the power-Doppler (color­Doppler energy, CDE) was introduced as a new mode of color-Doppler imaging. Although sonography has, as yet, an uncer­tain place among other differential diagnostic steps and is considered by many as a sec­ondary technique,2 HRUS and CDE available in most modem US machines enable pre­invasive work-up of breast lesions with more sensitivity and specificity, sparing thus many unnecessary biopsies of benign lesions. Tumor vascularization and power-Doppler Each tumor larger than a few millimeters depends, during its growth, on the prolifera­tion of new vessels in its periphery, and pro­duces substances (angiogenetic factors) that stimulate neoangiogenesis.3A In breast carci­nomas, an increased number of the vessels is evident. Their diameter is enlarged. The structure of its wall, as well as the architec­ture are aberrant (AV-shunts, sinusoids), with a consecutive abnormal function.5 The abnor­mality of such vessels is a clue to the features of Doppler signals gained from malignant breast masses, or from their close surround­ings. 6 B-mode, regardless of its high resolution, cannot visualize small intratumoral vessels because of their microscopic dimensions. The detection of such vessels on the basis of flow is thus a great advance, firstly, enabling a precise positioning of sample volume and acquisition of spectral flow signal and, sec­ondly, imaging of their distribution and architecture, especially with CDE. Although there are many inconsistencies and overlapping of results, and as malignan­cy cannot be ruled out only due to the absence of flow signal in the mass, the major­ity of authors agree that vessels are more numerous, and velocities higher in malignant than in benign masses. Some authors also report of the increase of pulsation indices.6-8 "Conventional" color-Doppler imaging (CDI, color-Doppler velocity, CDV) detects and displays blood velocity and its variance. Since 1993, a new color Doppler ultrasound technique has become available. It provides information of total amplitude or energy of signal, rather than velocity and direction of flow (Figure 1). A M p L 1 T u D E MEAN FREOUENCY Figure 1. shows typical Doppler signal spectrum: vari­ance is marked with arrows. Energy is proportional to the area under the curve under the curve, which is shown shadowed. The new mode is termed power-Doppler, color Doppler energy (CDE), "ultrasound angiography" or amplitude color-Doppler sonography, which is the most exact term.1 The flow is coded in hues of a single color, e.g. yellow, rather than blue and red (+green) as with standard CDI, and the color satura­tion of a pixel is related to the number of red blood cells in a unit of volume (voxel), regard­less of their vector sum. When the resultant velocity is zero, as within the areas of capil­laries randomly distributed in a voxel, related pixels on CDI will not be coded at all. On the contrary, CDE displays nearly all the amount Hig/J-reso/11/ion 11ltraso1111d and power-Doppler oj solid breasl lesions of circulating blood in these low-flow areas, which results in improved sensitivity.9,10 Thus, directional averaging in each pixel is substituted with the summing of amplitudes, and randomly distributed tissue capillaries altogether contribute to the signal strength. Hence, an overwhelming "blush" in good vas­cularized organs, such as kidneys, will be apparent.11 There is no angle-dependence of signal which is one of the basic principles (and limitations) of CDI. Patients and methods In the period from April to November 1997, we examined 25 women with breast lesions, aged 16 to 68 years (mean 42.2). The gray­scale morphology of the lesions was assessed with "classic 7.5 MHz" and high-resolution ultrasound machine, with their compressibil­ity and mobility additionally evaluated, then CDI performed (searching for eventually high velocities), and finally power-Doppler (CDE) examination carried out. Some patients were seen because of breast complaints (palpable mass, discomfort, nipple discharge), others were routinely examined prior to initiation of hormone replacement therapy. All but one were out-patients. Every patient had under­gone mammography, either in our hospital or in another institution, less than 30 days before ultrasound was recommended because of mammographically suspected abnormalities. Fina! diagnoses were obtained mainly by fine-needle aspiration cytology (FNAC) and, in 2 cases, by open surgical biopsy. All ultrasound examinations were done and images checked by one breast radiologist (Z.B.), and occasionally reviewed by another (I.D.). Scanning was carried out with three machines: General Electric-CGR RT 2800 equipped with linear-array 7.5 MHz transduc­er, and General Electric-Logic 400 MD and ­Logic 500 MD ultrasonic units with multifre­quent (7-13 MHz) transducers with a length of 38 and 50 mm, respectively. The latter two probes enable superior imaging in the near field, thus no distance silicone pad was required. It is especially suitable for radia] examination technique, with satisfactory visualization of pyramidal architecture and dueta! system. Color-Doppler velocity mode ("frequency mode") and color-Doppler energy mode ("amplitude mode", power-Doppler) are available in both. Besides the optimization of standard gray­scale scanning parameters, special attention was given to the optimization of Doppler pre­sets. Color Doppler velocity receiver gain was turned down until a few specks of color remained in the color box, i.e. background color "noise" was just suppressed. The color box was adjusted to include the lesion and some adjacent normal surrounding tissue. Thus defined region of interest was then scanned slowly until a persistent color signal was apparent. Power-Doppler gain was adjusted according to recommendations of Bude et a1.rn-n we would, however, like to stress that we did not strictly stick adhere to the articles referred to. We increased the gain until a clear and persistent color signal repre­senting intralesional vessel appeared, or, if such was absent, until the background became almost uniformly colored. The gray-scale criteria used in the evalua­tion of solid breast lesion were as follows: Typica/ benign lesions were smoothly marginal­ized, with linear borders and homogeneous fine-granulated echotexture, hypo-or hypere­choic, ovoid shaped with the long axis paral­lel to the chest wall (depth/width ratio, D/W>l), and with enhanced acoustic through transmission. Conversely, typical ,na/ignant features included ill-defined, spicu­lar or lobulated margins, of round or ovoid shaped with the long axis perpendicular to the chest wall (D/W <1, "taller than wide"), hypoechoic and sometimes heterogeneous 356 Bmh'Z etal. echotexture, posterior acoustic shadowing, sometimes with obvious central microcalcifi­cations, broken tissue planes or distorted breast architecture.13,14 If any of malignant characteristic mentioned above was present, the lesion was considered malignant, until praven otherwise. When ali criteria of benig­nity were strictly adhered to, the lesion was considered benign. Arbitrarily, when most of benign characteristics were present, the lesion was defined as "probably benign" or "indeterminate" .15 Similar to the methodology of Raza and Baum, 16 the appearances of vascular pattern in the lesion were categorized into 3 groups: (a) penetrating vessels -one or more blood ves­sels arising at the edge of the lesion, coursing toward the center, with an irregular branch­ing pattern, (b) peripheral vessels -one or more blood vessels of predominantly uniform appearance, parallel to the edge of a mass, linear or arcuate, without significant branch­ing, and (c) no detectable vessels -no vessels were reliably detected, or, in other words, color signals were not so constant to distin­guish them definitely from noise. Centrally located vessels were seen occasionally, but this vascular pattern was often indistinguish­able from (a), especially within small lesions, and therefore it was not included as a distinct category. Doppler images were obtained in peak­systolic phase when the vascular signal was the most excessively enhanced, tracing vas­cular architecture to the largest extent. Ali the examinations were performed with the lowest transmitted energies that allowed good visualization, as recommended in man­uals. Scanning performed in the most erratic manner that was possible not to insonate the same tissue volume for a too long tirne. Med­ica! Ethics Committee approved of the exami­nations referred to in this study. Results Of 25 solid nodules, 17 (68%) were benign and 8 (32%) were malignant. The negative-to­positive biopsy ratio was 2.13 : 1. Table 1. shows morphologic features detected in our patients by HRUS. Certain characteristics were seen in larger percentage with HRUS and than were detectable with conventional sonography.16,17 CDE was performed on ali masses includ­ed in this study. Six of 8 cancers showed pen­etrating vessels (Figure 2), one showed peripheral vessels, and in one case, no flow was detected. This latter one was a very small carcinoma (6 mm), which was diagnosed as carcinoma even with repeated FNAC. In the group of fibroadenomas, no detectable ves­sels were found in 6 cases. One or more peripheral vessels were seen in 4 cases and a penetrating vessel was found in one lesion. Six lesions were found to be benign breast tissue with some dysplastic changes. In 3 cases of this group, no significant flow was detected, in two cases, vessels were found at the periphery, and in one, circulation was observed arising from the intramammary lymph node. Table 2 is a concise presentation of the results. We detected flow in the mass in 15/25 (60%) cases, while in 10/25 (40%) lesions no flow was found. Penetrating ves­sels were most frequent in carcinomas and in 2 benign masses; but not ali cancers demon­strated vascularization. In benign lesions, the vessels, if detected, were situated predomi­nantly peripherally. Sometimes, in a hypere­choic boundary, they remind of (pseudo) cap­sule. In 10/17 (59%) of nodules, no flow was detected with CDE, although they were ali solid. Flig!t-resolutio11 ultrnsou11d n11d power-Doppler of so/hi /Jreast lesions Table l. Morphologic features detected with HRUS (bold figures) in comparison with "low frequency ultrasound". Some characteristics were seen in larger percentage with HRUS than with conventional sonography. These figures were marked with apostrophe (') Diagnose Carcinomas Fibroadenomas Other benign % lesions lrregularity of contours 88 88 18 36' 33 50' Inhomogeneity 75 88' 18 27' 33 67' Posterior attenuation 13 25' O 18' O 33' Laterni shadowing 13 25' 54 54 1616 Table 2. CDE features of solid breast lesions Vascular pattern Penetrant vessels Peripheral vesseles Vcssels undetected Tota! Carcinoma 6 1 1 8 Fibroadenoma 1 4 6 11 Other benign lesions 1 2 3 6 Ali diagnoses 8 7 10 25 Figure 2. Penetrating vessels visualised by CDE. Discussion Considering the aggressiveness of aspiration tissue-sampling techniques (FNAC, core­biopsy) and open surgical biopsy as well as their costs, additional characteristics differ­entiating between benign and malignant lesions that can be detected in the pre-inva­sive phase of diagnostic process, could elimi­nate some financial load and patients. suffer­ings. If negative predictive values (NPV) for malignant characteristics of the mass were high enough to substantiate recommenda­tions for surveillance rather than biopsy, ultrasound would become a more capable tool in diagnostics, not restricted to distin­guish only cystic from solid. Stavros et a!.15 classified prospectively 750 solid breast lesions as benign, indeterminate, or malig­nant using gray-scale sonography criteria, and calculated a NPV of 99.5% for malignan­cy. This is unexpectedly an encouraging result in spite of using state-of-the-art equip­ment and strict diagnostic criteria for benign masses; nevertheless, corroboration by other investigators is still required. The advantages of HRUS are undoubtedly proved 16,17 and our experiences are very similar. In our material, we detected irregularity of contours as a typical sign of malignancy15 in a larger number of carcinomas than other authors who employed low-frequency probes.16,17 As the spiculation is a sign with very high positive predictive value (PPV) for malignancy, 15 its accurate sonographic dis­closing is of great value. This is particularly relevant in the dense breast where mammog­raphy may be of limited value in the evalua­tion of the mass. However, in a considerable 358 BmicZ etal. number of benign lesions, irregularity of con­tours was displayed more frequently than with low frequency probes. This may result in false positive results, espedally when deal­ing with fibroadenomas. But, as they pre­dominate in younger population group and are not so characteristic for malignancy, nei­ther is there any overt spiculation, but only microlobulation of the contour,17 we prefer a non-aggressive (but not passive) approach, espedally if no risk factors in patient's histo­ry are present. We discovered heterogeneity in a large pro­portion of "other benign lesions", predomi­nantly as a heterogeneous group that inclu­des some regions of fibrocystic changes and areas of architectural distortion. Stavros et al. did not consider this feature as a separate category, but discussed about shadowing and punctate calcifications as signs of malignan­cy. Hence, we detected no clear (micro)calci­fications in any masses analyzed: we might have not paid sufficient attention to this fea­ture, probably also because microcalcifica­tions were present in only one third of carci­nomas. Undoubtedly, the detection of tiny caldfications has not been exclusively related to mammography; it can as reliably be done with HRUS, especially when situated in hypoechogenic mass. The lower their dimen­sion, the lower the sensitivity for their detec­tion.15 Posterior attenuation is the feature that has no decisive strength when dealing with breast tumors, unless confused with shadow­ing. It may serve as a clue for the diagnosis of fibrous dysplastic changes, which can explain some palpatory resistendes that are otherwise not suspected of malignancy. We were often faced with this feature, however, it was rather a practical difficulty in penetrat­ing to deeper parts of the voluminous breasts than a reliable diagnostic sign. Lateral shadowing was earlier referred to as a sign supporting benign diagnosis (fibroade­noma), but we detected it also in malignant tumors, and it was often asymmetric. The detection of this considerably unspedfic sign is not significantly influenced by HRUS, and Stavros at al. did not even point it.15 Predse imaging of the ductal system is one of the leading advantages of HRUS. Although fairly time-consuming and of limit­ed accuracy, when scrutinizingly performed radially, HRUS may compete with galactogra­phy/ductography at least in its lack of con­trast agent and invasiveness. On the other hand, it may be of valuable help in the char­acterization of an intraductal lesion detected by the latter. Thus, it is possible to detect pri­mary intraductal grnwth as well as duet exten­sion of proliferative process within or/and around the lumen which courses toward the nipple. This was to a very limited extent also possible with low-frequency US. The fre­quencies between 10-13 MHz are optimal for this task. Moreover, scrutinize scanning, with an optimal focus adjustment, may reveal small intraductal papillomas, microcalcifica­tions, and other intraductal masses with a possibility of predse needle guidance. In some cases, an intraductal location of calcifi­cation detected by HRUS, without presence of solid mass may be a decisive factor to con­sider them rather benign than suspected of malignancy. From our experience it is obvi­ous that we should be cautious not to overes­timate the significance of intraductal masses because, in many cases, they were proved to be an insignificant detritus, sites of atypical duet branching or just a tortuous duet seen in the scanned plane, as if the mass was con­tained within it. This may nullify advantages of HRUS by provoking many false positives resulting in additional FNACs or biopsies. When ductography cannot be obviated, the ducts dilated with contrast agent are better visualized with HRUS, and the lesion then punctured.18 We believe that the probe length of 38 mm, although adequate for scanning the sub­areolar region and axilla, may be too small for High-resolution ultrasound and power-Doppler of solid breast lesions the radial scanning technique of the breast, because of insufficient orientation in lobar/ductal anatomy; we therefare suggest a probe faotprint of 50 mm as ideal far this task. Until recently, investigations in the field of Doppler spectral analysis have not yielded unequivocal criteria far distinguishing malig­nant from benign solid breast lesions.6-8 Cos­grove et al.18 used a semiquantitative scoring system involving analysis of an average num­ber of vessels per square centimeter and aver­age density of color pixels. Although they faund color Doppler signals in 98% of can­cers, they did not prave correlation of color Doppler scores with the conventional prog­nostic indicators such as lymph node status or survival.19 Birdwell et al. employing power Doppler used similar methodology.20 They characterized breast masses with <10%, 10­25%, 25-50% and >50% of flow in a scanned area as avascular. They concluded that the presence of color in a solid breast mass was a non-specific finding, and that assessing of the extent of vascularity appears to be of lim­ited value in the evaluation of solid breast masses. The authors faund approximately equal numbers of malignant and benign masses among avascular lesions, and a quar­ter of malignant masses showed no color flow, although a sensitive method (CDE) was used. In their material, however, there was a significant number of small carcinomas as well as large fibroadenomas. The theory of the prevascular phase in tumor growth may explain the small amount of detected blood flow in the invasive carcinomas smaller than 2 cm, as well as good vascularity in large fibroadenomas.21-22 We can assume that the presence of acoustic shadowing may limit the acquisition of Doppler signal and may have accounted far the avascular assessment of carcinomas with surrounding fibrosis as the dominant morphologic feature. We studied the morphology and the pat­tem of distribution of vessels within the mass in an attempt to find the characteristics of malignant and benign lesions. In our study, carcinomas predominantly had pene­trating vessels. Benign lesions had no detectable vessels in 59% of cases (cysts were excluded from our material) or had vessels around the periphery of the mass. In fibroadenomas vessels, if detected, were situ­ated mainly in the peripheral parts. Some­times, in a hyperechoic boundary, they looked like (pseudo)capsules. In one larger fibroadenoma (34 mm), an overt penetrating circulation (with a borderline spectral find­ing) was shown, and in one palpable intra­mammary lymph node with a diameter of 1 cm, hilar vascular pattern, which was catego­rized as "penetrant", was depicted. Later, FNA revealed the real nature of the mass, and retrospectively, when reviewing MOD recordings, we concluded that the vessel that we describe as "penetrant" was, in fact, a nor­mal hilar vessel of the reactive lymph node. Unfartunately, we did analyze Doppler spec­tra in this case. In our work, we did not strict­ly analyze the morphology of the vessels, but in some carcinomas an examiner experienced in angiography would be amazed at a glance of a chaotic and irregularly branched vessels. The morphology was especially apparent when an individual vessel was kept well extended through the scanned plane, and the gain decreased so as to prevent the leakage of the color out of the lumen of the vessel. Such tortuous vessels depicted by standard color Doppler would be fairly confusing because of inconstant angle of insonation, and some­times aliasing, and because of power Doppler homogeneous coding. Also, slow flow just near the wall was not eliminated and the lumen was filled with color in its real width. We detected the flow in the mass in 15/25 (60%) cases, while in 10/25 (40%) lesions, no detectable flow was faund. In comparison to some recent studies, 20 there was a consider­able number of vascular lesions. Possibly, we were too rigid in eliminating some flow sig­ 360 Brnic Zet ni. nals, assuming that they were related to noise. We might have falsely extended our experiences with classic color Doppler, and in some situations, decreased the gain too much. Perception of weak tiny signals of flow through an overwhelming homogenous color may sometimes be exhausting; we therefore preferred to avoid it. Another possible reason may be the attenuation of the ultrasonic beam by some lesions with abundant fibro­sis, which obscure the acquisition of Doppler signal. As we had no strict criteria to differ peripheral from penetrating vessel, we put some lesions with strong signals, even just beneath the capsule, in the latter group to avoid false negative result which we consider more dangerous than false positive one. Motion artifacts were strong near the heart, especially when the breast was very small, and lesions situated deep close to the thoracic wall. Some patients were even unable of breathholding, or were also anx­ious and restless, which made the examina­tions more difficult. The use of ultrasonic contrast agent would theoretically help to overcome this problem, enabling the utiliza­tion of lower gain and higher pulse repetition frequency, but cost/benefit ratio remain questionable. Nevertheless, careful and patient scanning is, in the majority of cases, satisfactory to obtain images of acceptable quality. In many centers of our country and also elsewhere, HRUS and CDE, if available, should be applied prior to FNA or biopsy, in order to further characterize the undetermi­nate solid lesion and to increase the speci­ficity of the diagnostic process. It must be emphasized that this excellent sonographic technique, including radia! technique, with the best scanners and transducers, as well as strict observing of relevant criteria for benign lesions, is highly recommended. With this approach, the population with benign solid breast lesions that does not require invasive work-up, can be identified with considerable accuracy. This could result in improved care and reduction of patient's discomfort, morbidity and health care cost. Conclusions 1. HRUS can successfully help to distinguish many benign from malignant solid nod­ules in the breast. The chance to detect some malignant feature in a lesion are bet­ter with the application of HRUS than with conventional, lower frequency probes. 2. Assessment of interna! vascular architec­ture of the lesion is a new approach in Doppler analysis, different from spectral waveform analysis; it may possibly add new determinants of biological nature of breast lesions. Further prospective studies on larger patient population are required. 3. When combined with, and in addition to mammography and clinical examination, HRUS and CDE increase accuracy of preinvasive differentiation of solid breast nodules. References 1. Feig SA. Breast masses: mammographic and sono­graphic evaluation. Rad Cli11 Norih Am 1992; 30: 67-92. 2. Cilotti A, Bagnolesi P, Moretti, Gibilisco G, Bulleri A, Macaluso AM, et al. Comparison of the diag­nostic performance of high-frequency ultrasound as a first-or second-line diagnostic tool in non­palpable lesions of the breast. Eur Radio/ 1997; 7: 1240-4. 3. Folkman J. 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Quantifi­cation of tumor vascularity and flow with ampli­tude color Doppler sonography in an experimen­tal model: preliminary results. J U/trnso1111d Med J 996; 15: 827-33. 10. Rubin JM, Bude RO, Carlson PL, Bree RL, Adler RS. Power Doppler ultrasound: a potentially use­ful alternative to mean frequency-based color Doppler ultrasound. Radiologij 1994; 190: 853-6. 11. Bude RO, Rubin JM, Adler RS. Power versus con­ventional color Doppler sonography: comparison in the depiction of normal intrarenal vasculature. Radiology 1994; 192: 777-80. 12. Bude RO, Rubin JM. Power Doppler sonography. Radiology 1996; 200: 21-3. 13. Jackson VP. The role of US in breast imaging. Radiology 1990; 177: 305-11. 14. Harris JR, Lippman ME, Morrow M, Hellman S. Diseases oj the breas/. Hagerstown: Lippincott­Raven; 1996. p. 71-84. 15. Stavros AT, Thickman D, Rapp CL, Dennis MA, Parker SH, Sisney GA. Solid breast nodules: Use of sonography to distinguish between benign and malignant lesions. Radiology 1995; 196: 123-34. 16. Schlecht !, Hadijuana J, Hosten N, Oellinger H, Minguillon C, Lichtenegger W, et al. Ultraschal­luntersuchung der weiblichen brust: Vergleich von 7,5 und 13 MHz. Akt Radio/ 1996; 6: 69-73. 17. Schnarkowski P, Schmidt D, Milz P, KeB!er M, Reiser M. Vergleich zwichen herkommlicher und hochauflosender sonographie fiir die diagnostik von mammatumoren. Ultrnsha/1 Med 1996; 17: 190-4. 18. Sardanelli F, lmperiale A, Zandrino F, Calabrese M, Bonifacio A, Canavese G, et al. Breast intraduc­tal masses: US-guided fine-needle aspiration after galactography. Radiology 1997; 204: 143-8. 19. Cosgrove DO, Kedar RP, Bamber JC, et al. Breast diseases: color Doppler US in differential diagno­sis. Radiology 1993; 189: 99-104. 20. Birdwell RL, Ikeda DM, Jeffrey SS, Jefrey RB Jr. Preliminary experience with power Doppler imag­ing of solid breast masses. A111 j Radio/ 1997; 169: 703-7. 21. Folkman J, Watson K, lngber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989; 339: 58-61. 22. Brem S, Jensen H, Gullino P. Angiogenesis as a marker of preneoplastic lesions of the human breast. Ca11cer 1978; 41: 239-44. Radio/ Oncol 1998; 32(4): 363-5. Hypertrophic pyloric stenosis: Ultrasound diagnosis Goran Roic, Slavko Cop, Vesna Posaric, Suzana Ercegovic Department oj Radiology, Children's Hospital Zagreb, Croatia In the study, we analysed ultrasound (US) jindings oj 68 patients suspected oj having hypertrophic pyloric stenosis (HPS). In 44 out oj 45 patients with posilive US jindings, HPS was surgically conjirmed. In 82.6% patients (38 pts), pyloric muscle thickness was 5-7 111111, in 10.9% (5 pts), it was 3-5 mm, and only in 6.4% patients (3 pts), we perjormed barium study due to doubtjul values oj pyloric musc/e thickness (3.0­ 3.4 mm) detected by US; barium study conjirmed HPS in one patient and exc/uded in two palients. The pyloric musc/e thiclcness in ali patients with HPS was at least 1.5 times thicker comparing to antra/ muscle thickness. Ultrasound sensitivity was 98%, and specijicity 92%. Based on the results oj our study, ultra­sound examination oj the pylorus has proved to be highly recommendable as a routine method oj jirst choice in the diagnosis oj HPS. Key words : pyloric stenosis-ultrasonography; hyperthrophy Introduction With the advent of high resolution real-tirne ultrasound scanners, ultrasound has become a very important imaging technique, not only in the diagnosis of solid abdominal organ pathology, but also in the evaluation of hol­low gastrointestinal tract. t2 Hypertrophic pyloric stenosis is the best recognized use of ultrasound in the pediatric gastrointestinal tract.1, 3 Ultrasound has the advantage of pro­viding direct visualization of the pyloric mus­cle, and it also allows the measurements of muscle thickness.4-6 The ultrasonographic criteria for diagnosing pyloric stenosis vary from hospital to hospital, but most common Correspondence to: Dr Goran Roic, Department of Radiology, Children s Hospital Zagreb, Klaiceva 16, 10000 Zagreb, Croatia. belief is that pyloric muscle thickness of 3.5 mm or more and a pyloric canal length of 17.0 mm or more are diagnostic for HPS.2,7,8 Patients and methods In our study, we reviewed the findings of US performed in our hospital from 1990 to 1996 of 68 patients suspected of having HPS. The equipment used was ALOKA 1700 i ACUSON 128 XP, using curved and linear array transducers of 5 i 7-7.5 MHz. No partic­ular preparation of patients was needed. If the stomach was empty, patients were given fluid (tea or water) by bottle to allow ade­quate examination. In all patients transverse and longitudinal sonograms were made in a supine right posterior oblique position of the 364 RoicG et ni. p_atient's body aided by a rolled towel under his left side. We measured the outer diameter of the pylorus and thickness of the pyloric muscle itself on both, transverse and longitu­dinal sonograms, as well as pyloric canal length on longitudinal sonogram. We also considered the ratio between pyloric and antral muscle thickness. The measurements of antral muscle thickness were obtained on long axis seans with the antrum distended by fluid to avoid confusing a contracted antrum with an abnormally thickened muscle. The antral muscle thickness was measured from the outer border of the echoic submucosa to the outer border of hypoechoic muscle layer. No tirne limits for the examination were imposed, and diagnostic criteria of HPS called for the following criterion: 1. pyloric visualization, 2. pyloric muscle thickness > 3.5 mm, 3. pyloric canal length > 17.0 mm. Results During the period of 6 years, 68 patients sus­pected of having HPS were ultrasonographi­cally examined; of these, we established 45 diagnoses of HPS all of which, except one /false-negative/, were surgically confirmed. In 82.6% patients (38 pts) the diameter of the pyloric muscle was 5.0-6.0 mm, in 10.9% patients (5 pts) it was 3.5-4.0 mm and in 6.5% patients (3 pts) the diameter of the muscle was 3.0-3.4 mm. In all examined patients, the length of the pyloric canal exceeded 17.0 mm. In all patients, the pyloric muscle thickness was at least 1.5 times thicker than the antral muscle thickness. Only 3 patients underwent radiology, i.e. barium study, because of the borderline pyloric muscle thickness values of 3.0-3.4 mm. Two of these patients were false-positive and one false-negative (confirmed by barium study). Sensitivity of the US examination was 98 %, and specificity 92 %. Discussion The pylorus region can be easily located by US, specially when there is a positive finding of HPS. The gallblader is used as a landmark to locate the region of the pylorus. According to our experience, it is harder to \ocate nor­mal pylorus and exclude the diagnosis of HPS. In our hospital, we first locate the pylorus with a curved transducer of 5 MHz and then, for more detailed examination of pyloric region and the subsequent measure­ments, we use a linear array transducer of 7.0-7.5 MHz. Although the most important diagnostic criterion for HPS is the length of the pyloric canal 9-11, it is not always possible to show it in its entire length due to its awkward posi­tion. Problems in diagnosis may arise if the stomach is overdistended, because the pylorus may then be displaced which makes its identification and measurements more difficult.1•12 Thus, we believe that the thickness of the pyloric muscle measured on transverse and longitudinal sonograms is a better criterion for diagnosing HPS (Figure 1,2). It is also use­ful to follow gastric outlet ultrasonographical­ly. In order to be more accurate in diagnosing HPS, the thickness of antral muscle can be measured and compared with the pyloric muscle thickness. Normal pyloric muscle is usually <2 mm thick, and thickness between 2 and 3 mm may be seen in pylorospasm.13 In all patients with HPS in our study, the pyloric muscle thickness was at least 1.5 times thicker than the antral muscle thick­ness, ranging between 1.4 and 2.3 mm. According to our experience, the pyloric muscle thickness of the patients with HPS is in most cases 5.0-7.0 mm and in fewer cases 3.0 -3.4 mm. In doubtful muscle thickness values (3.0-3.4 mm), some other diagnostic criteria should be considered (the length of the pyloric canal) and, in other doubtful cases, the patient should be reexamined in Hy„erlrophic pyloric slenosis Figure 1. HPS-short-axis scan -central echogenic mucosa of pyloric canal surrounded by thickened pyloric muscle. Figure 2. 1-IPS-long-axis scan-abnormally thickened hypoechoic pyloric muscle (6mm). 24-48 hours or other diagnostic procedures should be perforrned (barium study). In conclusion, the real real-tirne ultra­sound is currently a method of choice; it is safe, painless diagnostic imaging technique for the diagnosis of HPS and, in experienced hands, it can almost completely replace con­ventional barium studies. The barium study should be reserved for those cases in which HPS is not considered the most likely cause of vomiting and in children with borderline measurements. References 1. Siegel MJ. Gastrointestinal tract. In: Siegel MJ, editor. Pedinlric sonogrnplzy. New York: Raven Press; 1985. p. 266-8. 2. Hayden CK Jr. Gastrointestinal tract. In: Babcock DS, editor. Neona/ni nnd pediatric sonogrnplzy. New York: Churchill Livingstone; 1989. p. 77-101. 3. Buonomo C, Taylor GA, Share JC, Kirks DR. Gas­trointestinal tract. ln: Practical pediatric imaging. New York: Lippincott-Raven 1998; p. 821-1007. 4. 4.Graif M, ltzchak Y, Avigad I, Strauss S. Ben-Arni T. The pylorus in infancy: overall sonographic assessment. Pediatr Radio/ 1984; 14: 14-7. 5. Haller JO, Cohen HL. I-lypertrophic pyloric steno­sis: diagnosis using US. Radiology 1986; 161: 335-9. 6. Keller 1-!waldmann D, Greiner P. Cornparison of preoperative sonography with intraoperative find­ings in congenital hypertrophic stenosis. J Pediatr Surg 1987; 22: 950-2. 7. Kovalivker M, Erez !, Shnider N, Glazer E, Lazar L. The value of ultrasound in the diagnosis of con­genital hypertrofic pyloric stenosis. Ciin Pediatr 1993; 32(5): 281-3. 8. Blumhagen JD, Maclin L, Krauter D, Rosenbaum DM, Weinberger E. Sonographic diagnosis of hypertrophic pyloric stenosis. AJR 1988; 150: 1367-70. 9. Blumhagen JD. The role of ultrasonography in the evaluation of vomiting in infants. Pediatr Radio/ 1986; 16: 267-70. 10. Blumhagen JD, Weinberger E. Pediatric gastroin­testinal ultrasonography. In: Llltraso1111d mmunl. New York: Raven Press; 1986. p. 99-140. 11. Stunden RJ, Le Quesne GW, Little KE. The improved ultrasound diagnosis of hypertrofic pyloric stenosis. Pediatr Radio/ 1986;16(3): 200-5. 12. Sawischuk LE, 1-!ayden CK Jr, Stansberry SD. Sonographic pitfalls in imaging of the antropyloric region in infants. Radiographics 1989; 9: 437-47. 13. O Keeffe FN, Stansberry SD, Swischuk LE, J-Jayden CK Jr. Antropyloric muscle thickness at US in infants: what is normal? Radiology 1991; 178: 827-30. Radio/ 011col l998; 32(4): 367-71. review Palliation of painful osseous metastases in patients with prostate cancer using Re-186-HEDP Susanne Klutmann, Karl H. Bohuslavizki, Ulrike Schulte, Sabine Kroger, Christian Bleckmann, Janos Mester, Malte Clausen Department oj Nuclear Medicine, Llniversity Hospital Eppendorf, Germany The skeleton is the second most common site oj metastases in patients with prostate cancer. While curative means are strongly limited in these patients their lije expectancy 111ay be stili severa/ years. Therejore, it is essential to improve quality oj lije of these patients. Sufficient therapy of painful osseous metastases is the main goal in patients with advanced prostate cancer. The primary approach to relieve bone pain is the application of peripheral or central analgesics. In case oj bone pain due to a single metastatic site local external beam radiation may provide pain relief in a reasonable amount of patients. In case of painful mul­tilocular metastases systemic application of radiopharmaceuticals may irradiate bone metastases while nor­mal tissue is spared from [3-irradiation. Due to their physical characteristics Re-186 and Sm-153 have been developed for palliative treatment of metastatic bone pain. The response rate amounts to about 70-80% oj ali patients treated. Pain relief may last for 1-6 months. Due to its /ow grade toxicity which is mainly domi­nated by a transient thrombocytopenia therapy can be repeated. Howeve1; Re-186-HEDP therapy does not alter lije expectancy. Key words: prostatic neoplasms; bone neoplasms-secondary; pain-therapy; rhenium, radioisotopes, Re-186-HEDP lntroduction Prostate cancer is the second most common malignancy in men in Western Europe. The incidence is 15-16 per 100000 habitants per year with increasing tendency. As much as 80% of patients with prostate cancer will develop bone metastases.1 In about one third Correspondence to: Dr. Karl H. Bohuslavizki, Depart­ment of Nuclear Medicine, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Ger­many. Phone: +49 40 4717 4047; Fax: +49 40 4717 6775. E-mail: bohu@medsph2.uke.uni-hamburg.de of ali patients osseous metastases are detect­ed at primary staging. Moreover, the skeleton is the only single site of metastases in a rea­sonable amount of patients.2 In case of multi­locular osseous metastases a complete remis­sion of prostate cancer is nearly impossible. Since osseous metastases are often associ­ated with bone pain effective pain relief is the primary goal when caring for patients with prostate cancer and multiple osseous metas­tases.3 Traditional therapeutic approach is the application of central or peripheral anal­gesics in combination with neuroleptics.4 368 Klut//lm111 Sel ni. Moreover, steroid medication, diphospho­nates, and hormona! drugs may complete analgesics effects. However, therapy with opioids is limited in many patients due to side-effects, i.e. nausea, vomitus and gas­trointestinal symptoms5 and thus, often asso­ciated with a loss of patient's quality of life.6 Skeletal pain confined to single site metas­tases usually responds to external beam radiotherapy in 70-80 %.4, 7 , 8 In case of multi­locular osseous metastases external beam radiation is helpful to avoid pathologic frac­tures or compression of the spinal cord.9, 10 However, hemibody or whole-body irradia­tion for pain relief is often limited by bone marrow suppression, gastrointestinal symp­toms and a radiation pneumonitis.11, 12 There­fore, an effective relief of pain with low side­effects and an improvement in patient's qual­ity of life is warranted in these patients. Osteotropic radionuclides The application of .-emitting osteotropic radionuclides is a promising method to selectively irradiate osseous metastases by sparing normal tissue from short-range irra­diation.13 Due to the osteoblastic character of osseous metastases the radionuclide is predominantly accumulated in malignant transformed cells which leads to a selective irradiation of bone metastases. The first agent used for this purpose, P-32-orthophos­phate, was replaced by Sr-89-chloride due to its severe bone marrow toxicity. Up to now Sr-89 is still the most commonly used agent for osteotropic radionuclide therapy.14 Sr-89 has a long physical half-life of 50,5 days with a maximum .-energy of 1,49 MeV (Table 1). Pain relief may occur 2-3 weeks after sys­temic application of 1,5-2,0 MBq/kg body­weight. However, Sr-89 has no y-emission and thus, posttherapeutic scintigraphy imag­ing is not feasible. Therefore, the aim of research was to develop alternative radianu­clides for palliative treatment of painful osseous metastases. Rhenium-186-HEDP Re-186-hydroxyethylidendiphosphonate as well as Sm-153 (Table 1) have recently been developed for the palliative treatment of painful osseous metastases.15 Re-186 has a therapeutic .-emission of 1,07 MeV associat­ed with a y-emission of 137 keV. Moreover, Re-186-HEDP and Tc-99m-HDP, that is com­monly used for diagnostic bone scintigraphy, have an almost exactly similar bone distribu­tion since both sorts of diphosphonates bridge to the hydroxyapatite of bone sub­stance. Therefore, pretherapeutic and post­therapeutic scintigraphy is possible which allows a control of Re-186 distribution as shown in Figure l. Re-186 has a short physi­cal half-life of 3,8 days when compared to Sr­ 89. This allows a single application of activi­ties of 1110 to 1850 MBq16, 17 with high tumor doses as well as an easy handling of radioac­tive waste, i.e. urine. About 50 % of the activity injected are excreted via the kidneys into the urine within the first 6 hours post application. Nearly 70 % of the activity are urinary eliminated within the first 24 hours post application. Apart from the distribution in osseous structures Re-186-HEDP is not accumulated in any other structures of the body. Pain relief is attained within two weeks after application of Re-186-HEDP and lasts for about 1-6 months. Response rates of Re-186-HEDP therapy of 70-80 % have been reported.2 , 17 , 18 Especially in patients with oral medication of non-opioids analgesics rheni­um-therapy led either to a reduction or to a stop of taking oral drug medication. Thus, the requirement for central analgesics may be delayed. Moreover, it is known that the clini­cal response is influenced by the size of osseous metastases. Pain relief mainly occurs Palliation of pai11ful osseous meatas/ases Table l. Characters of different radionuclides used for treatment of painful osseous metases P-32 Sr-89 Re-186 Sm-153 Physical half-life [ d} 14.3 50.5 3.8 P-emission [ke V] 1710 1490 1070 810 y-emission [keV] 0 0 137 103 Tracer phosphate chloride HEDP EDTMP Scintigraphicimaging 0 0 possible possible Table 2. lnclusion criteria of patienls for Re-186-HEDP therapy 20 Four or more osseous metastatic sites (al last one single site presents with bone pain) No diphosphonate therapy within 12 weeks No irradiation within 3 weeks No chemotherapy within 6 weeks No change of dosis of hormone therapy within 8 weeks Thrombocytes > 150000/µl Leukocytes > 4000/µl Creatinine < 1,3 mg/dl No clinical sign of cerebral involvement No heart insufficiency NYHA IV Karnofsky-Index > 70 % Life expectancy > 12 weeks No leve! III oder leve! IV toxicity of previous rhenium-therapy (only in case of re-treatment) in patients with small or medium-sized metastases, whereas large metastases with soft tissue infiltration often do not respond to radionuclide therapy.2 Therefore, the applica­tion of bone-seeking radiopharmaceuticals is a treatment option to early complete oral drug therapy. Due to the short physical half­life of Re-186 the treatment can be repeated after 4-6 months. Side-effects The main radiobiological side-effect of bone seeking radionuclides is their potential bone marrow toxicity. In contrast to Sr-89 which is associated with a prolonged bone marrow suppression, Re-186 has a relatively mild hematological toxicity. Thrombocytopenia plays the major role in its bone marrow sup­pressing effect. The decline of thrombocytes presents with a nadir about 3 weeks post application. Prior to treatment the decrease of platelet count can be estimated for an indi­vidual patient presenting for rhenium-thera­py.19 Thus, severe hematological side-effects can successfully be avoided. In general, a control of platelet counts in a two-week inter­val for the duration of two months is suffi­cient in posttherapeutic follow-up. Patient management Severa! days before the therapeutic admin­istration of Re-186-HEDP the patients under­go conventional bone scintigraphy with labeled diphosphonates, e.g. 600 MBq Tc-99m-HDP. In case of multilocular, osseous metastases with at least four metastatic sites and at least one single painful lesion rheni­um-therapy is indicated if the patient fulfills inclusion criteria20 as given in Table 2. Due to its potential bone marrow suppression 370 Klut111n1111 Set ni. Bone scintigraphyn Rhenium-therapy anterior posterior anterior posterior Figure l. 64-year-old patient with rnultilocular bone rnetastases of a prirnary prostate cancer. The patient clairned about pain of the left fernur and both scapulas. Left scintigrarn: Pretherapeutic conventional whole-body bone scintigraphy 3 hrs after application of 600 MBq Tc-99rn-HDP i.v. Note tracer accurnlations of the skull, the ribs, the sternurn, the bassin, both proximal fernurs, and the spina! colurnn corresponding to sites of osseous rnetastases. Right scintigrarn: Posttherapeutic whole-body scintigraphy 48 hrs after application of 1.3GBq Re-186-HEDP i.v. Note distribution of Re-186-HEDP corresponds to osseous metastatic sites. patients with thrombocytes below 150000/µl have to be excluded from therapy. In clinical routine, the blood count is defined directly prior to rhenium-application. If there are no contraindications, 1.3 GBq Re-186-HEDP with a total volume of about 2 ml are admin­istered via an intravenous line. 48 hrs post app!ication a whole-body scintigraphy with a scan-speed of 6 cm/min is obtained in order to evaluate the distribution of the bone-seek­ing Re-186-HEDP. Conclusion Palliative Re-186-HEDP therapy of multilocu­lar, painful osseous metastases in patients with prostate cancer is a sufficient therapeu­tic modality for pain alleviation with low tox­icity, thereby increasing patient's quality of life. However, life expectancy will not be affected. References 1. Jacobs SC. Spread of prostatic cancer to bone. Urology 1983; 21: 337 -44. 2. Palrnedo H, Bender H, Schomburg A, Griinwald F, Schoneich G, Zarnorra P, Reichrnann K, Dierke­Dzierzon C, Mallmann P, Biersack HJ. Schrnerztherapie mit Rhenium-186 HEDP bei multiplen Knochenmetastasen. Nucl Med 1996; 35: 63-7. 3. Quirijnen JMSP, Han SH, Zonnenberg BA, de Klerk JMH, van het Schip AD, van Dijk A, ten Kroode HFJ, Blijham GH, van Rijk PP. Efficacy of Palliatio11 of pain Jul osseous 111eatastases Rhenium-186-etidronate in prostate cancer patients with metastatic bone pain. J Nucl Med 1996; 37: 1511-5. 4. Schi:ineich G, Palmedo H, Heimbach D, Biersack HJ, Miiller SC. Advanced prostate cancer: sys­temic radiopharmaceutical therapy of metastatic prostate cancer with Rhenium-186 hydroxyethyli­dene diphosphonate. Onkologie 1997; 20: 316-9. 5. Schag CA, Cranz PA, Dim DS, Sim MS, Lee JJ. Quality of life in adull survivors of lung, colon and prostate cancer. Qual Lije Res 1994; 3: 127-41. 6. Schi:ineich G, Palmedo H, Dierke-Dzieron C, Miiller SC, Biersack HJ. Rhenium-186 HEDP: pal­liative radionuclide therapy of painful metastases. Preliminary results. Scand J Ural Neplzrol 1997; 31: 445-8. 7. Gilbert HA, Kagan AR, Nussbaum H, Rao AR, Satzman J, Chan P, Forsythe A. Evaluation of radi­ation therapy for bone metastases: pain relief and quality of life. Am J Roe11tge110l 1977; 129: 1095-6. 8. Hendrickson FR, Shehata WM, Kirchner AB. Radiation therapy for osseous metastasis. Int ] Radiat Oncol Biol Phys 1976; 1: 275-8. 9. Hoskin PJ. Radiotherapy in the management of bone metastases. In: Rubens RD, Fogelman I edi­tors. Bone metas/ases: diagnosis and trealment. Lon­don: Springer-Verlag 1991. p. 171-85. 10. Poulsen HS, Nielsen OS, Klee M, Rort M. Pallia­tive irradiation of bone metastases. Cancer Treat Rev 1989; 16: 41-8. 11. Perez CA, Cosmatos D, Carcia DM, Eisbruch AE, Poulter CA. Irradiation in relapsing carcinoma of the prostate. Cancer 1993; 71: 1110-22. 12. De Klerk JMH, Zonnenberg BA, van het Schip AD, van Dijk A, Han SH, Quirijen JMSP, Blijham GH, van Rijk PP. Dose escalation study of Rheni­um-186 hydroxyethylidene diphosphonate in patients with metastasic prostate cancer. Eur J Nucl Med 1994; 21: 1114-20. 13. De Klerk JMH, van Dieren EB, van het Schip AD, Hoekstra A, Zonnenberg BA, van Dijk A, Rutgers DH, Blijham GH, van Rijk PP. Bone marrow absorbed dose of Rhenium-186-HEDP and the relationship with decreased platelet counts. J Nucl Med 1996; 37: 38-41. 14. Blake GM, Zivanovic MA, McEwan AJ, Ackery DM. Strontium-89 therapy: strontium kinetics in disseminated carcinoma of the prostate. Eur J Nucl Med 1986; 12: 447-54. 15. Collins C, Eary JF, Donaldson G, Vernon C, Bush NE, Petersdorf J, Linvingston RB, Gordan EE, Chapman CR, Appelbaum FR. Samarium-53­EDTMP in bone metastases of hormone refractory prostate carcinoma: A phase !/II tria!. J Nucl Med 1993; 34: 1839-44. 16. Maxon HR, Schroder LE, Hertzberg VS. Rhenium­186 HEDP for treatment of painful osseous metas­tases. Results of a double blind cross over compar­ison with placebo. J Nucl Med 1991; 19: 1877-81. 17. Maxon HR, Thomas SR, Hertzberg VS: Rhenium­186 HEDP for treatment of painful osseous metas­tases. Semin Nucl Med 1992; 22: 33-40. 18. Zonnenberg BA, De Klerk HJM, van Rijk PP. Re­186 HEDP for treatment of painful bone metas­tases in patients with metastatic prostate or breast cancer. J Nucl Med 1991; 32: 1082-91. 19. De Klerk JMH, van het Schip AD, Zonnenberg BA, van Dijk A, Stokkel MPM, Han SH, Blijham GH, van Rij PP. Evaluation of thrombocytopenia in patients treated with Rhenium-186 HEDP: Guidelines for individual dosage recommenda­tions. J Nuc/ Med 1994; 35: 1423-8. 20. Halle L-H, Humke U, Trampert L, Ziegler M, Kirsch CM, Oberhausen E. Palliative Schmerzther­apie beim ossar metastasierten Prostatakarzinom mit dem osteotropen .-Stahler 186 Rhenium­Hydroxyethylidendiphosphonat (HEDP). Urologe 1997; 36: 540-7. Radio/ Onco/ 1998; 32(4): 373-83. rev1ew p53 -the paradigm of tumor-suppressor genes? Barbara Jezeršek and Srdjan Novakovic Institute oj Oncology, Ljubljana, Slovenia p53 is a tumor-suppressor gene the alterations oj which are among the most jrequent genetic changes detected in human neoplasms. Its product -p53 protein is a component oj severa/ biochemica/ pathways that are central to carcinogenesis: DNA transcription, genomic stability, DNA repair, celi cycle control, and apoptosis. The analysis oj the spectrum oj p53 mutations and insight into the p53 mediated biochemical pathways oj prograrmned celi death and celi cycle arrest, provide clues to the understanding oj molecular pathogenesis oj cancer and of mechanisms related to p53 mediated tumor suppression. The purpose oj the present article is to summarise the most important facts concerning p53 since understanding of the above listed processes might provide the potential molecular targets for the development of a rational cancer treat­ment. Key words: neoplasms; genes, suppresso1; tumor; genes, p53; protein p53 Historical background In 1979, Lane and Crawford, 1 as well as Linz­er and Levine2 independently discovered p53 as a nuclear 53kd phosphoprotein tightly associated with the large T antigen in the SV 40 tumor virus-transformed cells. Origi­nally, p53 protein came to be classified as a tumor antigen since it was suggested that the interaction of p53 with the large T antigen was important for transformation.1, 2 The p53 cDNA constructs isolated in this period were ali derived from tumor cells3 and were found to cooperate with the ras oncogene to trans­ 5 form rat fibroblasts in cell culture.4, So, p53 came to be classified as an oncogene. Finally, in late 1980s, ali the transfonning p53 cDNA clones were discovered to be mutant fonns of p53, while the wild-type gene isolated from normal cells failed to induce neoplastic trans­formation and even inhibited tumor celi growth or blocked the neoplastic transforma­tion. 6-10 Now p53 looks like being a tumor­suppressor gene, negatively regulating the celi cycle and requiring loss-of-function mutations for tumor formation. However, unlike other classical tumor-suppressor genes, at least some mutated p53 forms act as dominant transforming oncogenes.11 Structure and regulation Correspondence to: Barbara Jezeršek, Dept. of Tumor The p53 gene spans a moderately-sized seg­ Biology, Institute of Oncology, Zaloška 2, 1000 Ljubl­ ment of DNA, located on the short arm jana, Slovenia, Tei: +38661 323 063 ext. 29 33, Fax: +38661 1314 180; E-mail: snovakovic@onko-i.si (17p13) of chromosome 17 and is ultimately Jezeršek B and Novakovic S translated to a phosphoprotein consisting of 393 amino acids contained in 11 exons, the first of which is noncoding.12 Five evolution­ary conserved domains within the coding regions are supposed to be essential to the functional activity of p53.12 , 13 The N-terminal domain (residues 1-42) interacts with the subunits of the general transcription factors TFIID and TFIIH and acts as a transcriptional activator. This domain also binds the MDM-2 protein -a negative regulator of p53, and adenovirus ElB protein. The core domain (residues 100­ 300) harbors the sequence specific dsDNA binding function of p53, and encodes the binding site for SV4O large T antigen and, possibly, for the papillomavirus E6 protein. The C terminal domain (residues 300-393) has multiple functional activities, including nonspecific DNA binding and reannealing of complementary ssDNA oligonucleotides. Residues 320 to 355 are involved in oligomer­ization, and the very terminal C domain (residues 360-393) binds ssDNA ends and regulates specific DNA binding by the core domain.14-18 It appears that p53 alone assembles into inactive forms and requires activating factors to confer an effective sequence-specific DNA binding capacity. Such a regulation is exerted by the C-terminal end of p53 itself. Hupp et al. proposed a model according to which the C-terminus negatively regulated specific DNA binding by interacting with a region in another p53 molecule within the tetramer.19 This locked the tetramer in a conformation that was incapable of specific DNA binding. p53 contains multiple phosphorylation sites located at both the C and N-termini of the molecule. Eight different protein kinases are involved in p53 phosphorylation: p34cdc2 kinase, DNA-activated protein kinase, mitogen activated protein kinases, protein kinase C, casein kinase I and II, Raf-1 kinase, and Jun kinase.20-26 p34cdc2 kinase (an A-and B-cyclin dependent kinase) phos­phorylates at serine 315 and thus stimulates the specific binding of DNA to the consensus sequence of p53 and also causes a specific conformational change of the protein.20 DNA-activated protein kinase and mitogen activated protein kinases are involved in the phosphorylation of p53 at the N terminal domain, influencing the transcriptional activ­ity and the half-life of the protein.21, 22 Protein kinase C-dependent, direct or indirect, phos­phorylation of serine residues 372-381 at the C terminal of the p53 tetramer is a critical event for the transition from the latent to the active form of p53. Namely, the "open" con­figuration of the four phosphorylated C ends of the tetramer is a necessary prerequisite for the nonspecific DNA binding which, in turn, allows the consequent specific DNA binding 23 to p53 consensus motifs.19, , 27 The phospho­rylation of the serine 392 is dependent on casein kinase II, however, this site is less crit­ical for p53 activation.28 Another form of p53 regulation is exerted at the leve! of p53 protein stability. In normal cells, p53 shows a relatively short half-life (about 20 minutes) due to its rapid turnover, yet its half-life can be extended to hours fol­lowing some kinds of cellular stress or as a consequence of mutations involving the core domain. Stability of the protein is affected by its complex formation with a number of cell proteins that are capable of slowing-down or preventing its ubiquitin-pathway degrada­ tion.29, 30 It is still uncertain which physiological sig­nal activates p53 after an appropriate stimu­lus. Possible candidates are p3OO and the closely related transcription factor CBP that bind to N-terminal domain of p53. p3OO acetylates conserved lysine residues in the p53 C-terminal domain which results in the activation of specific DNA binding of p53_31 32 , Intracellular localisation, concentration, and state of phosphorylation of p53 are cell­cycle dependent. Activity of wild-type p53 p53 s11ppressar gene protein demands nuclear localisation of the protein which occurs close to the beginning of S phase. Following the beginning of DNA synthesis, p53 accumulates again in the cyto­plasm. 33 , 34 Function p53 protein is implicated in nearly ali forms of celi growth stimulation and inhibition. It may be required early in the induction of celi proliferation and is also a transcriptional reg­ulatory protein, capable of both stimulating and repressing gene expression. 35 p53 binds DNA in a sequence-specific manner and also influences gene expression indirectly by interacting with other transcription fac­tors.35 , 36 In certain celi types, over-expression 37 38 of p53 induces apoptosis.23 , , p53 may reg­ulate in vitro cellular senescence and, under the influence of certain cytokines, it cooper­ates in the induction of differentiation.39AO Transcription dependent pathway Severa! genes were found to be transcription­ally activated by p53, including MDM-2, p21, GADD45, cyclin Gl, BAX, FAS, transforming growth factor-alpha, muscle kreatinine kinase, and insulin-like growth factor-bind­ing protein 3.10A1-48 Following DNA damage, p53 protein rapidly accumulates in the nucleus. The C­terminal domain of p53 recognises the dam­aged DNA, and the accumulation of p53 is probably a consequence of conformational change of the protein which leads to reduced degradation by ubiquitin degradation path­way or, less likely, a consequence of increased synthesis of p53 protein.33 , 38A9 At the same tirne, no changes in p53 mRNA lev­els are observed.50 The p53 protein, in turn, activates down­stream genes whose products are involved in growth inhibition, e.g. p21 and GADD45. p21 is a cyclin-dependent kinase inhibitor that inhibits the activity of cyclin D-cdk 4/6 causing a hypophosphorylation of retinoblas­toma protein (Rb), thus preventing the release of E2F and blocking the Gl-S transi­tion.51,52 Transactivation of GADD45, the protein product of which binds proliferating cell nuclear antigen and inhibits S phase entry, may contribute to the p53 dependent cell cycle arrest pathway.53 Insulin-like growth factor-binding protein 3 gene which encodes a protein that binds insulin-like growth factor and thus inhibits its growth signalling is another p53 target gene that may function in this pathway.48 p53 also regulates the G2/M checkpoint of the celi cycle, yet the mechanism of p53 mediated G2/M control is 55 unknown. 54, The expression of MDM-2 protein is regu­lated by the leve! of wild-type p53 protein. The MDM-2 protein, in turn, forms a com­plex with p53 and decreases its ability to act as a positive transcription factor -which rep­resents a negative feedback loop to buffer changes in p53 levels.56, 57 Transcription independent pathway Modulation of cellular processes goes often via the mechanism of protein-protein interac­tions. In agreement with its multifunctional qualities, p53 protein associates with a group of vira! and cellular proteins that may play an important role in the p53 mediated and tran­ 36 scription independent pathway (Table 1).35 , Severa! basic transcription factors, includ­ing TATA binding protein, TATA binding pro­tein-associated proteins, TFIIH-associated factor p62 form a complex with p53.58-61 Binding of TATA binding protein to p53 pro­tein has been implicated to be responsible for p53 mediated transcriptional repression. The list of genes reported to be transrepressed by p53 consists of proliferating celi nuclear anti­gen, interleukin 6, Rb gene, multidrug-resi­tance (MDR) gene, p53, BCL-2, inducible Jezeršek B and Novakovi<' S Table l. Some of vira! and cellural proteins that associate with p53 Vira! proteins Cellular proteins human papilloma virus E6 heat-shock protein 70 simian virus 40 T antigen MDM-2 Epstein-Barr nuclear antigen ubiquitin-ligase E6-AP adenovirus ElB transcription factor WT-1 ni trie oxide synthase-2. 62-67 The binding of p53 to replication protein A also alludes to the possible direct role of p53 in DNA repli­cation and nucleotide excision repair.68 p53 in the nucleotide excision DNA repair The observations that p53 can selectively bind to severa! DNA helicases, including XPB and XPD, which are a part of transcription factor TFIIH, led to the hypothesis that p53 may play a direct role in modulating DNA nucleotide excision repair. 69, 7° Furthermore, p53 can also recognise severa! forms of dam­aged DNA (mismatched DNA, ssDNA ends).71 So, a new model emerged in which p53 may act as a sensor that binds to dam­aged parts and recruits the nucleotide exci­sion repair machinery by trapping TFIIH (i.e. the major component of the repair complex) at regions where it is needed which, in turn, facilitates the constitution of a functional "repairosome". 72 p53 mediated apoptosis The molecular mechanisms behind p53 induced apoptosis are only partially explained. The current idea is that DNA dam­age induces stabilization of the p53 protein which promotes DNA repair by assembling 71 the repair machinery.70, In case the DNA damage is unrepairable, p53 triggers cells to undergo apoptotic death to prevent propaga­tion of the cells carrying a mutation. Severa! activities of p53 have been identified that could participate in the process of pro­grammed cell death. Namely, p53 upregulates the expression of BAX and downregulates expression of BCL-2, all of which have been implicated in modulation of apoptosis.73 Another possible explanation far the induc­tion of apoptosis could be that the transacti­vation of insulin-like growth factor-binding protein 3 gene and thus increased insulin-like growth factor-binding protein 3 levels may presumably block an insulin-like growth fac­tor mediated survival signal and lead to apop­tosis.48 Finally, a whole series of new p53 induced genes related to redox control have been discovered that lead to the formation of reactive oxygen species, oxidative degradation of mitochondrial components and apoptotic cell death. 74 Beyond this, a transactivation independent function of p53 in the triggering of the apoptotic pathway has been implicated and may well be performed by a proline rich region located between residues 64 and 91 in p53 molecule. The proline rich region may provide a crucial accessory apoptotic signal, perhaps by interacting with a cellular SH3­domain-containing partner protein.75, 76 Briefly, the major role of p53 is being a monitor of cellular proliferation (guardian of the genome) and a determinant of response to DNA damage. Mutations The p53 mutations are found in the prepon­derance of human tumors and the functional p53 is last in approximately half of all human 77 malignancies. 52, The majority of p53 mutations are mis­sense point mutations giving rise to single p53 suppressor gene amino acid substitutions that abrogate the specific DNA binding activity.18 Concomi­tantly, the half-life of p53 extends from nor­mal 20 minutes (wild-type protein) to approx­imately 48 hours (mutant protein) resulting in nuclear accumulation of the mutant pro­tein.78 , 79 Most of the mutations are clustered in the most highly conserved domains of the gene spanned by four to nine exons. There are at least three mutation "hot spots" affecting the residues 175, 248, and 273.80 Although muta­tions of the p53 gene are most frequently acquired, they can also be inherited through the Li-Fraumeni syndrome. In these families, one mutant p53 allele is inherited, and the second allele acquires a mutation.81 p53 is not inactivated only through muta­tion, but also at the protein leve! through complexing with DNA tumor vira! oncopro­teins like the SV4O large T antigen, the aden­ovirus ElB protein, and the human papilloma virus E6 protein82 or cellular protein MDM­ 57 2. Detection of p53 mutations In respect to the fact that inactivation of p53 in tumor cells leads to the increased cellular proliferation and inhibition of apoptosis and concerning the observations that mutations of p53 gene are associated with advanced dis­ease, poor response to chemotherapy or radiotherapy, and short survival,83, 84 it is of great importance to determine the p53 status in every patient prior to treatment. Various approaches to the detection of p53 mutations have evolved in the last 19 years and each of them has certain advantages and certain dis­advantages. 77 , 35 The most informative method for the study of p53 mutations is the determination of the nucleotide sequence with either direct sequencing or indirect molecular analysis.85 Molecular sequencing is the only way to eval­uate the mutational event that inactivates the gene (there is no accumulation of the mutant protein) and allows for the unequivocal detection of alterations. On the other hand, the indirect molecular methods as denaturing gradient electrophoresis, single-strand con­formational polymorphism analysis or vari­ants as hydroxylamine and osmium tetrox­yde chemical cleavage, and pulse field gel electrophoresis, are more suitable for screen­ing and easier to perform. Yet, both methods share some drawbacks -namely, they cannot be at the moment performed in routine diag­nosis, tumor tissue is required, and care must be taken to avoid contamination from an excess of normal tissue.85-87 Immunohistochemical and immunocyto­chemical methods, under optimum condi­tions, are capable of detecting most missense mutations (which result in nuclear accumula­tion of the mutant protein) and can also iden­tify p53 stabilization without mutations (a consequence of the alteration of pathways regulating p53 expression). On the contrary, the mutations which do not induce p53 over­expression (nonsense mutations, frame-shift mutations, splice mutations, gene deletions, promoter mutations) will go undetected. Immunohistochemical results can be affected by the degradation of antigen during tissue processing and by the specificity of the anti­bodies used. Tumor tissue is needed, but the contamination by normal tissues is not a crit­ical factor. In sum, p53 immunostaining is stili an imperfect reflection of the prevalence 88 90 of p53 mutations.85, 86, ­ It remains controversial whether the mutant p53 protein can be detected in patients sera, since the results of various determinations are opposing. Namely, two groups of authors determined the serum lev­els of mutant p53 in patients with malignant lymphomas using a commercially available 92 ELISA kit,91, while another group of authors failed to do so using the same ELISA method in patients with lung cancer.87 Simi­ Jezeršek B and Novakovic S larly, Hassapoglidou using immunofluorimet­ric method could not detect mutated p53 pro­tein in sera of patients with cancer.93 Although p53 is a cellularly encoded pro­tein, it has been found to be immunogenic and capable of eliciting a p53 specific anti­body immune response. About one third of patients (the percentage varies for different types of cancer) with tumors that carry p53 missense mutations develop circulating p53 antibodies. These antibodies are not seen if there is no p53 accumulation in the tumor cell and, in case of lung carcinoma, they can appear before the cancer is detectable. The p53 protein may either be released during tumor cell necrosis, or otherwise translocates to the surface of the cell, inducing a B-cell response as a result of the breakdown of the immune system tolerance. Methods used for the determination of p53 antibodies include ELISA, immunoblot, and immunoprecipita­tion techniques. These methods can be per­formed routinely, they do not require tumor tissue, and can be used for follow up. There­fore, assessment of serum p53 antibodies is quite specific, but has low sensitivity (some mutations do not induce the production of p53 antibodies) in the detection of p53 muta­tions. 85,87,94-99 Therapeutic approaches Several therapeutic approaches are currently being assessed against the growth advantage and resistance to chemotherapy and radio­therapy observed in tumor cells with p53 mutations. The first approach is the investi­gation of active immunization against the potential tumor antigens carried by mutated p53, and indeed, it has been shown that it is possible to generate p53 specific CDS+ cyto­toxic T lymphocytes by immunizing mice with mutated p53 protein.1°° Furthermore, it was observed that a monoclonal antibody to p53, PAb 421, and a small peptide derived from p53 (the C-terminal domain) are able to restore the sequence specific DNA binding as well as growth suppression function of at least some mutant p53 proteins (by inducing a change in the configuration with a return to the active wild-type configuration).19,101-103 Among the recently proposed approaches, two are quite interesting. The first uses an adenovirus defective for ElB gene, which replicates only in the cells lacking functional p53 but not in the cells with wild-type p53, leading to selective destruction of tumor cells with mutant p53.104 The second, on the other hand, utilizes the transfer of a cytotoxic gene which is only activated in the presence of a mutant p53, resulting in a selective killing of tumor cells with p53 mutation.105 However, the most promising approach is p53 gene transfer in tumor cells carrying a p53 mutation. In tumor cells lacking func­tional p53, such a transfer can lead to tumor regression, as well as improve the cytotoxici­ty of antineoplastic agents, and the response to ionizing radiation.106-108 The most fre­quently used vectors for p53 gene transfer in animal models have been recombinant aden­oviruses, and less often retroviruses, which have a lower capacity of gene transfer in vivo. Interestingly, some tumor regressions were more important than expected. They were indicated by the percentage of p53 transfect­ed cells, suggesting a possible "bystander" effect, with destruction of non-transfected cells in the vicinity of transfected cells, as for suicide gene transfer.109 And finally, another idea was to try to identify the drugs that may trigger pro­grammed cell death through a p53 indepen­dent pathway. It has been suggested that taxol could be one of them, however, the clin­ical results with taxol were poorer in patients with mutant p53.no,m p53 s11ppressor gene Latest findings Even though p53 seems to play a central role in nearly all forms of celi growth stimulation and inhibition and was termed as the "guardian of the genome", it is becoming obvious that other proteins, as for example the recently discovered p33 and p75, also take an important part in the regulative mosaic. The nuclear protein p33 (a product of the tumor-suppressor gene INGl) forms a com­plex with p53 and cooperates in the negative regulation of cell proliferation by modulating p53 dependent transcriptional activa­tion.112,113 p73 is a protein that is closely related to p53, both structurally and functionally; how­ever, it is induced by different signals and thus plays a fundamentally different role in the maintenance of celi homeostasis. It can, at least when overproduced, activate p53 responsive genes and act as a growth sup­pressor.114,115 References 1. Lane DP, Crawford LV. T antigen is bound to a host protein in SV40-transformed cells. Nature 1979; 278: 261-3. 2. Linzer DI, Levine AJ. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embyonal carcinorna cells. Ce/11979; 17: 43-52. 3. Jenkins JR, Rudge K, Currie GA. Cellular imortal­ization by a cDNA done encoding the transforma­tion-associates phosphoprotein p53. 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Micronuclei in cytokinesis-blocked lymphocytes as an index of occupational exposure to antineoplastic drugs Vera Garaj-Vrhovac and Nevenka Kopjar Institute far Medica/ Research and Occupational Health, Zagreb, Croatia In order to investigate possible DNA damaging effects oj antineoplastic drugs, the micronucleus assay com­bined with Giemsa, DAPI and si/ver staining was performed. Blood samples were taken from nurses work­ing without adequate protection in oncological deparhnent on preparing and administration oj different antineoplastic drugs. Lymphocytes were cultivated in vitro at 37 ° C. To prevent cytokinesis, at 44h cytocha­lasine-B at afina/ concentration oj 3µg/ml was added. The results obtained indicate statistically significant increases in the tata/ number oj micronuclei in the exposed subjects compared to controls. DAPI staining has revealed signal-positive and signal-negative micronuclei while si/ver staining has revealed Ag-NOW and Ag-NOR-micronuclei. Compared to controls, the number oj signal-positive and Ag-NOR+ micronuclei in the exposed subjects were increased, indicating a greater susceptibility oj particular chromosomes to damage caused by antineoplastic agents. Key words: antineoplastic agents-adverse effects; occupational exposure; lymphocytes; micronucleus test Introduction Antineoplastic drugs are inhibiting or pre­venting growth of neoplasms, by checking the maturation and proliferation of malignant cells. They are risk factors for different cate­gories of workers who are occupationally exposed during the stocking, preparation, administration and disposal of such agents.1­4 Many of these, commonly used in cancer chemotherapy have proved carcinogenic, mutagenic and teratogenic in experimental Correspondence to: Vera Garaj-Vrhovac PhD, Neven­ka Kopjar, MSc. Institute for Medica! Research and Occupational Health, Ksaverska c. 2, HR-10001 Zagreb, Croatia. Tel:++385 1 467 31 88 (264); Fax: ++385 1 467 33 03; E-mail: vgaraj@mimi.imi.hr nkopjar@mimi.imi.hr animals and in vitro test systems.5-9 Careless handling of cytotoxic agents may lead to exposure of the personnel to amounts detectable by chemical or biological methods in the body fluids or cell samples of the sub­jects. The exposure is typically to mixed com­pounds over a longer period, and to low exposure levels with accidental peaks. There­fore, the use of biological exposure markers is appropriate for monitoring such exposure patterns. The biological markers/methods for exposure assessment are either non-specific (e.g. cytogenetic damage, point mutations or 32P-post-labelling adducts in peripheral blood lymphocytes, urinary mutagenicity) or specif­ic for a given compound (immunological methods, specific analytical methods).3, Garaj-Vrhovac Vand Kopjar N The in vitro micronucleus assay is used wide­ly as a useful endpoint in biomonitoring, and ecotoxicology, as well as for the assessment of in vitro and in vivo damage of chromoso­mal material caused by different mutagens.16­ The aim of this study was to investigate the cytogenetic damage resulting from the exposure to different antineoplastic drugs in nurses working in oncology department under inadequate protection. Genotoxic damage in lymphocytes was evaluated by a micronucleus assay. For sensitive detection of genome damage conventional Giemsa stain­ing was compared to DAPI and silver staining techniques. Materials and methods Subjects Samples of peripheral blood were taken from 20 healthy never-smoking subjects aged from 24 to 50 years (mean age 39.5 years). Ten of them were controls and ten were nurses working in oncological department of one hospital. Nurses were daily involved in the preparation and administration of different antineoplastic drugs for an average period of 17.3 years. Both experimental groups were previously interviewed to document a history of radiation exposure, chemical exposure and vira! infection within one month before the study. Micronucleus assay Blood samples were cultured at 37 ° C in vitro in F-10 medium (Gibco) supplemented with fetal bovine serum (Biological Industries, Israel), phytohaemagglutinin (Murex) and antibiotics (penicillin and streptomycin). Cul­tures were harvested at 72 h. To prevent cytokinesis, at 44 h Cytocha­lasin-B (Sigma) in the fina! concentration of 3 µg/ml was added to each sample, and the cells were harvested after a further incuba­tion of 28 h. The slides for scoring micronu­clei were prepared according to the modified method of Fenech and Morley. 22 After a brief treatment with physiological saline, cells were fixed with 3:1 mixture of methanol and acetic acid. They were dropped onto clean slides, dried at room temperature and after­wards stained using conventional Giemsa staining, DAPI and silver staining technique. Staining techniques Giemsa staining was performed by means of 5% buffered solution of Giemsa for 10 min­utes. After staining, the slides were washed and air-dried. DAPI (4',6-diamidino-2-phenylindol-dihy­drochloride) staining was performed accord­ing to Schweizer.23 Prior the staining, slides were preincubated in Me Ilvaine's buffer (cit­ric acid -disodium hydrogenphosphate) pH 7.0 for 10 minutes. The staining solution con­tained 1 µg of DAPI / ml of Me Ilvaine's buffer (pH 7.0). The staining procedure was carried out in dark and lasted for 10 minutes. Afterwards, slides were rinsed in Me Ilvaine's buffer, air-dried and mounted in 1:1 mixture of glycerol and Me Ilvaine's buffer. The preparations were observed under short-­wave-length blue light using an UG 1 filter for excitation. For the silver staining of NORs the method of Howell and Black24 , which uses gelatine as colloidal protector, was employed. To prepare colloidal developer, gelatine was dissolved in deionized water by stirring and gentle heating. When the gela­tine was dissolved, formic acid was added. To prepare silver nitrate solution, silver nitrate was dissolved in deionized water. To stain NORs, a colloidal developer and AgNO3 solution (50%) were mixed and pipet­ted onto the micronuclei preparations. The slides were covered with coverslips and Micronuclei -an index oj occupational exposure to antineoplastic drugs o e l. Binucleated lymphocytes of nurses occupationally exposed to antineoplastic drugs. (a) signal-positive micronu­cleus stained with DAPI, (b) signal-negative micronucleus stained with DAPI, (c) celi with two Ag-NOR· and one Ag­NOR+ micronuclei after silver staining, (d) Ag-NOR' micronucleus with two signals after silver staining Figur placed on a hot plate pre-heated to 70°C. After 1-2 minutes the solution turned yellow and then golden brown. At this stage slides were washed off, rinsed with deionized water and air-dried. Scoring of the slides Slides from the exposed and control subjects were scored by the same scorer. For the incidence of micronucleated lym­phocytes and determination of the frequency of micronuclei 500 binucleated cells per sub­ject were scored. The published criteria far MN determina­tion were fallowed: {l) binucleated cells con­taining any number of MN were scored; (2) fluorescence intensity per unit area of scorable MN was either equal in intensity or more or less intense than that of the main nuclei; (3) only MN that were distinctly sepa­rate from the main nuclei and located within binucleated cells with intact cytoplasmic and nuclear membranes were scored. Micronuclei were located far their DAPI fluorescence and NOR activity and successively classified as signal-positive and signal-negative as well as Ag-NOR+ and Ag-NOR MN. Garaj- Vrhovac Vand Kopjar N Table 1. Tota! number and distribution of micronuclei in lymphocytes of control group after Giemsa, DAPI and sil­ver staining SubjectNo. 1 2 3 4 5 6 7 8 9 10 No. of 496 498 497 496 498 498 498 497 497 498 binucleated 496 497 497 497 496 498 498 497 498 497 cells withoutMN No. of 3 2 2 4 4 2 2 3 3 2 binucleated 4 3 3 3 3 2 2 2 2 3 cells with 1 MN No. of 1 o 1 o 1 o o o o o binucleated o o oo 1 o o 1 o o cells with2MN Tota! No. of 5 2 4 4 6 2 2 3 3 2 MN per 500 4 3 3 3 5 2 2 4 2 3 binucelulated cells Giemsa staining technique 1 1 Statistical analysis The statistical significance of the results was determined using the x2 test. Results The results regarding the frequency, distribu­tion and total number of micronuclei (MN) for the control group are reported in Table 1, and for the exposed group in Table 2. All staining techniques have revealed sta­tistically significant increases in total number of micronuclei in the exposed group (P<0.05 using x2 test) compared to the controls. The distribution of micronuclei per 500 binucleat­ed cells in all the exposed subjects was also disturbed compared to the controls. Considering the presence or absence of bright DAPI, flourescent dots inside of MN signal-positive and signal-negative MN have been detected. Their total number and distri- DAPI staining Silver staining technique technique bution for the exposed and control groups are shown in Table 3. In all the exposed sub­jects we have observed an increased total number of micronuclei as well as an increased number of signal-positive micronu­clei compared to control. Related to the patterns of nucleolar orga­nizer (NOR) activity, Ag-NOR+ (MN that con­tain one or more NOR parts) and Ag-NOR­micronuclei (MN without NOR parts) have been noticed. Their total number and distrib­ution of in control and exposed subjects are shown in Table 3. In all the exposed subjects an increased total number of micronuclei as well as increased number of Ag-NOR+ micronuclei, compared to the controls, is observed. Figure 1 shows signal-positive and signal­negative micronuclei obtained after staining with DAPI (a,b) and Ag-NOR+ and Ag-NOR­micronuclei (c,d) obtained after silver stain­ing in subjects occupationally exposed to antineoplastic drugs. Micronuclei -an index of occupational exposure to antineoplastic drugs Table 2. Tota! number and distribution of micronuclei in lymphocytes of exposed group after Giemsa, DAPI and silver staining SubjectNo. 1 2 3 4 5 6 7 8 9 10 No. of 477 482 488 490 489 490 488 491 462 485 binucleated 488 478 480 482 485 480 490 483 474 484 cells withoutMN 12 No. of 13 16 12 10 10 8 11 8 32 binucleated 11 17 19 13 14 17 11 15 cells withlMN 15 No. of o 1 o o 1 2 1 o 5 2 binucleated 1 4 1 3 1 2 o 2 1 1 cells with2 MN No. of o 1 o o o o o 1 1 o binucleated o o o 1 o 1 o o 1 o cells with3 MN No. of o o o o o o o o o o binucleated o o o 2 o o o o o o cells with4 MN Tota! No. of 13 21 12 10 12 12 13 11 45 18 MN per 500 13 25 21 30 16 24 11 19 29 17 binucelulated cells Giemsa staining Discussion The primary source of human exposure to antineoplastic drugs results from their use in cancer therapy. However, persons involved in the manufacture, preparation and adminis­tration of drugs to patients and in nursing care of patients may also be exposed. The results of numerous studies on cytoge­netic endpoints performed on medica! per­sonnel exposed to antineoplastic drugs are conflicting, probably due to different degrees of exposure in different occupational set­tings, as well as due to different sensitivity of the indicators, to their different persistence Silver staining and different safe handling measures. 10,13,17,25,26 Based on their mode of action, antineo­plastic agents are divided into severa! cate­gories. Since most of these drugs exert their effects during a certain phase of the celi cycle (celi growth phase, celi division phase, rest­ing phase etc.), many treatment regimens combine two or more of these agents. This is the reason why are nurses usually exposed to a mixture of different antineoplastic drugs used in their daily preparation and adminis­tration procedures. The most frequently han­dled antineoplastic drugs in our study were: bleomycin, vinblastine, cyclophosphamide, Garaj-Vrhovne Vand Kopjar N Table 3. Tota! number and distribution of DAPI signal-positive and signal-negative micronuclei (MN) as well as Ag-NOR+ and Ag-NOR-MN for control and exposed subjects SubjectNo. 1 2 3 4 5 6 7 8 9 10 Exposed group 14 8 8 7 4 10 3 11 22 13 24 11 19 29 Tota! No. of MN 13 27 21 27 16 Control group To tal No. of MN 4 3 3 3 4 2 2 3 2 3 DAPI staining technique cisplatinum, 5-fluorouracil, adriamycin and mitomycin C. The results of our study have clearly indi­cated that occupationally exposure to anti­neoplastic drugs caused cytogenetic damage. The in vitro micronucleus assay combined by Giemsa, DAPI and silver staining techniques has revealed a significant increases in the number of micronuclei as well changes in their distribution in all the exposed subjects compared to control. It is known that micronuclei originate from either whole chromosomes or acentric chromosome fragments due to chromosomal breakage, or from lagging chromosomes which consequently are excluded from the main nuclei. Therefore, enumeration of MN can provide an index of chromosome loss from the main nuclei if whole chromosomes can be identified within them. The incidence of micronuclei observed could result from clastogenic as well as aneugenic effect on peripheral blood lymphocytes. It has been shown that lymphocytes are an extremely sensitive indicator of induced chromosome structural damage both in vivo and in vitro. Silver staining technique Approximately 90% of lymphocytes have a half-life of three years and thus can reflect damage incurred over a long period. Among the antineoplastic drugs used in our study some are known aneuploidy­inducing agents with spindle damaging effects (vinblastine) while others are clasto­gens with direct DNA damaging effects (bleomycin, mitomycin C). Considering the average duration of occupational exposure to those agents, an increased number of micronuclei observed in all the exposed sub­jects, compared to controls, is not surprising. These results are consistent with previous reports on the use of different endpoints with different antineoplastic drugs in vivo 1727 30 and in vitra.8,12 ,, , 28,29 , In this study, the micronucleus assay was performed in combination with conventional Giemsa staining and more specific DAPI and silver staining. Giemsa technique was com­pared to DAPI and silver because it is known that both techniques exhibit a considerable specificity in detecting particular chromoso­mal regions or distinct chromosomes. There­fore, they allow us to speculate about the ori­ Micronuclei -an index oj occupational exposure to antineoplastic drugs gin of micronuclei. It is known that DAPI staining produces intense fluorescence of the paracentromeric regions of chromosomes 1,9,16, of the 8 months before study entry, no evidence of cardiovascular disease, adequate organ func­tion including normal bone marrow function (leukocyte count >3,500/µL, platelet count >100,000/µL), renal function (creatinin <1.5 mg/dL or creatinin clearance >60 ml/min), and normal hepatic function (bilirubin < 38 mmol/L, liver transaminase level < 3 times the normal values, an albumen value>36 g/L, negative hepatitis B surface antigen). Patients with active cardiac disease, infec­tion, clinically significant pulmonary and neurologic dysfunction, cerebral metastases, any significant intercurrent illness, require­ment of systemic steroids, psychiatric history, or those who had undergone surgery, chemo­immuno-or radiation therapy were not eligi­ble. In the 4 weeks before entering the trials, the size of the metastatic lesions was deter­mined by computed tomography and/or Che111oi111111unolherapy oj colon ca11cer ultrasound and /or X-ray examinations and the serum levels of the carcinoembryonic antigen (CEA) was measured. The size of the lesions was detennined after every second treatment cycle. The patients with disease progression had the best supportive care with no additional second line chemothera­py. Treatment plan Patients received LV (Leucovorin Ben Venue Laboratories Bedford) 20 mg/m2/day intra­venously (i.v.) and half-an hour later 5-FU which was administered in 6-hr continuos intravenous infusion at a dose of 600 mg/m2/day diluted in 1,000 ml of normal saline. 5-FU was obtained commercially. Recombinant human IFN a-2a (Roferon; Hoffman La-Roche) was administered sub­coutaneusly (s.c.) in a dose of 6 MIU consecu­tively after each 5-FU administrations. Patients received ali three drugs for 5 consec­utive days. Cycles were repeated at 28-day intervals. The dose of 5-FU was modified for myelosupression, diarrhoea and stomatitis. In grade 3 or 4 toxicity the dose of 5-FU was reduced for 20%. Subsequent courses were delayed until haematological recovery to the following values: granulocytes, 1,500 /µL or higher and platelets 100,000 or higher, and resolution of all nonhematologic toxicity to Grade I or baseline. There was no reduction of the doses of LV and IFN. Response criteria Treatment response was evaluated after each 2nd cycle. The responders and those with sta­ble disease were subjected to regular clinical and radiological follow up every two months until progression, and then after 3 months until deaths. Tumor response was defined according to World Health Organisation (WHO) criteria.15 A complete response (CR) was defined as the disappearance of all known disease symptoms on two separate measurements performed in at least 4 week interval; partial response (PR) was defined as a ;c: 50% decrease in the sum of products of the largest perpendicular diameters of all measurable lesions for a minimum of 4 weeks, without the appearance of new lesions; stable disease (SD) was defined as a decrease by <50% or increase by <25%, with no new lesions; progressing disease (PD) was defined as a 25% or greater increase in mea­surable disease or appearance of new lesions. The duration of response was measured for patients with objective response from the onset of objective response to the tirne of pro­gression. The duration of survival was mea­sured from the first day of treatment until the date of death. Statistical Analysis The survival, duration of response and the tirne to progression were calculated using log-rank test, 16,17 while the calculations of the 95% confidence interval for response rate were made by Brookmeyer and Crowley's method.18 Results The characteristics of the 22 patients entered into this study are listed in Table 1. Seven patients received prior adjuvant therapy con­sisting of 5-FU and levamisol. The majority of patients had minimal cancer related symp­toms. Their ECOG PS was 0-1 except in 1 patient. Patients received 2 to 9 cycles of chemoimmunotherapy (mean 5 cycles). All patients were eligible and evaluable. Tumour response The overall response rate was 32% (95% confi­dence interval (CI) 14% to 52%) (Table 2). All 410 Štabuc B et al. Table l. Patients' characteristic Characteristic No. of patients Age, years Median 60 Range 31-78 Male/Female 11/11 ECOG performace status 0/1/2 13/8/1 Prior adjuvant therapy 7 Sites of disease: -Liver 12 -Lung 8 -Peritoneum 2 -Lymph nodes 3 No. of metastatic sites 1 19 2 2 3 1 responses were partial. There was no com­plete response. The median tirne to achieve initial response was 2.5 months (range 1.5 to 4.0). Most responding patients showed con- Table 2. Tumor response site, in the lung of one patient and in the soft tissue of the other. In one patient who had liver, lung and soft tissue metastases CR was observed in the lung and soft tissue whereas in the liver only PR was noticed. The patient who had experienced daily hectic fever to 38.5 °C caused by liver metastases, complete­ly resolved the fever within 2 weeks of the initiation of therapy. In the group of seven patients, treated with adjuvant chemothera­py, we did not see any response to chemoim­munotherapy. Survival Overall median survival tirne was 12.5 months and of responders 14.4 months (Fig­ure 1). Median follow up was 12 months. Among 22 patients, 20 patients died. Their survival ranged from 4 to 30 months. Two patients are still alive 26 and 36 months after beginning the study. The median tirne to pro­gression was 5.5 months in 20 patients. In the patients with objective response the median tirne to progression was 8.7 months. The two survivors have been without any Response No. of patients Response rate (%) Objective response 7 32 (95% C.I. 14-52%) CR o o PR 7 32 Stable disease 7 32 Pro_gi-essive disease 8 36 tinued improvement in their disease status, and the median tirne to achieve best clinical response was 4.0 months (range 1.5 to S.O). In addition, 32% of patients had stable dis­ease (range 3 to 14 months). Two of twelve patients with metastases involving liver only, two of eight patients involving lung only and two patients with the involvement of both, lung and an extrahepatic site responded. In two patients with lung and soft tissue metas­tases, CR was observed in one metastatic 100 90 % 80 u 70 r 60 s \' 50 40 v 30 20 1 10 o o 6 12 18 24 30 36 42 Survival Time in Months Figure 1. Probability of survival of the treatment group. Chemoimmunotherapy oj colon cancer sign of progression in the last 22 and 30 months, respectively. Toxicity As shown in Table 3, the side effects of chemotherapy were generally mild and not exceeding the toxicity grade of 3 to 4, neither causing toxicity related deaths nor requiring discontinuation of chemotherapy due to toxi­city. In one patient chemotherapy was post­poned for 14 days due to grade 2 leucopenia. One patient requested a