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Solutions that help ADIOLOGY ANO NCOLOGY Editorial office Radiology and OncologtJ June 2004 Institute of Oncology Vol. 38 No. 2 Zaloška 2 Pages 73-163 SI-1000 Ljubljana ISSN 1318-2099 Slovenia UDC 616-006 Plwne: +386 1 5879 369 CODEN: RONCEM Phone/Fax: +386 1 5879 434 E-mail: gsersa@onko-i.si Aims and scope Radiology and Oncology is a journal devoted to publication of original contributions in diagnostic and interventional radiologij, computerized tomography, ultrasound, magnetic resonance, nuclear medicine, radiotherapy, clinical and experimental oncologij, radiobiologij, radiophysics and radiation protection. Editor-in-Chief Editor-in-Chief Emeritus Gregor Serša Tomaž Benulic Ljubljana, Slovenia Ljubljana, Slovenia Executive Editor Editor Viljem Kovac Uroš Smrdel Ljubljana, Slovenia Ljubljana, Slovenia Editorial board Marija Auersperg Valentin Fidler Maja Osmak Ljubljana, Slovenia Ljubljana, Slovenia Zagreb, Croatia Nada Bešenski Be1aFomet Branko Palcic Zagreb, Croatia Budapest, Hungary Vancouver, Canada Karl H. Bohuslavizki Tullio Giraldi ]urica Papa Hamburg, Germany Trieste, Italy Zagreb, Croatia Haris Boka Andrija Hebrang Dušan Pavcnik Zagreb, Croatia Zagreb, Croatia Portland, USA Nataša V. Budihna Ltiszl6 Horvtith Stojan Plesnicar Ljubljana, Slovenia Pecs, Hungary Ljubljana, Slovenia Marjan Budihna Berta Jereb Ervin B. Podgoršak Ljubljana, Slovenia Ljubljana, Slovenia Montreal, Canada Malte Clausen Vladimir Jevtic Jan C. Roos Hamburg, Germany Ljubljana, Slovenia Amsterdam, Netherlands Christoph Clemm H. Dieter Kogelnik Slavko Šimunic Miinchen, Germany Salzburg, Austria Zagreb,_, Croatia Mario Corsi Jurij Lindtner Lojze Smid Udine, Italy Ljubljana, Slovenia Ljubljan_,a,Slovenia Ljubomir Diankov Ivan Lovasic BomtStabuc So fia, Bulgaria Rijeka, Croatia Ljubljana, Slovenia Christian Dittrich Marijan Lovrencic Andrea Veronesi Vienna, Austria Zagreb, Croatia Aviano, Italy Ivan Drinkovic LukaMilas Živa Zupancic Zagreb, Croatia Houston, USA Ljubljana, Slovenia Gillian Duchesne Metka Milcinski Publisher Association of Radiology and Oncology Affiliated with Slovenian Medica/ Association -Slovenian Association of Radiology, Nuc/ear Medicine Society, S/ovenian Society far Radiotherapy and Oncology, and Slovenian Cancer Society Croatian Medica/ Association -Croatian Society of Radiology Societas Radiologorum Hungarorum Friuli-Venezia Giu/ia regional groups of S.I.R.M. (Italian Society of Medica/ Radiology) Copyright © Radiology and OncologtJ. Ali rights reserved. Reader for English Mojca Cakš Key words Eva Klemencic Secretaries Milica Harisch Mira Klemencic Design Monika Fink-Serša Printed by Imprint d.o.o., Ljubljana, Slovenia Published quarterly in 700 copies Bank account number 02010-0090006751 Foreign currency account number 010-7100-900067 /4 NLB d.d., Podružnica Ljubljana Center, Ljubljana S. W.I.F. T. Code LJBASI2X Subscription fee far institutions EUR 100 (16000 SIT), individua/s EUR 50 (5000 SIT) The publication of this joumal is subsidized by the Ministry of Education, Science and Spori of the Republic of Slovenia. Indexed and abstracted by: BIOMEDICINA SLOVENICA CHEMICAL ABSTRACTS EMBASE / Excerpta Medica Sci Base This journal is printed on acid-free paper Radiologij and Oncology is available on the internet at: http://www.onko-i.si/radiolog/rno.html ISSN 1581-3207 Ljubljana, Slovenia ISSN 1318-2099 June 2004 UDC 616-006 Vol. 38 No. 2 CODEN: RONCEM CONTENTS ONCOLOGY Tumor markers in clinical oncology 73 Novakovic S Clinical utility of serine proteases in breast cancer Cufer T Cathepsins and their inhibitors as tumor markers in head and neck cancer Strojan P 93 Pharmacogenetics of thiopurines: can posology be guided by laboratory data? Stocco G, Martelossi S, Decorti G, Ventura A, Malusa N, Bartoli F, Giraldi T 101 Development of quantitative RT-PCR assays for wild-type urokinase receptor (uP AR-wt) and its splice variant uP AR-del5 * Farthmann J, Holzscheiter L, Biermann J, Meye A, Luther T, Kotzsch M, Sweep F, Schmitt M, Span P, Magdalen V 111 Ex vivo flow mammalian cell electropulsation Vernhes MC, Eynard N, Pierre Rols M, Ganeva V, Teissie J 121 Immunohistochemical expression of HER-2/neu in patients with lung carcinoma and its prognostic significance Petrusevska G, Ilievska-Poposka B, Banev S, Smickova S, Spirovski Z 131 New saccharide derivatives of indolo[2,3-b]quinoline as cytotoxic compounds and topoisomerase II inhibitors Godlewska J, Badowska-Rostonek K, Ramza J, Kaczmarek L, Peczyfzska-Czoch W, Opalski A 137 Regional comparison of cancer incidence Obralic N, Gavrankapetanovic F, Dizdarevic Z, Duric O, Šišic F, Selak I, Balta S, Nakaš B LETTER TO THE EDITOR Chemotherapy for small-cell lung cancer with paraneoplastic nephrotic syndrome Kagohashi K, Ohara G, Satoh H, Sekizawa K 153 SLOVENIAN ABSTRACTS 155 NOTICES 162 Radiol Oncol 2004; 38(2): 73-83. review Tumor markers in clinical oncology Srdjan Novakovic Department of Tumor Biology, Institute of Oncology, Ljubljana, Slovenia The subtle differences between normal and tumor cells are exploited in the detection and treatment of can­cer. These differences are designated as tumor markers and can be either qualitative or quantitative in their nature. That means that both the structures that are produced by tumor cells as well as the structures that are produced in excessive amounts by host tissues under the influence of tumor cells can function as tumor markers. Speaking in general, the tumor markers are the specific molecules appearing in the blood or tissues and the occurrence of which is associated with cancer. According to their application, tumor markers can be roughly divided as markers in clinical oncology and markers in pathology. In this review, only tumor markers in clinical oncology are going to be discussed. Current tumor markers in clinical oncology include (i) oncofetal antigens, (ii) placental proteins, (iii) hor­mones, (iv) enzymes, (v) tumor-associated antigens, (vi) special serum proteins, (vii) catecholamine metabo­lites, and (viii) miscellaneous markers. As to the literature, an ideal tumor marker should fulfil certain criteria - when using it as a test for detection of cancer disease: (1) positive results should occur in the early stages of the disease, (2) positive results should occur only in the patients with a specific type of malignancy, (3) positive results should occur in all patients with the same malignancy, (4) the measured values should correlate with the stage of the disease, (5) the measured values should correlate to the response to treatment, (6) the marker should be easy to meas­ure. Most tumor markers available today meet several, but not all criteria. As a consequence of that, some criteria were chosen for the validation and proper selection of the most appropriate marker in a particular malignancy, and these are: (1) markers' sensitivity, (2) specificity, and (3) predictive values. Sensitivity ex­presses the mean probability of determining an elevated tumor marker level (over the "cut-off value") in a tumor-bearing patient. Specificity expresses the mean probability that a normal tumor marker value derives from a tumor-free individual. The predictive value shows the applicability of a tumor marker in a mixed group of patients. Many theoretical applications exist for tumor markers in clinical oncology. Clinically important utilization of markers includes (i) early detection of the tumor, (ii) differentiating benign from malignant conditions, (iii) evaluating the extent of the disease, (iv) monitoring the response of the tumor to therapy, and (v) pre­dicting or detecting the recurrence of the tumor. Since no ideal tumor markers with adequate sensitivity and specificity currently exist, they are only exceptionally used in screening (prostate specific antigen - PSA). Nevertheless, tumor markers can play a crucial role in the detection of an early disease relapse and assess­ment of response to therapy in selected groups of patients. In monitoring the patients for disease recurrence, tumor marker levels should be determined only when meaningful treatment is possible. Key words: neoplasms-diagnosis; tumor markers, biological What exactly are tumor markers? The result of malignant transformation is a malignant cell that, in each cycle of cell divi­sion, generates a new malignant cell. In this process, the malignantly transformed cells ac­quire some new properties through which they differ from nonmalignant cells of the same origin. The acquired properties can be either the changes in cellular morphology, physiology, or the changes in cell growth (be­havior).1 These subtle differences between normal and malignant cells are therefore be­ing exploited in the detection of malignant cells (or malignancy in general), and the sub­stances that are being determined in this process are termed tumor markers. According to the fact that the differences be­tween tumor cells and normal ones appear on various levels (see above), also tumor markers differ one from another and represent a rather broad conception that comprises both various substances and various cellular processes. Consequently, membrane antigens, hormones, enzymes, polyamines, nucleosides, products of oncogenes, products of tumor suppressor Table 1. Utilization of tumor markers in oncology I) FOR FOLLOW-UP OF THE DISEASE 1) determination in body fluids a) to monitor the treatment response b) to detect early the disease recurrence c) to evaluate the extent of disease d) to differentiate benign from malignant conditions e) as screening method for some types of cancer 2) immunoscintigraphy and lymphoscintigraphy 3) immunohistochemistry a) to set the diagnosis b) to determine the prognosis c) to predict the treatment response II) FOR TREATMENT 1) direct cytotoxicity of specific monoclonal antibodies (MoAb) a) binding of complement to specific MoAb b) binding of cytotoxic cells to specific markers-receptors 2) binding of drugs to specific MoAb 3) binding of toxins to specific MoAb 4) binding of radioactive isotopes to specific MoAb 5) inhibition of growth factor receptors Received 25 February 2004 Accepted 28 March 2004 Correspondence to: Srdjan Novakovic Ph.D., Institute of Oncology, Zaloška 2, 1000 Ljubljana, Slovenia. Tel. +386 1 522 5118; Fax. +386 1 5879 434; E-mail: sno­vakovic@onko-i.si This paper was presented at the "3rd Conference on Experimental and Translational Oncology", Kranjska gora, Slovenia, March 18-21, 2004. genes, or DNA ploidy and proportion of cells in S phase of the cell cycle (proliferative activity) can be considered as tumor markers.2,3 Different fields in oncology utilize different tu­mor markers according to their needs and their techniques of follow-up: tumor markers in clin­ical oncology differ from the ones in molecular biology, and these differ from the ones in im­munohistochemistry, in physiology, etc. The application of tumor markers Theoretically, the possibilities for the applica­tion of tumor markers in oncology are nu­merous, but the utilization depends upon the sensitivity and specificity of the marker and upon reliability of other methods that are be­ing used for the same purpose (Table 1). Tumor markers in clinical oncology The standard definition of tumor markers in clinical oncology comprises predominately the substances that are produced by malig­nant cells or the substances that are produced by other cells under the influence of malig­nant cells and that can be determined in body fluids. Tumor markers can be either newly synthesized substances (that are not com­monly produced by normal healthy cells) or the substances that can be found in normal organisms in much lower concentrations.2 The determination of tumor markers in clini­cal oncology is helpful in many processes: in the process of diagnose setting and prognose prediction, in determining the disease extent and planning the treatment, as well as in the early detection of disease recurrence or metastasis. However, the markers in clinical oncology are nowadays, due to their incom­petences, only exceptionally applied as screening methods for the detection of malig­nant diseases. Different markers are used for different purposes – namely, some of them are more appropriate for the follow-up of the disease and the others for the early detection of the disease recurrence.3-5 In addition to the above applications, tumor markers can also serve as predictors of a disease outcome. The follow­up of malignant disease before, during and after treatment, and careful processing of the data, namely, gives us a valuable information about the nature of malignancy, and thus al­so about the patient’s prognosis. In general, extremely high concentrations of tumor markers are predictors of poor outcome.6 Determination of tumor marker in patient’s sera In body fluids, tumor markers are found in low concentrations and for their determina­tion highly sensitive technology is needed. The techniques that are being used are more or less based on the same principle – i.e. the determination of antigen-antibody complex­es. Most widely used techniques are the ra­dio-immune assay, the enzyme-immune as­say, and the luminometric-immune assay, which differ in the compound bound to the detection antibodies, and in the method of detection of the formed complexes.7 Sensitivity and specificity of tumor markers The ideal tumor marker should be (i) present only in tumor cells, (ii) specific for the organ and type of tumor, (iii) assessable in the sera of all patients with the same type of tumor, (iv) assessable in the serum at the very begin­ning of tumor development and its concen­trations in the serum should correlate with the tumor burden. Besides, the serum con­centrations of the marker should be regarded as a valid predictor of disease in patients with the specific type of tumor.2 Up till now, no antigenic structure is known that would be present only in tumor cells – and that means that the antibodies against certain tumor markers crossreact also with other anti­genic structures. We can therefore conclude that no tumor marker and no methods of fol­lowing up the presence of malignant cells are 100% specific. When assessing these results, we have to bear in mind that not only a malig­nant disease causes elevated levels of tumor markers, but that there are also other factors that affect their concentration. The most com-mon incompetences of tumor markers are (i) inadequate specificity for the type of malig­nancy, (ii) production of markers in high con­centrations in nonmalignant diseases (differ­ent inflammatory processes, benign tumors, nonmalignant diseases of the liver and pan­creas), (iii) production of markers in different physiological conditions (pregnancy, menstru­ation, lactation), and (iv) production in per­fectly healthy tissues.2,3,7 To determine as accurately as possible the role and applicability of a specific tumor marker and the method in a specific type of malignancy, some new terms were intro­duced – including the sensitivity and speci­ficity. The sensitivity of a marker reports the proportion of patients bearing a specific type of tumor in which the serum (urine, plasma, cerebrospinal liquor) level of marker is ele­vated. The more patients with the same type of tumor have an elevated level, the more sen­sitive is the marker, and the lower is the ex­pected number of false negative determina­tions. The specificity of a marker represents the proportion of patients who do not have a certain type of malignancy and in whom the marker level is normal. That means that the lower is the number of patients not bearing a certain type of tumor and having an elevated marker level, the higher is the specificity, and the lower is the expected number of false neg­ative determinations. In case there are more tumor markers ele­vated in one type of malignancy, it is possible to increase the sensitivity of detection by combining these markers’ determinations, but we have to bear in mind that, in this way, the specificity of detection is decreased. By combining properly the marker determina­tions, the specificity of detection can be only slightly lowered, while the sensitivity is sig­nificantly increased. The prerequisite for a proper combination of different markers is their high specificity and complementarity for the type of tumor. An example of such proper combination is the determination of ßHCG (human chorionic-gonadotropin) and AFP (alpha fetoprotein) in non-seminoma germ tumors. Each of the above-mentioned markers has a specificity of more than 90% and the sensitivity of approximately 60% for this type of tumors. Due to their complemen­tarity (meaning that they are elevated in dif­ferent patients), the sensitivity of the combi­nation of the two markers reaches approxi­mately 95%.8-10 Classification of tumor markers Tumor markers can be classified in several ways: according to their chemical structure, their tissue of origin, types of malignancies in which they are elevated, etc. The most com­mon classification tries to combine their bio­chemical properties, tissue of origin, and functionality. According to this classification, we distinguish: oncofetal proteins, hormones and/or carcinoplacental antigens, enzymes, tumor-associated antigens, special serum pro­teins, and miscellaneous markers.3,11 Oncofetal proteins Oncofetal proteins are antigens that are nor­mally produced during the embrional develop­ment. In adults, their production is limited or completely absent (stopped). Elevated concen­trations in adults result from reactivation of certain genes that control cellular growth and are directly connected to malignant process. Carcinoembryonic antigen (CEA) is a typical representative of this group and one of the first known tumor markers. During embrion­al development, it is produced in epithelial cells of the gastrointestinal tract, liver, and pancreas. CEA is important for the follow-up of patients with colorectal cancer because 65% of all patients (including those with lo­calized disease and stage I), and as much as 100% of patients with metastatic disease have elevated serum levels of CEA.2,4 Besides, this marker is convenient also for the follow-up of patients with other malig­nancies – especially breast, ovarian, pancre­atic, lung, liver, and endometrial cancer.12,13 Serum concentrations between 4 - 10 ng/ml can be found either in the patients with ma­lignant or in the patients with benign dis­eases, and even in some heavy smokers, while concentrations above 10 ng/ml speak more in favor of a malignancy.14 Elevated serum concentrations can be found also in the patients with bronchitis, gastritis, duode­nal ulcer, liver diseases, pancreatitis, colorec­tal polyposis, etc.14 Alpha-fetoprotein (AFP) is known for ap­proximately as long as CEA. It was discov­ered in 1963 in the sera of mice with hepato-cellular carcinoma.15 AFP is a glycoprotein produced in yolk sac, in the epithelial cells of the gastrointestinal tract and liver during em-brional development.16 In pregnancy, AFP enters amniotic fluid through fetal blood, and passes the placenta, thus going into maternal blood. In healthy adults, AFP can be found in the blood in very minute concentrations. Normal serum concentrations appear approx­imately 9 months after birth. Elevated serum AFP levels (above 10 ng/ml) in adults can be found in the patients with acute viral hepati­tis, liver cirrhosis, obstructive icterus, and in some malignant diseases, as pancreatic can­cer, lung cancer or gastric cancer. The main role of AFP is to follow up the patients with hepatocellular carcinoma (95 to 100% speci­ficity and sensitivity) in whom the concentra­tions above 1200 ng/ml practically confirm the diagnosis of primary liver (hepatocellular) cancer, and the patients with non-seminoma germ tumors (specificity 60%).16,17 Hormones Malignant formations can alter the synthesis and secretion of various hormones. Quantitative and qualitative alterations of the synthesis and hormone secretion can there­fore be the indicators of a malignant process and can be monitored as tumor markers. Quantitative alterations occur when tumors develop in the tissue of endocrine glands, thus influencing the normal production of hormones by either increasing or decreasing it. This group comprises hormones of malig­nant endocrine tumors as parathyroid hor­mone, insulin, prolactin, catecholamines and oth­ers. Qualitative changes take place when ma­lignantly transformed cells of some organs (lungs, breast, stomach, central nervous sys­tem, ovaries) start producing hormones – i.e. the so called ectopic hormone production (e.g. calcitonin and parathyroid hormone in breast cancer, lipotropin in carcinoid tumors, calcitonin, insulin, parathyroid hormone in thymic malignomas).2,3 Among all hormones, human chorionic go-nadotropin (ßHCG) is one of the most applica­ble tumor markers. This is a protein with the molecular mass of 45 KD. It belongs to the group of carcinoplacental antigens – proteins that are synthesized in placenta during preg­nancy (most early pregnancy tests are based on the detection of ßHCG) and can be found in adults only exceptionally.18 Elevated serum concentrations of ßHCG can be found in al­most all female patients with germ tumors with trophoblastic component (choriocarci­nomas), hydatidiform moles, and in the ma­jority of male patients with germ tumors. ßHCG has a very short serum half-life (36-48 hours) and is therefore functional to follow up treatment response, as well as to predict prognosis. In combination with AFP, ßHCG is an excellent marker for monitoring the pa­tients with germ tumors. On the other hand, approximately 10% of germ tumors do not synthesize tumor markers; hence, they can­not be used for diagnostic purposes and in the follow-up of these patients. Slightly ele­vated levels of ßHCG can be found also in the patients with breast, gastric, lung, liver, or colorectal cancer, but are irrelevant in clinical monitoring of these patients.19 Enzymes Certain enzymes that are produced more in­tensely if a malignant process is occurring in the organism can also be used as tumor markers. Prostatic acid phosphatase is an enzyme that is produced in normal prostatic tissue. Elevated serum concentrations (above 3 ng/ml) can be observed in the patients with prostatic cancer and usually correlate with an advanced phase of the disease when the tu­mor penetrates the prostatic capsule. The de­termination of prostatic acid phosphatase is therefore convenient for the discrimination of benign (hypertrophy) from malignant processes.20 Alkaline phosphatase exists in the form of iso-enzymes that are synthesized in the liver, bones or placenta. Elevated serum concentra­tions in patients with malignant disease usu­ally indicate a metastatic spread of the dis­ease into the liver and/or bones, and/or the presence of primary bone tumors (osteosar-coma).20 Neuron specific enolase (NSE) is a cytoplas­mic glycolytic enzyme that was primarily de­tected in the cells of neuroectodermal origin and in neuronal cells. The latter were proved to be in the tumor tissue of the tumors with neuroectodermal or neuroendocrine differen­tiation.21-23 Among other (nonspecific) markers in this group, we should not leave out lactic dehydro­genase (LDH) that is quite often elevated in the sera of patients with malignant lymphomas and germ tumors (seminomas), . glutamyl transpeptidase (GGT) that indicates cholestasis (often elevated because of liver metastasis), and thymidine kinase (TK) that helps to evalu­ate the disease spread in the patients with leukemia, lymphoma, brain tumors, small cell lung cancer, and breast cancer. Tumor-associated antigens This is a heterogeneous group of markers that comprises various membrane structures of tumor cells. Updated technology exploits the possibility of producing specific monoclonal antibodies against certain antigenic structure that is most characteristic for the type of tu­mor cells. Therefore, the markers in this group are more specific for the type of malig­nancy than the others and quite often their serum concentrations reflect more accurately the growth or regression of the tumor mass. Carcinomic antigen 15-3 (CA 15-3) is pro­duced in the secretory epithelium (of the breast, lungs, gastrointestinal tract, uterus, etc.) and can be found frequently in the ex­cretions of healthy adults. Elevated serum concentrations of this marker (above 30 U/ml) are detected predominately in the pa­tients with breast cancer; however, raised lev­els of CA 15-3 can be observed also in other malignancies as lung, prostatic, ovarian, cer­vical, and gastrointestinal cancer.13, 24 Hence, CA 15-3 is no specific marker either for the organ or for the type of tumor. Despite that, CA 15-3 is a good indicator of treatment re­sponse and disease course in breast cancer patients (whose tumors produce that anti­gen). The serum level of CA 15-3 can be in­fluenced also by different non-tumoral dis­eases of the breast, by benign breast tumors, but the values rarely exceed 40 U/ml. Raised concentrations can be observed in approxi­mately 8% of pregnant women between 38th and 40th week of gestation.3 Concomitant determination of CA 15-3 and CEA in breast cancer patients increases the sensitivity (reduces the number of false nega­tive determinations), while retaining a sub­stantial specificity of the procedure (rather low number of false positive determinations). Carcinomic antigen 125 (CA 125) is a charac­teristic marker for ovarian cancer (it is elevat­ed in more than 80% of patients with non-mu­cinous ovarian cancer). During embrional de­velopment, CA 125 is produced in celomic epithelium, Muellerian ducts, epithelial cells of the pleura, pericardium, and peri­toneum.25,26 In adults, CA 125 can be found in mucosa of the cervix uteri and in the lung parenchyma, however, it is not produced by healthy ovarian tissue. Elevated concentra­tions of CA 125 (above 35 U/ml) can be found not only in the patients with ovarian cancer but also in the patients with benign or malig­nant gynecological diseases (endometriosis, ovarian cysts, endometrial cancer, cervical cancer) as well as in the patients with non-gy­necological malignancies (lung cancer, pro-static cancer, peritoneal malignant mesothe­lioma). In the group of patients with ovarian cancer, CA 125 is most reliable and applica­ble in the follow-up of patients with epithelial and undifferentiated ovarian cancer.27 Carcinomic antigen 19-9 (CA19-9) is a glycol-ipid and actually represents a modified Lewis’s hapten from the blood group system. CA 19-9 is frequently elevated in the serum of patients with gastrointestinal tumors. The marker is slightly more specific for pancreat­ic and liver cancer, yet quite often raised con­centrations can be found in patients with col-orectal, gastric, and ovarian cancer. In rela­tively high concentrations, it can be detected in healthy adults in the prostatic fluid, gastric fluid, amniotic fluid, and in the excretions of the pancreas and duodenum. Therefore, only determinations in the serum or plasma are ra­tional because there the concentrations will be elevated only in case of disease.28 Prostate specific antigen (PSA) is a serine pro­tease first isolated from the tissue extract of the prostate and sperm. It is produced in the prostate tissue and excreted in the prostate fluid where it can have very high concentra­tions. The role of this serine protease is to pre­vent coagulation of sperm. In healthy persons, very minute amounts of PSA enter the blood­stream so that its concentration in serum is rather low. In the patients with prostatic dis­ease, the amounts of PSA that enter blood­stream increase significantly (especially in case of prostatic malignancy), thus generating high serum concentrations. This marker is substantially specific for prostatic cancer and its serum concentrations reflect very well the tumor burden. Due to its high sensitivity and extraordinary correlation with tumor burden we prefer applying PSA to prostatic acid phos­phatase in the follow-up of prostatic cancer patients.20,29 Besides, the method (together with other procedures) is being utilized in the screening of prostatic cancer in the group of males over 50 years old who have more risk factors (e.g. prostatic cancer in patient’s fa­ther, breast cancer in patient’s mother or sis­ter, obesity, prostatic cancer in more than one generation). With the determination of differ­ent forms of PSA (i.e. total, bound or free), and with a proper evaluation of free to total PSA ratio, it is possible to predict quite confidently if the patient suffers from a benign or malig­nant prostatic disease.30 Special serum proteins This group comprises various proteins. One of the best known is feritin that binds iron in-tracellularly and is responsible for detoxica­tion (e.g. binding of free radicals). Under nor­mal circumstances, high concentrations of feritin can be found in the liver, spleen, and bone marrow. Normal serum level of feritin ranges from 8 to 440 ng/ml. Raised concen­trations can be observed in the patients with acute leukemia, lymphomas (especially Hodgkin’s lymphomas), lung, liver, and pro-static cancer.2 Thyroglobulin is an intracellular glycopro­tein responsible for the production and stor­age of thyroxine. In low concentrations, it can be found in the sera of most healthy persons (0-75 ng/ml), while extremely high concentra­tions can be detected in the patients with well differentiated follicular (rarely anaplastic) thyroid carcinoma. Conversely, in the pa­tients with medullary thyroid carcinoma, the serum levels of thyroglobulin do not follow the development and course of malignancy.2,3 Beta-2-microglobulin is a protein that is identi­cal with the HLA light chain and thus appears on the cell membrane of almost all differentiat­ed cells. Raised serum concentrations can be observed in the patients with lung, breast, pan­creatic, colorectal cancer, as well as lymphomas and chronic lymphoid leukemia (CLL).2,3 S-100 protein was first isolated from bovine brain. Normal serum concentration of this marker is below 0.3 ng/ml. In addition to be­ing a good indicator of traumas to CNS, S-100 can be applied as tumor marker in the pa­tients with neurinoma, glioblastoma, astrocy­toma, and meningeoma. It has a special role as prognostic factor and in the follow-up of the patients with malignant melanoma (<0.3 ng/ml - 85% 3 years' survival; 0.3-0.6 ng/ml ­50% 3 years' survival; >0.6 ng/ml - 10% 3 years' survival).31,32 Miscellaneous markers This group involves markers the production of which correlates perfectly with the changes in velocity of cellular proliferation. It is a het­erogeneous group of substances which are not specific for the type of tumor but general­ly indicate the presence of a malignant process. The group comprises polyamines, nucleosides and tissue polypeptide antigen (TPA). Polyamines like spermine, spermidine and putrescine were detected in elevated concen­trations in urine in cases of a rapid regenera­tion of cells of certain tissue. Nucleosides as dimethylguanosine and pseudouridine are components of RNA that (like polyamines) enter the circulation in larg­er amounts in cases of enhanced cellular pro­liferation. Tissue polypeptide antigen (TPA) is likewise a nonspecific marker of enhanced cellular prolif­eration. The molecular mass of this polypep-tide is approximately 180 KD and the molecule is composed of different cytokeratin units – i.e. of cytokeratin 19 (44%), cytokeratin 18 (36%), and cytokeratin 8 (30%). During embrional de­velopment, it is produced in various embrional tissues as well as in the placenta. In adults, TPA is a part of cellular membranes (cytoskeleton) of normal and tumor cells. It is synthesized during S phase of the cell cycle and its concen­trations reflect the velocity of cellular prolifera­tion. A more rapid cellular proliferation de­mands a more rapid synthesis of TPA, and consequently, larger amounts of this polypep-tide enter the circulation. Therefore, TPA is a common (universal) marker that goes together with pathological cellular proliferation (that is usually present in malignant transformation) regardless of the type or localization of the tis­sue. Unlike in other markers, the serum con­centrations of TPA poorly reflect the tumor burden. Normal serum concentrations of TPA are below 90 U/ml and concentrations higher than 120 U/ml can be either a consequence of a malignant or of a benign process.2 Biological factors that affect serum concen­trations of tumor markers Regarding the facts, that no tumor marker is ideal and that the substances that are being applied as tumor markers are not synthe­sized exclusively as a consequence of malig­nancy, I present some of the most common factors that affect serum concentrations of tumor markers (Table 2). When to determine tumor markers? Tumor markers are determined in such a way that we gain as much as possible of clinically useful data. We have to prepare an approxi­mate concept that includes the determination of markers (i) prior to surgery or any kind of treatment (chemotherapy, radiotherapy, bio­logical therapy, or hormonal therapy); (ii) after the surgery, during the treatment, and after the treatment once in 3 to 6 months' period during the first and second year, then once yearly or at regular controls; (iii) in case of sus- Table 2. Factors (in addition to malignant disease) that affect serum concentrations of tumor markers False positive results presence of inflammatory processes; benign liver diseases and consequential disturbancies in metabolism and excretion (AFP, TPA, CEA, CA 19-9, CA 15-3); disturbancies of renal function (beta-2-microglobulin, calcitonin, PSA, CEA, CA 19-9, CA 15-3); extensive tumor necrosis; as a consequence of diagnostic and therapeutic procedures (digitorectal examination, mamography, surgery, radio and chemotherapy); as a consequence of different physiological conditions (pregnancy - ßHCG, CA 125, CA 15-3, MCA, AFP, menstrual cycle - CA 125). False negative results complete absence of production (e.g. CA 19-9 in Le(a-b-) persons); insufficient expression of a certain antigenic determinant (or production in only some of tumor cells); insufficient blood circulation in the tumor; production of immune complexes with autoantibodies; rapid degradation and clearance of antigens; pected relapse or disease progression; (iv) pri­or to introduction of a novel treatment; (v) at least 3 weeks after the introduction of a novel treatment; (vi) 2 to 3 weeks after the determi­nation of raised concentrations of the marker. The proposed concept of tumor marker de­terminations is applicable in the majority of tu­mor markers. Certainly, the demands in clini­cal oncology are quite often different and we have to adjust the dynamics of determinations in accordance with the type and properties of the tumor, and with the planned methods of treatment. In other words, the above men­tioned concept represents only an approximate model that has to be further modified in each patient specifically regarding the fact that each patient is a control to himself (monitoring of the dynamics of serum concentrations). Conclusion Determination of tumor markers for moni­toring the course of malignant disease is an established and often an irreplaceable onco-logical laboratory method. Tumor markers are reliable predominately in monitoring the treatment response, as well as in early detec­tion of disease recurrence (prior to develop­ment of clinically notable signs). Due to their incompetences, tumor markers' determina­tions can be only exceptionally applied as screening methods or as the sole diagnostic tool; however, in combination with other di­agnostic methods, they play an important role in the diagnostic process and in treat­ment planning. Besides, by combining vari­ous tumor markers we can achieve a greater specificity and sensitivity in the follow up of one type of malignancy. The simplicity and noninvasiveness of the method for the deter­mination of tumor markers enable monitor­ing the disease also in the patients not eligi­ble for other types of diagnostic procedures. On account of individual differences in the serum concentrations of each individual tumor marker, we recommend multiple determina­tions of tumor markers and monitoring the dy­namics of serum concentrations (even in cases when serum concentrations are below the cut­off values). It would be ideal to determine the level of tumor markers in each patient before treatment, several times between the treatment (depending upon the type of treatment, type of malignancy, and the sort of tumor marker), and after the treatment. Tumor markers should be monitored also for a certain period after the treatment has been finished, best at regular con­trol examinations (once in six months or once yearly). This kind of follow up will enable a time­ly detection of disease recurrence even in asymptomatic patients. From a single determi­nation of tumor markers we can find out whether the malignancy has developed or not and, if it has, what is its extent, but only if the concentrations are very high. In patients who are undergoing just symptomatic (palliative) therapy, the determination of tumor markers no longer makes sense. If the patient had normal concentrations of tumor markers prior to any kind of treatment, and if these concentrations did not change (increase or decrease) at disease progression or regression, it is very unlikely that concentrations will be elevated in case of disease recurrence. In these patients, tumor marker lev­els need not to be followed either during treat­ment or after treatment. Hence, it is important to make the first determination of tumor mark­ers prior to any treatment in order to disclose the group of patients in whom tumor marker deter­minations are not sensible. References 1. Hill RP, Tannock IF. Introduction: cancer as a cel­lular disease. In: Tannock IF, Hill RP, eds. The ba­sic science of oncology. New York: Mc Graw-Hill, 1992: 1- 4. 2. Rubin P, ed. Clinical oncology: A multidisciplinary 7th approach for physicians and students, ed. Philadelphia: W.B. Saunders, 1993: 791. 3. Novakovic S. Pregled pomembnejših tumorskih oz-nacevalcev v klinicni onkologiji. Ljubljana: Onkološki inštitut, 2000: 64. 4. Clinical practice guidelines for the use of tumor markers in breast and colorectal cancer. Adopted on May 17, 1996, by the American Society of Clinical Oncology. J Clin Oncol 1996; 14: 2843-77. 5. Bast RC, Ravdin P, Hayes DF, Bates S, Fritsche H, Jessup JM, et al. 2000 update of recommendations for the use of tumor markers in breast and col-orectal cancer: Clinical practice guidelines of the American Society of Clinical Oncology. J Clin Oncol 2001; 19: 1865-78. 6. Perkins LG, Slater DE, Sanders KG, Prichard GJ. Serum tumor markers. Am Fam Physician 2003; 68: 1075-82. 7. Malkin A. Tumor markers. In: Tannock IF, Hill RP, eds. The basic science of oncology. New York: Mc Graw-Hill, 1992: 196-206. 8. International germ cell consensus classification: A prognostic factor-based staging system for metastatic germ cell cancers. International Germ Cell Cancer Collaborative Group. J Clin Oncol 1997; 15: 594-603. 9. Mazumdar M, Bajorin DF, Bacik J, Higgins G, Motzer RJ, Bosl GJ. Predicting outcome to chemotherapy in patients with germ cell tumors: The value of the rate of decline of human chorion-ic gonadotropin and alpha-fetoprotein during therapy. J Clin Oncol 2001; 19: 2534-41. 10. Einhorn LH. Treatment of testicular cancer: A new and improved model. J Clin Oncol 1990; 8: 1777-81. 11. Novakovic S, Serša G. Tumor markers in clinical oncology. In: Benulic T, Serša G, Kovac V, eds. Advances in Radiology and Oncology. Ljubljana: Radiologia Iugoslavica, 1992: 214-20. 12. Ballesta AM, Molina R, Filella X, Jo J, Gimenez N. Carcinoembryonic antigen in stanging and follow­up of patients with solid tumors. Tumour Biol 1995; 16: 32-41. 13. Jezeršek B, Cervek J, Rudolf Z, Novakovic S. Clinical evaluation of potential usefulness of CEA, CA 15-3, and MCA in follow-up of breast cancer patients. Cancer Lett 1996; 110: 137-44. 14. Fletcher RH. Carcionembryonic antigen. Ann Inter Med 1996; 104: 66-74. 15. Abelev GI. Study of the antigenic structure of tu­mors. Acta Int Cancer 1963; 19: 80-5. 16. Johnson PJ. The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma. Clin Liver Dis 2001; 5: 145-59. 17. Yuen MF, Cheng CC, Lauder IJ, Lam SK, Ooi CG, Lai CL. Early detection of hepatocellular carcino­ma increases the chance of treatment: Hongkong experience. Hepatology 2000; 31: 330-5. 18. Fowler JE, Platoff GE, Kubrock CA, Stutzman RE. Commercial radioimmuno assay for beta subunit of human chorionic gonadotropin: Falsely positive determinations due to elevated serum luteinizing hormone. Cancer 1982; 49: 136-9. 19. Bosl GJ, Bajorin DF, Sheinfeld J, Motzer RJ, Chaganti RS. Cancer of the testis. In: DeVita VT, Hellman S, Rosenberg SA, eds. Cancer. Principles 6th and Practice of Oncology, ed. Philadelphia: Lippincott, Williams & Wilkins, 2001: 1491-518. 20. Cooper HC, Robinson MRG, Whelan P, Ferro AM. Tumor markers in prostate cancer. Cancer 1992; 70: 225-9. 21. Schmechel D, Marangos PJ, Zis AP, Brightman M, Goodwin FK. Brain enolase as specific markers of neuronal and glial cells. Science 1978; 199: 313-5. 22. Carney DN, Ihde DC, Cohen MH, Marangos PJ. Serum neuron-specific enolase: a marker for dis­ease extent and response to therapy of small-cell lung cancer. Lancet 1982; I: 583-5. 23. Wick MR, Brend MD, Scheithauer W, Kovacs K. Neuron specific enolase in neuroendocrine tu­mors of the thymus, bronchia and skin. Am J Pathol 1983; 79: 703-7. 24. Geraghty JG, Coveney CE, Sherry F, O’Higgins JN, Duffy JM. CA 15-3 in patients with locoregional and metastatic breast carcinoma. Cancer 1992; 70: 2831-4. 25. Tuxen MK, Soletormos G, Dombernowsky P. Tumor markers in the management of patients with ovarian cancer. Cancer Treat Rev 1995; 21: 215-45. 26. Novakovic S, Serša G, Krošl G, Plesnicar S. CEA, MCA and CA 125 tumor markers in pregnant women. Radiol Iugosl 1990; 24: 79-84. 27. Jacobs IJ, Skates SJ, MacDonald N, Menon U, Rosenthal AN, Davies AP, et al. Screening for ovar­ian cancer: A pilot randomized controlled trial. Lancet 1999; 353: 1207-10. 28. Steinberg W. The clinical utility of the CA 19-9 tu-mor-associated antigen. Am J Gastroenterol 1990; 85: 350-5. 29. Woodrum LD, Brawer KM, Partin WA, Catalona JW, Southwick CP. Interpretation of free prostate specific antigen clinical research studies for the detection of prostate cancer. J Urol 1998; 159: 5-12. 30. Catalona WJ, Partin AW, Slawin KM, Brawer MK, Flanigan RC, Patel A, et al. Use of the percentage of free prostate-specific antigen to enhance differ­entiation of prostate cancer from benign prostatic disease: A prospective multicenter clinical trial. JAMA 1998; 279: 1542-7. 31. Ghanem G, Loir B, Morandini R, Sales F, Lienard D, Eggermont A, Lejeune F. On the release and half-life of S100B protein in the peripheral blood of melanoma patients. Int J Cancer 2001; 94: 586­90. 32. Jury SC, McAllister JE, MacKie MR. Rising levels of serum S100 protein procedure other evidence of disease progression in patients with malignant melanoma. Br J Derm 2000; 143: 269-74. Radiol Oncol 2004; 38(2): 85-92. review Clinical utility of serine proteases in breast cancer Tanja Cufer Institute of Oncology, Ljubljana, Slovenia The serine protease uPA and its inhibitor PAI-1 are involved in the degradation of tumor stroma and base­ment membrane. The independent prognostic value of serine protease urokinase-type plasminogen activator uPA and its inhibitor PAI-1 in breast cancer has been almost uniformly confirmed in numerous individual studies as well as in a meta-analysis, including 18 data sets of more than 8,000 patients. According to these observations, the risk of relapse in node negative patients with low levels of uPA and PAI-1 is less then 10%; these patients could be spared from toxic adjuvant systemic therapy. Clinically relevant and even more im­portant is the information that uPA and its inhibitor PAI-1 may also have a predictive value for response to either hormonal or cytotoxic therapy in early breast cancer. According to our data obtained from alto­gether 460 operable breast cancer patients, uPA and PAI-1 may have a predictive value for the response to hormone therapy, but not to chemotherapy. The high PAI-1 levels were associated with a higher risk of re­lapse in the patients without adjuvant systemic therapy (HR 2.14; C.I. 95%= 0.48-9.56; p=0.321) and in the patients treated with chemotherapy (RR 2.48; C.I. 95%= 1.35-4.57; p=0.003). However, in the patients treat­ed with adjuvant hormone therapy, either alone or in combination with chemotherapy, the prognostic val­ue of uPA and PAI-1 was diminished. Moreover, high levels of both uPA and PAI-1 were associated with a lower risk of relapse (HR 0.79; p=0.693 and HR 0.26 p= 0.204, respectively). On the basis of currently available evidence, serine protease uPA and its inhibitor PAI-1 are certainly the markers that improve a proper selection of candidates for adjuvant systemic therapy and may also be the markers that could facili­tate treatment decision in each individual patient, which is of utmost importance. Key words: breast neoplasms; urinary plsminogen activator; plsminogen activator inhibitor 1; prognosis Received 24 March 2004 Accepted 8 April 2004 Correspondence to: Tanja Cufer, MD, PhD, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 656; Fax: +386 1 5879 454. E-mail: tcufer@onko-i.si This paper was presented at “3rd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. Introduction Breast cancer is the most common malignan­cy in females all over the world; more than a million of women diagnosed with this disease each year. Tremendous improvements in the treatment of this disease have been made dur­ing the last decades and the survival rates im­proved from around 50% to more than 70% in operable disease. This improvement is mostly due to early detection of the disease and to the introduction of adjuvant systemic therapy.1 According to the meta-analysis, in which data obtained from more than 50,000 patients par­ticipating in different studies were included, adjuvant systemic therapy was found to re­duce the risk of death by approximately one third in all operable breast cancer patients, ir­respective of their individual risk of death based on traditional prognosticators, such as lymph node involvement, tumor stage, tumor grade, etc.2,3 The absolute benefit is the same in all patient sub-groups and only the relative benefit is much higher in the high risk sub­group of patients. According to the so-called classical clinico-pathological data, the majori­ty of patients are categorized into average and high-risk groups and treated by some kind of adjuvant therapy, although it is well known that less than 50% of all patients and only 30% of patients with node- negative disease are to develop metastases without any systemic treatment. Therefore, there is an urgent need to identify new prognostic markers that will enable us to categorize patients according to their individual risk of relapse more precisely. On the other hand, there is a continuous search for new biological markers, which could not only prognosticate the faith of the disease, but would rather predict the response of each individual patient to the particular therapy. This approach ensures that both the individual patient and the society as a whole benefit, by minimizing the treatment-related side effects and maximizing cure rates. There are a lot of new, prospective prognostic and predictive factors under investigation and ser­ine proteases are among them.4 Prognostic factors in breast cancer A prognostic factor for breast cancer is de­fined as any measurement available at the time of diagnosis or surgery and associated with disease-free or overall survival in the ab­sence of systemic adjuvant therapy. Based on the knowledge of prognostic factors, the risk of relapse for each individual patient treated solely by local therapy could be estimated. Nowadays, tumor size, lymph node status, histological type and grade, mitotic figure count, and hormone receptors are considered the standard prognostic factors according to which the adjuvant therapy is planned. The prognostic value of these, so-called standard prognosticators in early breast cancer, has been confirmed in multiple studies and their value as category I prognostic factors has been recognized at a 1999 Consensus Conference held under the auspices of the College of American Pathologists.5 According to these factors, only about 10% of patients are considered to have minimal, less than 10% risk of relapse and are therefore not of­fered adjuvant systemic therapy.6,7 There are additional new prognostic fac­tors under investigation, which may better separate patients according to their own risk of relapse and spare the patients with low risk of relapse from unnecessary toxic treat­ment. Undoubtedly serine protease, uroki-nase-type plasminogen activator uPA and its inhibitor PAI-1 are among them. The prog­nostic value of both uPA and PAI-1 has al­ready been confirmed at the highest level I ev­idence, in the meta-analysis and in the prospective randomized trial.8,9 Prognostic value of uPA and PAI-1 The serine protease uPA and its inhibitor PAI-1 are involved in the degradation of the tumor stroma and basement membrane. A critical balance of uPA, its cell surface recep­tor uPA-R, and PAI-1 are the prerequisites for efficient focal proteolysis, adhesion, and mi­gration, and hence, the subsequent tumor cell invasion and metastases.10 In 1988, Duffy with co-workers was the first to report that high primary tumor enzymatic activity of uPA is associated with poor survival of breast can­cer patients.11 Shortly, the same authors did not only confirm this finding in a larger study, but also demonstrated the independ­ent prognostic impact of uPA on the disease-free survival and overall survival.12 This in­formation was confirmed and even strength­ened by the finding of the German group that not only the enzymatic activity, but to an even greater extend also the tumor tissue antigen level of uPA is of prognostic rele­vance.13 The prognostic impact of uPA has since been confirmed by several investiga­tors14-24; and PAI-1 was also found to be an independent prognostic marker in breast can­cer (Table 1).14-16,18-25 Surprisingly, high levels of PAI-1 were found to be associated with a higher risk of recurrence and a shorter overall survival in breast cancer. The data from basic research may help to explain the finding that PAI-1 does not act as a true uPA inhibitor, but rather as a proteolitic factor. PAI-1 was found to be indispensable for optimal focal proteol­ysis, adhesion, and migration, and subse­quent tumor cell.10 The independent prognostic value of serine protease uPA and its inhibitor PAI-1 in breast cancer has been confirmed in a meta-analysis, including 18 data sets of 8,377 patients with a median follow-up of 6.5 years.8 In this meta-analysis, uPA, PAI-1 as well as the combina­tion of both were found to be the strongest prognostic factors in breast cancer, next to lymph node involvement, irrespective of the type of surgery, year of surgery, publication status of data sets, as well as menopausal sta­tus and lymph node status.26 The independent prognostic value of uPA and PAI-1 was also confirmed in a prospective randomized multi-center therapy trial in node-negative breast cancer (“Chemo N0”), randomizing high­uPA/PAI-1 patients to adjuvant CMF or obser­vation only, which was performed in 13 German academic centers and in our center in Slovenia between 1993-1999.9 In this study, uPA and PAI-1 were prospectively determined in detergent extracts of primary tumor tissue using commercially available ELISA assays (American Diagnostica Inc., Greenwich, CT). The first scheduled interim analysis of this Table 1. Prognostic impact of uPA and PAI-1 in early breast cancer – overview of selected references Author (year) Country Factors Patients (N0) Follow up Reference analyzed (median, months) Duffy (1988)11 Ireland uPA 52 (25) 17 Cancer 62:531 Jänicke (1993)13 Germany uPA, PAI-1 247 (101) 30 BCRT 24:195 Grřhndahl-Hansen (1993)15 Denmark uPA, PAI-1 119 (12) 102 Cancer Res 53: 2513 Foekens (1994)16 Netherlands uPA, PAI-1 657 (273) 48 J Clin Oncol 12:1648 Fernö (1996)17 Sweden uPA 688 (265) 42 Eur J Cancer 32:793 Kim (1998)18 Japan uPA, PAI-1 130 (130) 53 Clin Cancer Res 4:177 Kute (1998)19 USA uPA, PAI-1 168 (168) 58 BCRT 54:147 Knoop (1998)20 Denmark uPA, PAI-1 429 (178) 61 Br J Cancer 77:932 Harbeck (1999) 21 Germany uPA, PAI-1 316 (147) 77 Breast Cancer Res Treat 54:147 Bouchet (1999)22 France uPA, PAI-1 499 (233) 72 J Clin Oncol 17:3048 Foekens (2000)23 Netherlands uPA, PAI-1 2780 (1405) 88 Cancer Res 60:636 Jänicke (2001)9 Germany uPA, PAI-1 556 (556) 32 JNCI 93:913 Konecny (2001)24 USA / Germany uPA, PAI-1 587 (283) 26 Clin Cancer Res 7:2448 Look (2002)8 Europe uPA, PAI-1 8377 (4676) 79 JNCI 94:116 study confirmed the independent prognostic impact of both uPA/PAI-1 for the disease-free survival; within the frame of this study, the previously optimized cut-offs for uPA and PAI­1 used to distinguish between low and high uPA and PAI-1 were validated.21 According to the data obtained in the frame of this prospec­tive study, the low-risk group, identified by uPA/PAI, encompasses as much 50% of node-negative patients, which is much more than 10% of node-negative patients, categorized in­to the low-risk group according to the clinical-pathological criteria. The risk of relapse in the patients with low levels of uPA and PAI-1 was found to be only 6.7% at 3-years, and these pa­tients could be spared from toxic adjuvant sys­temic therapy, especially chemotherapy. The uniformly found prognostic value of uPA and PAI-1 in multiple individual studies as well as in a prospective randomized trial and meta-analysis is quite unique among prognostic markers. For most of the so-called established and very well recognized prog­nostic factors, such as tumor size, histological type and grade, hormone receptor status as well as mitotic index, the data are far from be­ing so consistent. In addition, the standardi­zation of the method of determining, inter­preting, and reporting the uPA and PAI-1 lev­els was possible, which is a prerequisite for a clinically useful marker. The immunoenzy­matic assays are standardized, international quality assurance of the kit is guaranteed, 27 and the optimal cut-off values have been val­idated.21 In addition, the extracts, prepared from as little as 100 µg tumor tissue, corre­sponding to about 1 µg protein extract, suf­fice for testing. All these facts make a serine protease uPA and its inhibitor PAI-1 an ideal prognostic factor for routine clinical use. Predictive factors in breast cancer To optimize treatment approaches and to im­prove the results of treatment, new biological markers, which will help us not only to predict the course of disease, but will also be able to predict the response to specific therapy in each individual patient, the so-called predic­tive markers are urgently needed. A predictive factor is any measurement associated with re­sponse or lack of response to a particular ther­apy. If, in addition to prognostic value, a mark­er also has a predictive value for response to a particular therapy, its prognostic strength could be increased or diminished, which de­pends on the fact whether worse or better prognosis correlates with treatment efficacy. After four decades of systemic treatment, we are still faced with only two established predictive factors in breast cancer: hormone receptor status for response to hormonal therapy and HER-2 status for response to HER-2 antibody trastuzumab. Thus, the pa­tients whose tumors strongly express hor­mone receptors are likely to respond to ta­moxifen or other hormonal manipulates, while the patients with receptor-negative dis­ease do not benefit from hormonal therapy.3 Similarly, HER-2 overexpression in primary tumors predict a better response to trastuzumab.28 There are also some new, pu­tative markers for the response to either hor­monal therapy or different chemotherapeutic agents, such as HER-2, p53 and BCL-2, which are under investigation. However, the data on their predictive value are still insufficient and even contradictory4, which enables us to use them as a guide in the selection of systemic therapy. According to our data,29 as well as the data published by Munich and Rotterdam group,30,31,32 serine protease uPA and its in­hibitor PAI-1 may also have a predictive value for the response to either hormonal or cyto­toxic chemotherapy in early breast cancer. Predictive value of uPA and PAI-1 The latest observations, based on the data provided by the patients mostly treated by some kind of adjuvant systemic treatment, showed a possible loss of the prognostic val­ue of uPA and PAI-117,29-32, which indicates that the level of proteases in the primary tu­mor could also predict a response to systemic therapy. In addition, the data from meta-analysis show that the bad prognostic impact of high levels of either uPA or PAI-1 was most pronounced in the sub-groups of patients with node-negative disease and in the sub­groups of patients treated before nineties which are the sub-groups that did not receive adjuvant systemic treatment in such an ex­tent as the sub-groups of patients with node-positive disease, and the sub-groups of pa­tients treated in the last decade. So far, findings on the predictive value of uPA and its inhibitors for response to hor­monal therapy are limited and, to some ex­tent, even contradictory. Preclinical data ob­servations that estrogens as well as antiestro-gens modulate the expression of uPA and tu­mor cell growth in vitro33-35 suggested that the levels of serine proteases in the primary tumor could be predictive for the efficacy of hormonal manipulations in breast cancer. In a large group of 235 patients with metastatic breast cancer, high levels of both uPA and PAI-1 in primary tumor predicted a poor re­sponse to tamoxifen.36 On the contrary, the observations made in the frame of the Munich group30,31 and Swedish group17 pointed out that high levels of uPA17,30 and PAI-130 or the combination of uPA/PAI-131 in the primary tumor may predict a better re­sponse to the adjuvant tamoxifen treatment. However, in a large study conducted in an­other Swedish center, PAI-1 was not found to predict any benefit of adjuvant tamoxifen treatment.37 Similarly, in the largest data set obtained from two different centers (Munich and Rotterdam), no significant interaction be­tween the combination of uPA/PAI-1 and the efficacy of adjuvant tamoxifen was found in altogether 3,424 patients.32 Our data, ob­tained from 460 early breast cancer patients, speak in favor of the predictive value of high uPA and PAI-1 for a good response to hor­monal therapy in adjuvant setting.29 The data on the possible predictive value of serine proteases for the response to chemotherapy are even more scarce and maybe less contradictory. In the first pub­lished study no significant influence of PAI-1 levels on the response to the neoadjuvant an-thracycline based chemotherapy in locally ad­vanced breast cancer was found.38 Recently, the results from Munich pointed out that high levels of PAI-1 alone30 or the combination of uPA/ PAI-131 may predict for a better re­sponse to adjuvant chemotherapy. This ob­servation was confirmed in a large data set obtained from two different centers (Munich and Rotterdam); in a collective of 3424 pa­tients the benefit from the adjuvant chemotherapy was found to be significantly higher in the subgroup of patients with high levels of uPA/PAI-1.32 According to our data, obtained from 460 operable breast cancer patients, uPA and PAI-1 may have a predictive value for the re­sponse to hormone therapy, but not to chemotherapy.29 In our study, the high uPA levels were found to be associated with a higher risk of relapse in the patients without any adjuvant systemic therapy and in the pa­tients treated with adjuvant chemotherapy (HR 1.37 and HR 1.44, respectively; non-sig­nificant). The high PAI-1 levels were also as­sociated with a higher risk of relapse in the patients without adjuvant systemic therapy (HR 2.14; C.I. 95%= 0.48-9.56; p=0.321) and in the patients treated with chemotherapy (RR 2.48; C.I. 95%= 1.35-4.57; p=0.003) (Table 2). However, in the patients treated with ad-juvant hormone therapy, either alone or in combination with chemotherapy, the prog­nostic value of uPA and PAI-1 was dimin­ished, even more, high levels of both uPA and/or PAI-1 were associated with a lower risk of relapse (HR 0.79; p=0.693 and HR 0.26 p= 0.204, respectively) (Table 2). The 3­ Table 2. Risk of relapse according to uPA and PAI-1 levels in sub-groups of patients with different adjuvant systemic therapies (adopted from reference 29). Prognostic factor Without ST HT HT or ChT&HT ChT ChT or ChT&HT (in ng/mg protein) (n=52) (n=141) (n=252) (n=156) (n=267) uPA p=0.71 p=0.693 p=0.914 p=0.381 p=0.194 (<3vs =3) HR=1.37 HR=0.79 HR=1.04 HR=1.44 HR=1.50 95%CI (0.27-7.1) 95%CI (0.24-2.56) 95%CI (0.50-2.18) 95%CI (0.64-3.25) 95%CI (0.81-2.77) PAI-1 p=0.321 p=0.204 p=0.8294 p=0.003 p=0.002 (<14 vs =14) HR=2.14 HR=0.26 HR=0.64 HR=2.48 HR=2.22 95%CI (0.48-9.56) 95%CI (0.03-2.06) 95%CI (0.25-1.68) 95%CI (1.35-4.57) 95%CI (1.35-3.66) ST=adjuvant systemic therapy; HT= adjuvant hormone therapy; ChT=adjuvant chemotherapy year DFS rates were not found to be influ­enced by uPA and/or PAI-1 in the patients treated with hormonal therapy, whereas in the patients treated with adjuvant chemotherapy and in the small group of pa­tients without any adjuvant therapy, the bad prognostic impact of high uPA or/and PAI-1 levels is obvious (Figure 1). Unfortunately, the data on the predictive value of serine protease uPA and its inhibitor PAI-1 are not as univocal as are the data on the prognostic value of these two markers. According to their mechanism of action, it Figure 1. A: 3-year DFS of the patients with low and high values of uPA in the sub-groups of patients treat­ed with different adjuvant systemic therapies (adopt­ed from reference 29). B: 3-year DFS of the patients with low and high values of PAI-1 in the sub-groups of patients treated with different adjuvant systemic ther­apies (adopted from reference 29). could be expected that uPA and PAI-1 does not play a major role in the prediction of the response to either hormonal therapy or chemotherapy in large tumors presented in the metastatic disease. However, serine pro­teases may have an impact on the growth and spread of micrometastatic disease under ad-juvant systemic therapy. The above data, al­though obtained in the frame of the retro­spective observations and contradictory in some way, are informative enough to strengthen our believes that serine protease uPA and its inhibitor PAI-1 may have predic­tive value for the response to either hormon­al therapy or chemotherapy in early breast cancer. To make any firm conclusions on the predictive value of proteases, the data from a larger data sets need to be obtained and ana­lyzed by means of different statistic tools, like interaction terms used, and the assessment of the predictive value of these markers in the frame of prospective clinical trial should be made. The ideal way to evaluate the predic­tive value of any marker is to set a prospec­tive randomized study with no treatment arm. However, due to ethical reasons, such a study is not feasible in adjuvant breast cancer treatment any more. Conclusion A part of breast cancer research is still fo­cused on finding prognostic markers which would help us to identify better the patients who could be spared adjuvant systemic ther­apy, but there is also an even more urgent need to identify new predictive markers that would help us to understand better which treatment option may be of benefit to each individual patient. On the basis of currently available data, serine protease uPA and its inhibitor PAI-1 are certainly the markers that improve a proper selection of patients for ad-juvant systemic therapy and could be used as a new prognostic tool that will reduce the risk of over-treatment with better identifica­tion of patients who need adjuvant systemic therapy. In addition, according to the en­couraging preliminary data, serine protease uPA and its inhibitor PAI-1 might also be the markers that will improve the treatment de­cision in each individual patient in the near future. References 1. Coleman MP, Gatta G, Verdecchia A, et al. Eurocare-3 summary: cancer survival in Europe at the end of the 20th century. Ann Oncol 2003; 14 Suppl 5: V128-V149. 2. Early Breast Cancer Trialists’ Collaborative Group. Polychemotherapy for early breast cancer: an overview of the randomized trials. Lancet 1998; 352: 930-42. 3. Early Breast Cancer Trialists’ Collaborative Group. Tamoxifen for early breast cancer: an overview of the randomized trials. Lancet 1998; 351: 1451-67. 4. Hamilton A, Piccart M. The contribution of molec­ular markers to the prediction of response in the treatment of breast cancer: A review of the litera­ture on HER-2, p53 and BCL-2. Ann Oncol 2000; 11: 647-63. 5. Fitzgibbons PL, Page DL, Weaver D et al. Prognostic factors in breast cancer. College of American Pathologists consensus statement 1999. Arch Pathol Lab Med 2000; 124: 966-78. 6. Goldhirsch A, Wood WC, Gelber RD, et al. Meeting Highlights: Updated international expert consensus on the primary therapy of early breast cancer. J Clin Oncol 2003; 21: 1-9. 7. National Institutes of Health Consensus Development Conference Statement: Adjuvant Therapy for Breast Cancer, November 1-3, 2000. J Natl Cancer Inst 2001; 93: 979-89. 8. Look MP, van Putten WLJ, Duffy MJ, et al. Pooled analysis of prognostic impact of uPA and PAI-1 in 8,377 breast cancer patients. J Natl Cancer Inst 2002; 94: 116-28. 9. Jänicke F, Prechtl A, Thomssen C, et al. for the German Chemo N0 Study Group. Randomized ad-juvant therapy trial in high-risk lymph node-nega­tive breast cancer patients identified by urokinase-type plasminogen activator and plasminogen acti­vator inhibitor type I. J Natl Cancer Inst 2001; 93: 913-20. 10. Schmitt M, Wilhelm OG, Reuning U, et al. The plasminogen activation system as a novel target for therapeutic strategies. Fibrinolysis 2000; 14: 114-32. 11. Duffy M, O’Grady P, Devaney D et al. Urokinase­plasminogen activator, a marker for aggressive breast carcinomas. Cancer 1988; 62: 531-3. 12. Duffy MJ, Reilly D, O’Sullivan C et al. Urokinase­plasminogen activator, a new and independent prognostic marker in breast cancer. Cancer Res 1990; 50: 6827-9. 13. Jänicke F, Schmitt M, Hafter R et al. Urokinase-type plasminogen activator (u-PA) antigen is a pre­dictor of early relapse in breast cancer. Fibrinolysis 1990; 4: 69-78. 14. Jänicke F, Schmitt M, Pache L, et al. Urokinase (uPA) and its inhibitor PAI-1 are strong, inde­pendent prognostic factors in node-negative breast cancer. Breast Cancer Res Treat 1993; 24: 195­208. 15. Grřhndahl-Hansen J, Christensen IJ, Rosenquist C, et al. High levels of urokinase-type plasmino-gen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res 1993; 53: 2513-21. 16. Foekens JA, Schmitt M, van Putten WLJ, et al. Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J Clin Oncol 1994; 12: 1648-58. 17. Fernö M, Bendahl PO, Borg Ĺ, et al. Urokinase plasminogen activator, a strong independent prog­nostic factor in breast cancer, analyzed in steroid receptor cytosols with a luminometric immunoas­say. Eur J Cancer 1996; 32A: 793-801. 18. Kim SJ, Siba E, Kobayashi T, et al. Prognostic im­pact of urokinase-type plasminogen activator (PA), PA inhibitor type-1 and tissue-type PA anti­gen levels in node-negative breast cancer: a prospective study on multicenter basis. Clin Cancer Res 1998; 4: 177-82. 19. Kute TE, Grřhndahl-Hansen J, Shao SM, et al. Low cathepsin D and low plasminogen activator type 1 inhibitor in tumor cytosols defines a group of node negative breast cancer patients with low risk of re­currence. Breast Cancer Res Treat 1998; 47: 9-16. 20. Knoop A, Andreasen PA, Andersen JA, et al. Prognostic significance of urokinase-type plas­minogen activator and plasminogen activator in­hibitor-1 in primary breast cancer. Br J Cancer 1998; 77: 932-40. 21. Harbeck N, Thomssen C, Berger U, et al. Invasion marker PAI-1 remains a strong prognostic factor after long-term follow-up both for primary breast cancer and following first relapse. Breast Cancer Res Treat 1999; 54: 147-57. 22. Bouchet C, Hacčne K, Martin JP, et al. Dissemination index based on plasminogen acti­vator system components in primary breast can­cer. J Clin Oncol 1999; 17: 3048-57. 23. Foekens JA, Peters HA, Look MP, et al. Urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res 2000; 60: 636-43. 24. Konecny G, Untch M, Arboleda J, et al. HER-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer. Clin Cancer Res 2001; 7: 2448-57. 25. Borstnar S, Vrhovec I, Cufer T. Prognostic value of plasminogen activator inhibitors in breast cancer patients. Int J Biol Marker 2002; 17: 96-103. 26. Look MP, van Putten WLJ, Duffy MJ, et al. Pooled analysis of prognostic impact of uPA and PAI-1 in breast cancer patients. Tromb Haemost 2003; 90: 538-48. 27. Sweep CGJ, Geurts-Moespot J, Grebenschikov N, et al. External quality assessment of trans-euro­pean multicentre antigen determinations (ELISA) of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) in human breast cancer tissue extracts. Br J Cancer 1998; 78: 1434-41. 28. Cobleigh MA, Vogel Cl, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastat­ic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999; 17: 2639. 29. Cufer T, Borstnar S, Vrhovec I. Prognostic and pre­dictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer. Int J Biol Marker 2003; 18: 106-15. 30. Harbeck N, Alt U, Krüger A, Berger U, et al. Prognostic impact of proteolytic factors (uPA, PAI­1, cathepsins B, D, L) in primary breast cancer re­flects effects of adjuvant systemic therapy. Clin Cancer Res 2001; 7: 2757-64. 31. Harbeck N, Kates R, Schmitt M. Clinical relevance of invasion factors uPA and PAI-1 for individual­ized therapy decisions in primary breast cancer is greatest when used in combination. J Clin Oncol 2002; 20: 1000-9. 32. Harbeck N, Kates RE, Look MP, et al. Enhanced benefit from adjuvant chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (n=3424). Cancer Res 2002; 62: 4617-22. 33. Levenson AS, Svoboda KM, Kwaan HC, Jordan VC. Agonist activity of antiestrogen-receptor com­plexes to regulate urokinase plasminogen activa­tor (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression in breast cancer cells. Cancer Letters 1998; 125: 215-20. 34. Xing RH, Mazar A, Henkin J, Shafaat AR. Prevention of breast cancer growth, invasion, and metastasis by antiestrogen tamoxifen alone or in combination with urokinase inhibitor B-428. Cancer Res 1997; 57: 3585-93. 35. Abidi SMA, Howard EW, Dmytryk JJ, Pento JT. The influence of antiestrogen on the release of plasminogen activator (uPA) by MDA-MB-231 and MCF-7 breast cancer cells. Clin Exp Metastasis 1998; 16; 235-41. 36. Foekens JA, Look MP, Peters HA, et al. Urokinase-type plasminogen activator and its inhibitor PAI-1: Predictors of poor response to tamoxifen therapy in recurrent breast cancer. J Natl Cancer Inst 1995; 87: 751-6. 37. Billgren MA, Rutqvist LE, Johansson H et al. The role of cathepsin D and PAI-1 in primary invasive breast cancer as prognosticators and predictors of treatment benefit with adjuvant tamoxifen. Eur J Cancer 2000; 30: 1374-80. 38. Pierga JY, Lainé-Bidron C, Beuzeboc P et al. Plasmonogen activator inhibitor-1 (PAI-1) is not related to response to neoadjuvant chemotherapy in breast cancer. Br J Cancer 1997; 76: 537-40. Radiol Oncol 2004; 38(2): 93-100. review Cathepsins and their inhibitors as tumor markers in head and neck cancer Primož Strojan Institute of Oncology, Department of Radiation Oncology, Ljubljana, Slovenia The invasion and metastasizing of tumor cells is closely connected with the disintegration of basement mem­branes and extracellular matrix. The carriers of these processes are different proteolytic enzymes, among them cysteine and aspartic cathepsins B, H, L and D as well, a group of ubiquitous lysosomal proteases, and en­dogenous inhibitors of the former, cystatins. The aim of the present review was to collect the current knowl­edge on the predictive and prognostic value of cathepsins and their inhibitors in squamous cell carcinoma of the head and neck. In this particular tumor type, the UICC/AJCC TNM-classification system and histopatho-logical characteristics of the tumors were found inadequate to reliably predict either the response to therapy or patients’ survival. Moreover, to date, no factor within the wide spectrum of biochemical and histological factors has yet been identified as reliably predicting the natural course of the disease or its response to ther­apy. To construct a prognostically meaningful tumor profile, new markers are intensively investigated. Key words: head and neck neoplasms; carcinoma, squamous cell; cathepsins, cystatins; prognosis Introduction During the past decades it has been shown that proteases of various classes can act as tu­mor progression factors in all stages of malig­nant progression. They can create a local envi­ronment that is supportive to the growth, sur­vival, and progression of a tumor through the Received 20 March 2004 Accepted 13 April 2004 Correspondence to: Primož Strojan M.D., Ph.D., Institute of Oncology, Department of Radiation Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone: +386 1 5879 110; Fax: +386 1 5879 400; E-mail: pstrojan@onko-i.si This paper was presented at the “3nd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. modulation of growth factor pathways, cell-cell adhesions, and cell-matrix adhesions.1 Cathepsins are lysosomal proteolytic en­zymes. With regard to their chemical composi­tion, cathepsins are glycoproteins, and the ma­jority of them belong to the group of endopep­tidases. In cancer, the most studied cathepsins are those of the cysteine and aspartic classes, cathepsins B, H, L and cathepsin D. Endogenous inhibitors of cysteine cathepsins belong to cystatins, which are subdivided into three families, i.e. stefins, cystatins, and kininogens, and thyropins, whereas the natu­rally occurring inhibitor of aspartic protease, cathepsin D, has not been found yet in men.2 Their contribution to the progression of breast, lung, and colorectal cancer has been most ex­tensively investigated at a preclinical level and as markers predicting the response to various treatment regimens and prognosis.3,4 Head and neck cancer Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most prevalent cancer worldwide, with a global yearly incidence of 500,000.5 Despite the evolution and refinement of multimodal treatments for the head and neck cancer in the last 20 years, 5-year survival rates have not improved significantly, remain­ing at the 50% level.6 Patients grouping with the use of conventional UICC/AJCC TNM stag­ing system and established histopathological characteristics of the primary tumor as well as its metastases on the neck in operated patients allow significant prognostic variation among the individuals within any of these groups. Compared to the carcinoma of the breast, lung or colorectum, the SCCHN falls into a far less investigated group of cancers. Apart from the studies focused on the activity or level of cathepsins and their inhibitors as determined in matched pairs of tumor tissue and normal mucosa 7, there is only a limited number of re­ports in literature assessing their predictive or prognostic value in this particular type of can­cer. In the present review, clinical data on the predictive and prognostic value of cathepsins B, H, L, and D, and their endogenous inhibitors stefin A, stefin B, and cystatin C in SCCHN have been assembled. The results of our own investigations are also presented and their ap­plicability in routine clinical practice discussed. Clinical relevance of cathepsins and their inhibitors in squamous cell carcinoma of the head and neck Markers for diagnosis The largest pertinent study was reported by Krecicki and Siewinski8, who measured serum cathepsin B-like activity in 110 sam­ples from patients with laryngeal carcinoma. Enzyme activity was significantly higher in malignant samples compared to healthy con­trols, whereas no difference was found be­tween the latter and the group with non-ma­lignant, mainly infectious diseases. In cancer patients, no false-negative serum values of cathepsin B-like activity were obtained. The sensitivity of the assay was calculated to be 100% and the specificity 97.5%.8 The method­ology used in this study was criticized by Bongers et al.9 They claimed that cathepsins B-like activity as determined by Krecicki and Siewinski is better referred to as “serum-pro­tease-activity”. However, when serum-pro­tease-activity was compared between pa­tients with SCCHN and non-cancer controls using the same methodology, and after the adjustment for alcohol and tobacco consump­tion, no difference was observed between the two groups. We used enzyme-linked im­munoassay (ELISA) and also found no alter­ations in serum cathepsin B between cancer patients and healthy controls, as was the case with cathepsin L.10 When the same group of patients was tested for cathepsins H and D, a significant increase of both enzymes was found in the sera of patients with cancer.11,12 The diagnostic value of the inhibitors of cysteine proteases was first studied by Siewinski et al.13 Using their method they were able to discriminate between total in­hibitory activity (free molecules and enzyme-inhibitor complexes) and that of active (en-zyme-free) and latent (enzyme-inhibitor com­plexes) forms of specific papain-like cysteine inhibitors; however, the method had no po­tential to assess the contribution of individual inhibitors. A significantly higher total in­hibitory activity (and of latent fraction) and lower activity of active fraction of the in­hibitors were found in cancer patients com­pared to healthy controls or patients with in­flammatory diseases. On the contrary, our ELISA measurements allowed quantification of specific inhibitors but were not able to rec­ognize different forms of the inhibitor mole­cules. Significantly higher stefin A 10 and cys­tatin C 14 levels were measured in the pa­tients’ sera than in controls, whereas levels of stefin B were significantly lower.10 Due to accompanying alterations of cathepsins and their inhibitors in non-malig­nant conditions1, it seems that their screening perspectives are quite limited. Furthermore, considerable overlap of their concentration ranges between patients and healthy controls or those with benign diseases further reduces their diagnostic strength. Predictive markers for lymph node metastasis The presence of lymph node metastases is the single most adverse independent prognostic factor in SCCHN that, in comparison to node negative patients, reduces a 5-year overall survival rate by about 50%.15 The use of tu-mor-related histopathological factors in pre­dicting lymph node metastases or advanced imaging techniques is not a reliable method. Furthermore, one should be aware that up to two-thirds of clinically N0 necks are classi­fied as node-free on histopathological exami­nation after surgery (pN0) and that some morbidity resulted even after most strictly limited dissections on the neck, and vice ver­sa, a substantial proportion of clinically posi­tive necks are actually pN0.16 The predictive value for lymph node metastasis most often correlated with in­creased Cathepsin D immunohistochemical staining. Grandour-Edwards et al.17 reported on 34 patients with oral cavity, oropharyngeal and hypopharyngeal SCCs. In node negative group, 13/15 (87%) tumors were found to be cathepsin D negative, whereas 11/19 (58%) pN+ tumors stained positive for cathepsin D. When adjusted for tumor stage and grade, cathepsin D positivity was nearly twice as likely to be associated with node metastasis.17 In two other studies, including exclusively oral cavity tumors, cathepsin D immunoreac­tivity correlated significantly with the pN-stage of the disease 18,19, whereas in the study of Resnick et al, limited to laryngeal tumors, no such relationship was found.20 Of the oth­er cathepsins, a statistically insignificant trend of higher levels of intensity of im­munoreactivity in pN+ group compared to pN0 group of oral cavity tumors was also re­ported for cathepsin B 18 and cathepsin L.21 The cathepsin inhibitors were studied by our team only. In the subgroup of patients with operable tumors (various subsites) and clinically positive neck nodes at presentation, stefin A and stefin B concentrations emerged as significant predictors of lymph-node in­volvement with tumor cells, i.e. pN-stage (Figure 1).22 Differentiating between the pa­tients with nodes enlarged due to inflamma­tion and those with metastatic nodes, a portion of patients could be spared more aggressive therapy and treatment-related side effects. On the other hand, in the patients with clinically undetectable nodes at diagnosis, stefins had no potential in predicting pN-stage of the dis­ease. However, clinical relevance of this find­ing is limited if surgery is technically correctly performed because no adjuvant therapy is in­dicated in pN0 subgroup, whereas pN+ pa­tients are highly curable with a moderate-dose of postoperative radiotherapy, i.e. =95% cure rate at a dose of 50 Gy.23 Predictive markers for response to therapy and for recurrent disease When assessing the efficiency of particular therapy by monitoring the presence of tumor cells in the body, surgery-based therapies and non-surgical therapies should be evaluated separately regarding the differences in mech­anisms of tumor cell eradication. Unfortunately, no study was found to have analyzed the predictive value of cathepsins and their inhibitors for response and for dis­ease recurrence to non-surgical therapies, i.e. Figure 1. Distribution of tumor concentrations of stefins between patients with histopathologicaly determined negative and positive necks, as measured in a group with clinically palpable nodes at presentation. The top and the bottom of the box represent the 25th and 75th percentiles, respectively, and the ends of the bars represent the rang. The line in the box is the median value. n, number of samples. (Reproduced by kind permission of Radiology & Oncology from Strojan P et al., Radiol Oncol 2002; 36: 145-6.) radiotherapy and/or chemotherapy. For the patients successfully treated with surgery for larynx carcinoma, Krecicki and Siewinski re­ported a constant decline in serum cathepsin B-like activity, which normalized within four months of the operation.8 In the subgroup of patients in whom the treatment failed, the mean serum values of cathepsin B-activity dropped in the first month after surgery, but rapidly increased in subsequent assays. The elevation occurred in all cases at least two months before clinical evidence of metastases or a recurrent tumor became apparent.8 According to our experience with more heterogeneous group of tumors treated with surgery and postoperative radiotherapy, cy­tosolic level of stefin A 24,25 and pretreatment serum level of cathepsin L 10 were found pre­dictive for disease outcome, which, in turn, re­flected the tumor response to applied therapy. In the same population the additional sample of serum was collected during regular follow­up visits 7 to 407 days (median, 59 days) after the completion of all therapies. No correlation was found between post-treatment concentra­tions of any of the studied cathepsins or in­hibitors and the time of serum sampling. However, when only those patients with a time interval of more than 45 days (n = 14) from the completion of therapy to the serum sampling were considered, a trend of gradual decline in stefin B (unpublished data) and cys­tatin C concentrations was observed with the increasing time delay (Figure 2).14 It appears that the decrease in enzyme and inhibitor ac­tivity in the treated area after the resection of the gross tumor burden was followed by a transient elevation of their serum levels, like­ly due to the inflammatory response of the tis­sues confined in the irradiation field during postoperative radiotherapy. The cutoff time of 45 days concurs well with the duration of ra­diomucositis as seen in clinics, gradually sub­siding in 4–6 weeks following the end of a ra­diotherapy course.26 Markers for prognosis The prognostic relevance of cathepsin D was studied most frequently (Table 1). It showed a universal trend of higher survival probability in the patients with low cytosolic or serum levels of the enzyme 24,27-29, and low intensity level of immunoreactivity.19 The prognostic reliability of cathepsin D was proved in three 27,30,31 out of four 32 studies that utilized mul­tivariate analysis. The same relation between enzyme expression and survival was ob- Figure 2. Relationship between post-therapy concentration of stefin B and cystatin C, and the time interval from the completion of therapy to serum sampling in non-relapsed patients with a time delay of more than 45 days (n = 14). served for cathepsin B 10,25,33 and cytosolic in cathepsin H.11,24 However, due to univari-levels of cathepsin L 24,25, whereas higher lev-ate setting of survival analysis, the prognostic els of cathepsin L in the serum was identified information collected from these studies as prognostically superior 10, as was the case leaves much to be desired. Table 1. Prognostic relevance of cathepsins and their endogenous inhibitors in squamous cell carcinoma of the head and neck Author (Ref.) Cathepsin D Maurizi et a.27 Lazaris et al.30 Seiwerth et al.31 Kawasaki et al.19 No.of patients 63 64 61 78 Tumor site Larynx Larynx Larynx Oral cavity Method IRA IHC IHC IHC Prognostic significance , MVA , MVA , MVA , UVA Cathepsin B Russo et al.33 68 Larynx EA , UVA Cathepsin H 11 Strojan et al. 18 All sites ELISA , UVA Cathepsin L 24 Budihna et al. 10 Strojan et al. 23 35 All sites All sites ELISA ELISA , UVA , UVA Stefin A 25 Strojan et al. 90 All sites ELISA , MVA Stefin B 25 Strojan et al. 90 All sites ELISA , MVA Cystatin C 34 Strojan et al. 82 All sites ELISA , MVA IRA, immunoradiometric assay; IHC, immunohistochemical analysis; EA, enzyme activity; ELISA, enzyme-linked immunosorbent assay; MVA, multivariate analysis; UVA, univariate analysis; , correlation of high levels with poor prognosis; , correlation of low levels with poor prognosis. Figure 3. Prognostic significance of the combination of cystatin C and stefin A concentrations: disease-free sur­vival and disease-specific survival. LR, Low-risk group (high stefin A and high cystatin C, n=23); MR, Medium-risk group (high stefin A and low cystatin C or low stefin A and high cystatin C, n=41); HR, High-risk group (low stefin A and low cystatin C, n=18). The results on the prognostic value of cathepsin inhibitors were provided by our only.22,24,25,28,34 team In operable SCCHN, higher cytosolic concentrations of stefin A, stefin B and cystatin C strongly correlated with longer survival probability in univariate survival analysis, which concurs with the concept of protective role of high levels of cysteine protease inhibitors in tissue ho­mogenates. In multivariate analysis, only stefin A and cystatin C retained their inde­pendent prognostic information. However, when comparing the prognostic strength of stefin A with that of cystatin C, the latter lost its significant prognostic power. In addition, the combination of the two inhibitors, stefin A and cystatin C could further stratify the risk of adverse event (Figure 3).34 No prog­nostic information was provided from the serum levels of any of the studied in­hibitors.10,14 Conclusions Although the clinical utility of cathepsins and their endogenous inhibitors in the manage­ment of SCCHN remains open to investiga­tion, it is evident that they exhibit potential in the clinical setting, particularly as markers for lymph node metastasis, for monitoring the presence of tumor cells in the body, and for prognosis. In future studies, larger num­bers of patients with clinically more homoge-nous characteristics should be included and a comparison between various analytical proce­dures should be focused on. It seems, howev­er, that clinical value of tumor marker profil­ing in SCCHN patients would be further im­proved by combining the predictive informa­tion from several markers with unrelated or mutually opposing biological roles. Acknowledgment The author thanks to Professor Janko Kos for his critical reading of the manuscript. The work was supported by the Ministry of Education, Science and Sport of the Republic of Slovenia Grant J3-4308. References 1. Bank U, Krüger S, Langner J, Roessner A. Review: Peptidases and peptidase inhibitors in the pathogenesis of diseases. Adv Exp Med Biol 2000; 477: 349-78. 2. Strojan P. Cathepsins and their endogenous in­hibitors in clinical oncology. Radiol Oncol 1996; 30: 120-33. 3. Kos J, Lah TT. Cysteine proteinases and their endogenous inhibitors: target proteins for prog­nosis, diagnosis and therapy in cancer (review). Oncol Rep 1998; 5: 1349-61. 4. Kos J, Werle B, Lah T, Brunner N. 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Ann Otol Rhinol Laryngol 1995; 104: 685-94. 21. Nikitakis NG, Rivera H, Lopes MA, Siavash H, Reynolds MA, Ord RA, et al. Immuno-histochemical expression of angiogenesis-relat­ed markers of oral squamous cell carcinoma with multiple metastatic lymph nodes. Am J Clin Pathol 2003; 119: 574-86. 22. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Cysteine protease in­hibitors stefin A and stefin B in operable carci­noma of the head and neck. Radiol Oncol 2002; 36: 145-51. 23. Withers HR, Peters LJ, Taylor JMGP. Dose-re­sponse relationship for radiation therapy of subclinical disease. Int J Radiat Oncol Biol Phys 1995; 31: 353-9. 24. Budihna M, Strojan P, Šmid L, Škrk J, VrhovecI, Župevc A, et al. Prognostic value of cathep-sins B, H, L, D and their endogenous inhibitors stefins A and B in head and neck carcinoma. Biol Chem Hoppe-Seyler 1996; 377: 385-90. 25. Strojan P, Budihna M, Šmid L, Svetic B, Vrhovec I, Kos J, et al. Prognostic significance of cysteine proteinases cathepsins B and L and their en­dogenous inhibitors stefins A and B in patients with squamous cell carcinoma of the head and neck. Clin Cancer Res 2000; 6: 1052-62. 26. Cooper JS, Fu K, Marks J, Silverman S. Late ef­fects of radiation-therapy in the head and neck region. Int J Radiat Oncol Biol Phys 1995; 31: 1141-64. 27. Maurizi M, Almadori G, Cadoni G, Scambia G, Ottaviani F, Ferrandina G, et al. Cathepsin D concentration in primary laryngeal cancer: cor­relation with clinico-pathological parameters, EGFR status and prognosis. Int J Cancer (Pred Oncol) 1996; 69: 105-9. 28. Šmid L, Strojan P, Budihna M, Škrk J, VrhovecI, Žargi M, et al. Prognostic value of cathepsins B, D and stefin A and B in laryngeal carcinoma. Eur Arch Otorhinolaryngol 1997; 254: 150-3. 29. Strojan P, Budihna M, Smid L, Vrhovec I, Skrk J. Urokinase-type plasminogen activator, plas­minogen activator inhibitor type 1 and cathep-sin D: and lysis of their prognostic significance in squamous cell carcinoma of the head and neck. Anticancer Res 2000; 20: 3975-82. 30. Lazaris AC, Lendari I, Kavantzas N, Kandiloros D, Adamopoulos G, Davaris P. Correlation of tumor markers p53, bcl-2 and cathepsin-D with clinicopathologic features and disease-free sur­vival in laryngeal squamous cell carcinoma. Pathol Int 2000; 50: 717-24. 31. Seiwerth S, Štambuk N, Konjevoda P, Mašic N, Bura VM, Klapan I, et al. Immunohistochemical analysis and prognostic value of cathepsin D determination in laryngeal squamous cell carci­noma. J Chem Inf Comput Sci 2000; 40: 545-9. 32. Leto G, Tumminello FM, Gebbia N, Bazan V, Tomasino RM, Dardanoni G, et al. Differential expression levels of urokinase-type plasmino-gen activator and cathepsin D in locally ad­vanced laryngeal squamous cell carcinoma: clinical implications. Int J Biol Markers 2001; 16: 245-9. 33. Russo A, Bazan V, Gebbia N, Pizzolanti G, Tumminello FM, Dardanoni G, et al. Flow cyto-metric DNA analysis and lysosomal cathepsins B and L in locally advanced laryngeal cancer. Cancer 1995; 76: 1757-64. 34. Strojan P, Oblak I, Svetic B, Šmid L, Kos J. Cysteine proteinase inhibitor cystatin C in squamous cell carcinoma of the head and neck: relation to prognosis. Br J Cancer 2004; 90: 1961-8. Radiol Oncol 2004; 38(2): 101-9 Pharmacogenetics of thiopurines: can posology be guided by laboratory data? Gabriele Stocco1, Stefano Martelossi2, Giuliana Decorti1, Alessando Ventura2, Noelia Malusŕ3, Fiora Bartoli1, Tullio Giraldi1 1 Department of Biomedical Sciences, University of Trieste; 2 Research Children Hospital ‘Burlo Garofolo’, Trieste; 3 Department of Prevention, Sanitary Services Agency Number 1, Trieste, Italy Background. The purpose of this study was to investigate the relationships between the presence of muta­tions in the TPMT gene, the consequent reduced enzymatic activity, and the clinical toxicity of the treatment with thiopurine antimetabolite drugs. Materials and methods. The study was performed on 44 patients with inflammatory bowel disease treat­ed with AZA. DNA was extracted from blood samples collected from each patient, and genotyping was per­formed using specific polymerase chain reaction assays in order to detect the three more frequent mutations of the gene. Enzymatic activity was measured on red blood cell lysates by HPLC. Results. Among the subjects, 4 (9.0%) were heterozygous for mutations in the TPMT gene; no subject was homozygous for mutations in the TPMT gene. A complete concordance between TPMT mutated genotype and reduced enzymatic activity could be determined. The incidence of toxicity in the subjects with a mutat­ed genotype was not different from that observed in the patients with a normal TPMT gene. Conclusion. Genotyping methods provide a simple and reliable DNA-based strategy to identify TPMT ho-mozygotes that should avoid thiopurines administration. However, it seems that the most common, less dan­gerous forms of thiopurine toxicity could be caused by factors different from TPMT gene mutations examined. Key words: inflammatory bowel diseases – drug therapy; 6-mercaptopurine; azathioprine; genotype; phenotype Received 30 April 2004 Accepted 10 May 2004 Correspondence to: Prof. Giraldi Tullio, Department of Biomedical Sciences, University of Trieste, Via L.Giorgieri 7,9, 34127 Trieste, Italy. Tel: +39 0405583539; Fax. +39 040577435; Email: giraldi@units.it This paper was presented at the “3rd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. Introduction 6-mercaptopurine (6MP) is an antimetabolite drug that oncologists have been using for more than 30 years to treat acute lym­phoblastic leukemia in children. It is a pro-drug that undergoes a complex metabolism1: it requires an intracellular activation to its thioguanine nucleotides (TGNs) in order to exert cytotoxic effects. Particularly relevant for the cytotoxicity of 6MP, and thus of TGNs, is the interference with de novo purine biosynthesis, and the modification of DNA structure after incorporation of TGNs that produces an alteration of the function of DNA processing enzymes.2,3 In vivo biotransformation of thiopurines also leads to their metabolic inactivation, ei­ther by oxidation to thiouric acid catalyzed by xanthine oxidase (XO), or by methylation of the thiol moiety of the molecule by thiop-urine-S-methyltransferase (TPMT). The treatment of patients with thiopurines can cause various adverse effects that can be so severe (even life-threatening) to require the cessation of therapy. The most common ad­verse effects that have been described in­clude nausea, bone marrow suppression, hepatitis and pancreatitis. The toxicity of the treatment with thiopurines, and the occur­rence of bone marrow toxicity in particular, have been ascribed to a genetically deter­mined deficiency of the enzyme TPMT which is held responsible for the metabolic deacti­vation of the drug.4-6 TPMT exhibits genetic polymorphism in all large ethnic groups stud­ied to date7-10; approximately one individual in 300 inherits two mutant TPMT alleles and is TPMT deficient, and about 10% are het­erozygous at the TPMT gene locus and have intermediate enzyme activity. It has been re­ported that subjects who inherit a deficiency in TPMT exhibit intolerance to thiopurines medications, including 6MP and azathioprine (AZA). Unless TPMT-deficient patients are treated with 10- to 15- fold lower doses of these medications, they develop a profound haematopoietic toxicity that precludes the ad­ministration of thiopurines and also of other chemotherapeutic agents and that can be fa­ tal.4,5,10,11 Moreover, some reports indicate that, in ALL patients who are treated with 6MP carrying a mutant allele for TPMT and who receive intracranial irradiation, there is a greater risk of developing secondary fatal tu­mours.12 More than 10 non-functional mutant alleles for TPMT have been reported, and reliable poly­merase chain reaction (PCR) based assays were developed to detect the three most prevalent mutant alleles: TPMT*2, TPMT*3A and TPMT*3C.13 These variants result from the fol­lowing point mutations in the TPMT open read­ing frame: G238C transversion for TPMT*2 al­leles and the G460A and A719G transitions for TPMT*3 alleles. While variant alleles other than TPMT*2 and TPMT*3 may lead to a reduced en­zyme activity, the frequency of these variant al­leles is likely to be very low. Indeed, genotyping for TPMT*2 and TPMT*3 mutant alleles yielded 95% concordance between genotype and phe­notype in different populations.13-17 Currently, there is a great debate about the opportunity of suggesting genetic testing for TPMT when prescribing 6MP aiming to iden­tify, before treatment, the subjects with a higher risk of severe toxicity. The question of whether to add on an advisory gene testing to the 6MP package label is now before FDA; 6MP is the first drug to be evaluated as possi­bly requiring a gene test before use.18 It has to be noted that thiopurine medica­tions are employed for some ‘off label’ uses, in particular to treat inflammatory disease like ulcerative colitis, Crohn’s disease and rheumatoid arthritis.19-21 There are indica­tions that the number of prescriptions of thiopurines could be 10 times higher for these patients as compared to cancer patients, which constitutes the registered indication for the use of these agents.18 Although thiop-urines are used to treat inflammatory dis­eases at a dose lower than that employed for cancer therapy, the treatment is usually much more prolonged, providing a strong argument in favor of testing TPMT genotype, in order to prevent the appearance of serious adverse ef­fects in both types of patients. In the last two years, a collaborative study run by the Department of Biomedical Sciences of the University of Trieste and the Burlo Garofolo Children’s Research Hospital of Trieste is being implemented in order to examine the occurrence of thiopurine in­duced adverse effects and the presence of mutations in the TPMT genes. The samples are being obtained from the hemato-oncology and gastroenterology clinics that use thiop-urines to treat leukaemia and inflammatory bowel disease (IBD), respectively. This paper reports the current results about the relation­ship between the outcome of the treatment with AZA used as an immunosuppressive drug to treat IBD, and TPMT genotype in chil­dren population or young adults. Materials and methods Patients Between July 2002 and July 2003, 44 patients with IBD were enrolled. The average age at the time of analysis was 16.4 years (range 4­38); among these patients, 20 (45.4%) were fe­male. They all received AZA at an average dose of 2 mg/kg/day (range 1-5 mg); the aver­age length of the treatment with AZA was of 20.6 months (range 0.5-63). Blood sample preparation Blood samples of the patients were obtained in Vacutainer Tubes, using EDTA as an anticoag­ulant. Total genomic DNA was isolated using a commercial kit (Talent, Trieste Italy) according to the suppliers’ instructions. Collected DNAs were dissolved in distilled water to a final con­centration of 20 ng/µl, as determined by UV spectrophotometry; these solutions were used as template in the PCR reactions. Erythrocyte lysates were prepared from the blood samples to measure TPMT activity according to the procedure already de­scribed.22 Erythrocytes (RBC) were collected by centrifugation at 800xg for 10 minutes at 4°C, washed twice with two volumes of an isotonic sodium chloride solution (0.9% w/v), and lysed with distilled water added to the fi­nal volume of 10 ml. TPMT enzyme assay TPMT activity was measured with the HPLC assay previously described.22 This assay is based on the in vitro conversion of 6MP to 6­methylmercaptopurine (6MMP), using S-adenosyl-L-methionine (SAM) as the methyl donor. Briefly, RBC lysates were purified from bivalent cations that could interfere with the assay by an incubation of 1 hour at 4°C with the chelating resin Chelex 100 (BIO­RAD, Richmond, CA, USA). The purified lysates were than incubated in the presence of 6-MP and SAM for one hour at 37°C. 6­MMP produced during the reaction was sepa­rated by an HPLC system Hewlett Packard HP Agilent 1100 including a G1311A Quaternary Pump, G1315A Diode Array Detector, G1313A Autosampler, G1322A Vacuum Degasser. The analytical column was a C18 reverse phase 250 mm long (VARIAN, Palo Alto, CA, U.S.A.); the mobile phase con­sisted of a solution of acetic acid 0.1% and 15% acetonitrile, with a flow rate of 1 ml/min. The detection wavelength was 290 nm, and the quantitative determination was per­formed comparing the area of the 6MMP peak with a standard curve of the compound dissolved in water. All chemicals were ob­tained from Sigma-Aldrich (Milan, Italy). TPMT genotyping The genotype of each individual at the TPMT*2, TPMT*3A, TPMT*3B and TPMT*3C alleles was determined using previously described poly­merase chain reaction (PCR)-based as­says.13,23,24 The mutations present in the vari­ants of the alleles TPMT*3 (G460A and A719G) were assayed by restriction fragment length polymorphism (RFLP) analysis. TPMT exon 7 and exon 10 were amplified in two separate re­actions by the use of primers that hybridized with the sequences flanking each of these poly­morphic nucleotides. The sequences of the primers employed to amplify exon 7 were P460F: AGG CAG CTA GGG AAA AAG AAA GGTG and P460R: CAA GCC TTA TAG CCT TAC ACC CAG G23, for exon 10 were P719F: AAT CCC TGA TGT CAT TCT TCA TAG TAT TT and P719R: CAC ATC ATA ATC TCC TCT TCC.24 The exon 7 amplicon was digested for 1 hour at 60°C with MwoI restriction enzyme (New England Biotechnologies, Beverly, MA, U.S.A). MwoI restriction site was present in the wild type allele, but not in the mutant allele. The exon 10 amplicon was digested for 1 hour at 37°C with Acc I (New England Biotechnologies, Beverly, MA, U.S.A). AccI restriction site was present in the mutant allele, but not in the wild-type allele. The unpurified products of the en­zymatic reaction where recognized by elec­trophoresis in a 2-per cent agarose gel stained with ethidium bromide. An allele specific PCR was used for the analysis of the G238C muta­tion (TPMT*2). DNA was amplified in two spe­cific reactions, one containing a forward primer wild-type specific (P2W: GTA TGA TTT TAT GCA GGT TTG) and one a mutant specific primer (P2M: GTA TGA TTT TAT GCA GGT TTC); the reverse primer (P2C: TAA ATA GGA ACC ATC GGA CAC) was the same in both re­actions.13 Unpurified PCR products were ana­lyzed after electrophoresis in a 2-per cent agarose gel stained with ethidium bromide. A DNA fragment was amplified with P2M and P2C primers when C238 (mutant) was present, or with P2W and P2C primers when G238 (wild type) was present. The primers were purchased from Invitrogen (Milan, Italy). Results TPMT genotype distribution The distribution of the TPMT genotype in 44 patients with IBD so far enrolled in the study is reported in Table 1. Among these subjects, 40 had a wild type TPMT genotype, while 4 Table 1. TPMT genotype in 44 patients with IBD Genotype N Normal (wild type/wild type) 40 (90.9 %) Heterozygous (wild type/mutant) 4 (9.1%) Homozygous (mutant/mutant) 0 Total 44 (100.0 %) The considered mutations for the TPMT gene are G238C, G460A and A719G, identified as described in the experimental section. Table 2. TPMT activity in patients with IBD according to TPMT genotype. TPMT activity (nmol h-1 ml-1 RBC) Wild type (n=23) 11.2 +- 0.1 Heterozygous (n=4) 6.0 +- 0.4 * TPMT activity is expressed as nmol of 6MMP pro­duced during 1 hour of incubation by 1 ml of RBCs and is reported as mean +- SE. * Means significant­ly different, t student’s test, p<0.0001. subjects were heterozygous for a mutation in the TPMT gene. Three patients displayed a TPMT*3A mutated allele, and one patient dis­played a TPMT*2 mutated allele; no patients were homozygous for TPMT variant alleles. TPMT activity TPMT activity was measured in 27 out of these 44 subjects enrolled in the study, and the results obtained are reported in Table 2. The average activity among these patients was 10.4 nmol/hour/ml RBC; 23 of these sub­jects had a normal TPMT gene, whereas 4 had a mutated TPMT gene. The average TPMT ac­tivity in the subjects with a normal gene was 11.2 nmol/hour/ml RBC, whereas in those with a mutated gene was 6.0 nmol/hour/ml RBC (p < 0.0001 t student’s test). Clinical toxicity of AZA treatment and correlation with TPMT genotype The toxicity of the treatment in the 44 sub­jects is reported in Table 3. During AZA ad-ministration, 22 patients (50%) developed side effects. Because of this toxic event, the dosage was reduced in 8 patients and the treatment was suspended in 14 cases. In 13 subjects (29.5%), the bone marrow toxicity manifested as lymphopoenia or thrombocy­topoenia. In 9 patients, other side effects were observed: pancreatitis, n = 2 (4.5%); he-patotoxicity, n = 2 (4.5%); infections, n = 3 (6.8%), neuropathy, n = 2 (4.5%). Among the 4 subjects with a mutated TPMT allele, 2 re­sponded normally to therapy whereas 2 de­veloped neuropathy. Discussion Thiopurine drugs play an important role in the treatment of leukemias and of some chronic inflammatory diseases. However, the use of these drugs is limited due to serious adverse effects, among which bone marrow suppression may require even the cessation of therapy. Individual differences in suscepti­bility to AZA/6MP have been observed, and they were attributed to variable intracellular concentrations of the cytotoxic metabolites of the drugs, TGNs. It is known that TPMT defi­ciency, caused by a frequent genetic poly­morphism, might induce a profound bone marrow suppression in the patients receiving thiopurines because it causes a reduced methylation of 6MP and the consequent ac­cumulation of TGNs toxic metabolites. About 10% of Caucasians inherit a mutant allele of the TPMT; they also have a reduced TPMT ac­tivity; on the other hand, the subjects ho­mozygous for the variant alleles of TPMT are encountered at the much lower rate of about 1 per 300 Caucasians, and have no measura­ble TPMT activity. In the 44 subjects exam­ined in the present study, no subject ho­mozygous for TPMT mutated gene was iden­tified, presumably because of the insufficient number of subjects genotyped; at the same time, 4 subjects (9.1%) were found to be het­erozygous for a mutated allele of the TPMT gene. The overall frequency of defective alle­les obtained in the present study is compara­ble with that reported in the Italian popula­tion25 and also in other populations of Caucasian origin by other researchers.13,24,26 As far as phenotype is concerned, TPMT activity has been so far measured in 27 of the 44 subjects enrolled. The average value of the enzymatic activity measured in the patients with a mutated TPMT genotype was signifi­cantly lower than that determined in the re­maining group not carrying the mutations considered. This finding is in agreement with the studies showing a strong correlation for TPMT between genotype and phenotype in patients with IBD.13-17 This finding also con­firms the view that genotyping for the identi­fication of the considered TPMT mutations may allow an approach, effective in identify­ing the subjects with a reduced inherited TPMT activity. Several studies have been recently pub­lished reporting the investigations about the relationship between TPMT reduced enzymat­ic activity, observed in IBD patients with in­herited mutated alleles of TPMT gene, and bone marrow toxicity, which occurred after Table 3. Correlation between TPMT genotype and the toxicity of AZA treatment in patients with IBD TPMT genotype n Wild type Heterozygous Homozygous Without adverse effects 22 (50.0 %) 20 (45.5 %) 2 (4.5 %) 0 With adverse effects 22 (50.0 %) 20 (45.5 %) 2 (4.5 %) 0 Total 44 40 (90.9 %) 4 (9.1 %) 0 The adverse effects considered are bone marrow toxicity (lymphopoenia, thrombocytopoenia, hepatotoxicity, pancreatitis, infections and neuropathy). Radiol Oncol 2004; 38(2): 101-9. thiopurine treatment.27,28 The results reported in these studies show in some instances a sig­nificant increase in the occurrence of profound bone marrow toxicity in the subjects with a mutated TPMT gene; on the other hand, other side effects (such as hepatotoxicity, pancreati-tis, mild lymphopenia and reduced platelet counts) appeared not to be related to TPMT mutations. In particular, in a group of 41 pa­tients with Crohn’s disease displaying signifi­cant myelotoxicity following the treatment with thiopurines, only 10% were homozygous for the considered TPMT mutations which lead to a severe enzymatic deficiency, and 17% were heterozygous for the same mutated alle­les. These mutations, which are associated with the reduced TPMT activity, were over­represented in this group as compared with a general population, although in most patients, the bone marrow toxicity was not associated with the genotype corresponding to low TPMT activity.27 Similar results were published also in a further study, reporting the TPMT geno­type of 50 patients with IBD treated with thiopurines who suffered from adverse effects that required to discontinue the drug adminis­tration: five patients (10%) were heterozygous for TPMT mutated alleles, and one (2%) was homozygous. Even if the single subject with the homozygous mutated genotype developed bone marrow suppression after AZA treat­ment, the treatment toxicity could not be relat­ed to a reduced TPMT activity in most of the patients.28 When the adverse effects of AZA in the IBD patients were examined in the group of 44 patients considered in the present investi­gation, the toxicity of the treatment was in general moderate (grade II or III). The most frequent toxicity encountered was bone mar­row toxicity (13 subjects, 29.5%), and none of these patients had any of the considered mu­tated TPMT alleles. Four subjects were het­erozygous for a TPMT mutation; among them, two responded normally to the thera­py, whereas two developed an idiosyncratic form of myalgia and arthralgia, which re­quired to discontinue the treatment. Myalgia and arthralgia are more associated with with Type I hypersensitivity reaction than direct drug toxicity, and are unlikely to be associat­ed with TPMT mutated alleles.29 These results thus appear to agree with those showing that the more common forms of myelosuppres­sion during AZA administration to the IBD patients have causes different from genetic factors such as the considered mutations leading to a reduced TPMT activity.27,28 Drug toxicity is a multifactorial phenome­non involving multiple biological and envi­ronmental processes, including drug interac­tions. As far as AZA is concerned, almost all the subjects treated with this drug receive al­so other medications, which might be respon­sible for the interactions leading to adverse effects. Drugs like aminosalycilates are com­monly used to treat IBD in combination with AZA; at the same time, they have been re­ported to reduce the metabolic inactivation of thiopurines, through TPMT inhibition.30,31 Moreover, genetic factors different from TPMT mutated alleles, such as the polymor­phism of genes whose transcripts are in­volved in the detoxification of xenobiotics, could also influence the pharmacokinetics of thiopurines, and consequently, their clinical efficacy. In this connection, a study is being carried on by the authors aiming to examine the relationships between the clinical toxicity of the treatment with AZA and polymor­phisms in the genes for P-glycoprotein32, a transporter involved in the extrusion of xeno­biotics from cells, and polymorphisms in the genes for glutathione-S-transferase, the en­zyme responsible for the conjugation of drugs with glutathione.33 High throughput techniques are currently available for the investigation of the relation­ships between the response to drug treatment and the genetic characteristic of the patients. Microarray technology allows the study of the expression of thousands of genes in a single sample, and has been employed to study the molecular basis of diseases, including various cancers 34, and the molecular mechanisms of drug action.35 One of the authors (S.G.) spent a short period as visiting scholar at St.Jude Children’s Hospital in Memphis (TN, USA) in order to learn the basis of microarray data analysis. These methods were applied to the study of thiopurine cytotoxicity and gene ex­pression aiming to find new markers of drug action that could assist the clinician in deter­mining the patients’ individual sensitivity to the effectiveness and toxicity of a drug. In this study, the expression of genes involved in the small GTPase signaling pathways were determined in relation to the effectiveness of the drug, expressed as the magnitude of the decrease of white blood cell number in the 4 days following a single drug administration. Interestingly, the gene expression of Rac1 small guanosine triphosphatase seems to be related to the efficacy of thiopurines: indeed, 6MP is less effective in the subjects with a higher expression of this protein 24 hours af­ter the treatment. This finding is in agree­ment with a recent observation, indicating a new possible mechanism for thiopurines tox­icity, consisting of the inhibition of a Rac1, a critical regulator in mammalian T cells.36 These results allow to conclude that geno-typing provide a simple and reliable DNA-based strategy to identify TPMT homozygotes who should avoid AZA/6MP administration at conventional dosages. However, it seems that the most frequent, even if less dangerous, forms of thiopurine toxicity in IBD patients could be attributable to factors different from a mutated TPMT genotype. Although TPMT genotyping may be useful to identify subjects with a risk of very severe toxicity, clinicians should still need to monitor carefully the pa­tients treated with these toxic medications, in order to detect other common forms of toxici­ty. Further studies are needed to enlight the ge­netic characteristics of subjects experiencing toxic effects after thiopurine treatment, which may lead to the identification of additional ge­netic markers of toxicity that could assist the clinician in the treatment of patients with these toxic medications. Acknowledgments The data reported in this paper are preliminary results of the experimental work carried out by one of us (G.S.) for his post-graduate training. The same author (G.S.) wishes to thank Dr. William E. Evans for the support and encour­agement during his stay at St. Jude Children’s Research Hospital, Memphis, TN, U.S.A. References 1. Lennard L. The clinical pharmacology of 6-mer­captopurine. 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Thiopurine methyltransferase activi­ty: new conditions for reversed-phase high-per­formance liquid chromatographic assay with­out extraction and genotypic-phenotypic corre­lation. J Chromatogr B 2002; 773: 119-27. 23. Masahiro H, Tomoko I, Omori F, Agatsuma Y, Mizugaki M. Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population. Mutation Res 2000; 448: 91-5. 24. Otterness D, Szumlanski C, Lennard L, Klemetsdal B, Aarbakke J, et al. Human thiop-urine methyltransferase pharmacogenetics: Gene sequence polymorphism. Clin Pharm Ther 1997; 62: 60-73. 25. Rossi AM, Bianchi M, Guarnirei C, Barale R, Pacifici GM. Genotype-phenotype correlation for thiopurine S-methyltransferase in healthy Italian subjects. Eur J Clin Pharmacol 2001; 57: 51-4. 26. Tai HL, Krynetski EY, Yates CR, Loennechen T, Fessing MY, Krynetskaia NF, Evans WE. Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most preva­lent mutant allele associated with loss of cat­alytic activity in Caucasians. Am J Hum Genet. 1996; 58: 694-702. 27. Colombel JF, Ferrari N, Debuysere H, Marteau P, Genere JP, Bonaz B, et al. Genotypic Analysis of thiopurine S-methyltransferase in patients with Crohn’s disease and severe myelosuppression during azathioprine therapy. Gastroenterology 2000; 118: 1025-30. 28. Gearry RB, Barclay ML, Burt MJ, Collett JA, Chapman BA, Robert RL, Kennedy MA. Thiopurine S-methyltransferase (TPMT) geno­type does not predict adverse drug reactions to thiopurine drugs in patients with inflammatory bowel disease. Aliment Pharmacol Ther 2003; 18: 395-400. 29. Gardiner SJ, Begg EJ, Barclay ML, Kirkpatrick CMJ. Genetic polymorphism and outcomes with azathioprine and mercaptopurine. Adverse Drug React Toxicol Rev 2000; 19: 293-312. 30. Lewis LD, Benin A, Szumlanski CL, Otterness DM, Lennard L, Weinshilboum RM, Nierenberg DW. Olsalazine and 6-mercaptop-urine-related bone marrow suppression: A pos­sible drug-drug interaction. Clin Pharm Ther 1997; 67: 464-75. 31. Dewit O, Vanheuverzwyn R, Desager JP, Horsman Y. Interaction between azathioprine and aminosalicylates: an in vivo study in pa­tients with Crohn’s disease. Aliment Pharmacol Ther 2002; 16: 79-85. 32. Choi KH, Chen CJ, Kriegler M, Robinson IB. An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (p-glycoprotein) gene. Cell 1988; 53: 519-29. 33. Lee AU, Farrell GC. Mechanism of azathio­prine-induced injury to hepatocytes: roles of glutathione depletion and mitochondrial in­jury. J Hepatol 2001; 35: 756-64. 34. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D, Mahfouz R, Behm FG, et al. Classification, subtype discovery, and predic­tion of outcome in pediatric acute lymphoblas-tic leukemia by gene expression profiling. Cancer Cell 2002 ;1: 133-43. 35. Cheok MH, Yang W, Pui CH, Downing JR, Cheng C, Naeve CW, Relling MV, Evans WE. Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nat Genet. 2003; 34: 85-90. 36. Tiede I, Fritz G, Strand S, Poppe D, Dvorsky R, Strand D, Lehr HA, Wirtz S, Becker C, et al. CD28-dependent Rac1 activation is the molecu­lar target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest 2003; 111: 1133-45. Radiol Oncol 2004; 38(2): 111-9. Development of quantitative RT-PCR assays for wild-type urokinase receptor (uPAR-wt) and its splice variant uPAR-del5 * Juliane Farthmann1, Leon Holzscheiter1, Julia Biermann1, Axel Meye2, Thomas Luther3, Matthias Kotzsch3, Fred Sweep4 , Manfred Schmitt1, Paul Span4, Viktor Magdolen1 1Klinische Forschergruppe der Frauenklinik, TU München, München; 2Klinik für Urologie und 3Institut für Pathologie, TU Dresden, Dresden, Germany; 4Department of Chemical Endocrinology, University Medical Centre, Nijmegen, The Netherlands The receptor for the serine protease urokinase-type plasminogen activator, uPAR (CD 87), plays an impor­tant role in tumor cell invasion and metastasis of solid malignant tumors. uPAR is a highly glycosylated, glycan lipid-anchored membrane protein, consisting of three homologous domains. Each individual domain is encoded by two exons: DI by exons 2+3, DII by exons 4+5, and DIII by exons 6+7. Beside the wild-type (wt) uPAR mRNA, two splice variants either lacking exon 5 (uPAR-del5) or both exons 4 and 5 (uPAR­del4/5) have been described. Previously, we studied expression of the mRNA variant uPAR-del4/5 and uPAR mRNA encompassing exons 2, 3, and 4 (i.e. uPAR-wt plus uPAR-del5) applying real-time RT-PCR assays for quantification of the mRNA concentration. In the present paper, we established two additional specific, robust and highly sensitive RT-PCR assays, based on the LightCycler technology, to specifically quantify either uPAR-wt or its splice variant, uPAR­del5. Expression of uPAR-wt and uPAR-del5 was analyzed in different human malignant cell lines (ovari­an cancer cell lines OVMZ-6 and OVMZ-10; breast cancer cell lines MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435, and aMCF-7; brain tumor cell line LN 18) as well as in a set of 174 breast cancer tissue sam­ples. uPAR-del5 mRNA was found to be expressed very frequently at a rather low level (typically less than 1% of uPAR-wt mRNA). In tumor tissue from breast cancer patients, a statistically significant correlation between uPAR-del5 and uPAR-wt mRNA (r = 0.779; P < 0.001) was observed. There was no association between the expression level of either mRNA and clinical parameters such as nodal status, tumor size and grade. In estrogen receptor negative tumors, a significantly higher uPAR-del5 expression was found (P = 0.023). The two developed quantitative RT-PCR assays described here may aid further analysis of the function and clinical relevance of uPAR-wt and one of its splice variants, uPAR-del5, in malignant tumors. Key words: neoplasms; urinary plasminogen activator; RNA, messenger; reverse transcriptase poly­merase chain reaction; RNA splicing Introduction Tumor cell dissemination and formation of metastases is facilitated by the interaction of diverse proteolytic systems, including serine proteases, cysteine proteases, and matrix metalloproteinases.1 These proteases enable tumor cells to degrade the extracellular ma­trix and to cross natural boundaries.2 The re­ceptor for the serine protease urokinase-type plasminogen activator (uPAR, CD 87) is es­sentially involved in this process as it focuses the proteolytic activity of uPA to the cell sur­face. Furthermore, it interacts with a broad variety of other ligands, including vitronectin or integrins, and by this modulates prolifera­tion, cell adhesion and migration, invasion, and angiogenesis.3,4 High tumor levels of uPA and/or its inhibitor PAI-1 have been shown to be a predictor for poor prognosis of patients with solid tumors, including breast, gastric, esophageal, ovarian, colorectal or hepatocel­lular cancer.4,5 Different therapeutic ap­proaches have been employed to obstruct the uPA/uPAR system, using small molecules, such as antibodies or modified toxins.6-9 uPAR is a highly glycosylated, glycan lipid (GPI-)-anchored membrane protein, consisting of three structurally homologous domains (DI, DII, DIII).10 In the past, a number of glycosyla­tion variants and different molecular forms of uPAR antigen such as soluble uPAR, uPAR-DI, and uPAR-DII+III has been described and ana- Received 20. March 2004 Accepted 5 April 2004 * a report from the Receptor and Biomarker Group of the European Organisation for Research and Treatment of Cancer This paper was presented at the “3nd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. Correspondence to: Viktor Magdolen, PhD, Klinische Forschergruppe der Frauenklinik der TU München, Klinikum rechts der Isar, Ismaninger Str. 22, D-81675 München, Germany. Phone: +49-89-4140-2493; Fax: +49-89-4140-7410; E-mail: viktor.magdolen@lrz.tum.de 11) lyzed (for a summary see Luther et al.. In some cases, certain (novel) functions or activi­ties could be assigned to these variants: endo-proteolytic processing of CD87 with removal of DI is, e.g., a likely pathway for controlling cell adherence and migration 3,12,13, as the ex­tent of glycosylation of DI strongly contributes to the affinity for its ligand uPA.14 Furthermore, splice variants of uPAR have been identified, i.e. an uPAR mRNA splice variant lacking exon 5 (uPAR-del5) as well as a variant lacking exons 4 and 5 (uPAR­del4/5).11,15 Since splice variants of genes of­ten display a different expression pattern and biological role compared to that of the wild-type genes, especially in tumor tissue 16 , we previously studied the expression of the mRNA variant uPAR-del4/5 in a representa­tive set of breast cancer tissues applying a real-time RT-PCR assay for quantification of the mRNA concentration.11 The mRNA variant uPAR-del4/5 was, in fact, expressed very fre­quently in breast cancer tissue and, strikingly, higher uPAR-del4/5 expression was signifi­cantly associated with shorter disease-free sur­vival of breast cancer patients. Thus, these re­sults suggest that uPAR-del4/5 mRNA may serve as a prognostic marker in breast cancer. The aim of the present study was to establish a highly sensitive real-time RT-PCR assay based on LightCycler technology for the uPAR-del5 mRNA variant in order to be able to analyze the expression pattern of this alternatively spliced mRNA in solid malignant tumors. Material and methods Cell lines and cell lysates, uPAR ELISA Human ovarian cancer cell lines OVMZ-6 and OVMZ-10, human breast cancer cell lines MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435, and aMCF-7 as well as the human brain tumor cell line LN 18 were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air in DMEM medium (Invitrogen, Karlsruhe, Germany), supplemented with 10% fetal calf serum (Invitrogen) and 1% peni­cilline-streptomycine (Biochrom, Berlin, Germany), 1% arginine-asparagine (Sigma, Deisenhofen, Germany) and 1% HEPES buffer (Invitrogen). Cells were harvested from monolayer dishes after two days. Total RNA from the cells was extracted using Trizol Reagent (Invitrogen). cDNA was synthesized using AMV cDNA First Strand Synthesis Kit (Roche Diagnostics, Penzberg, Germany). cDNAs from the cell lines were diluted 1:15 and aliquoted at -20 °C. For uPAR antigen detection, 2 x 106 cells were cultured for two days on monolayer dishes, then harvested, resuspended in phos­phate-buffered saline and sedimented by cen­trifugation (200 x g, 10 min, RT). Cells were disrupted by two freezing and thawing cy­cles, followed by a solubilization step (10 min, in 100 µl per 106 cells sample buffer con­taining 0.2% Triton X-100) and stored at -20 °C. uPAR antigen was determined in cell lysates by uPAR IIIF10 ELISA as described by Kotzsch and co-workers (2000). Protein con­tent was determined using the Micro BCA protein assay kit (Pierce, Rockford, IL). uPAR antigen levels in cell lysates are expressed as ng per mg of total protein. Patients - tissue selection The study adhered to national regulations of The Netherlands on ethical issues and was approved by the local ethical committee. Tumor tissue was obtained from patients with unilateral breast cancer after surgical resection of the primary tumor. Patients who had re­ceived neo-adjuvant treatment, or who had a previous diagnosis of cancer or who had a car­cinoma in situ, were excluded. Furthermore, patients with recurrent disease within one month after surgery or with distant metastases at time of diagnosis were excluded as well. After surgery, performed between November 1987 and December 1997 in participating hos­pitals of the Comprehensive Cancer Center East in The Netherlands, a representative part of the tumor was selected by a pathologist, frozen in liquid nitrogen, and sent to the Department of Chemical Endocrinology for routine determination of estrogen (ER) and progesterone (PgR) receptor status by ligand binding assay.17,18 Remaining frozen tissue or tissue powder (after dismembration) prepared from this tumor was kept in liquid nitrogen. For the present study, samples were selected based on the availability of tissue stored in the tumor bank. Patients- cDNA synthesis Total RNA was isolated from approx. 20 mg of tissue powder using the RNeasy mini kit (Qiagen, Hilden, Germany) with on-column DNase-I treatment as previously described.18 Reverse transcription was performed using the Reverse Transcription System (Promega Benelux BV, Leiden, The Netherlands) ac­cording to the manufacturer’s protocol. After annealing of random hexamers for 10 min at 20 °C, cDNA synthesis was performed for 60 min at 42°C, followed by an enzyme inactiva­tion step for 5 min at 95 °C. cDNAs were di­luted 1:3 and aliquoted. Quantitative real-time RT-PCR of uPAR-del5, uPAR-wt, and the housekeeping gene G6PDH RT-PCR primers and hybridization probes were obtained from TibMolBiol (Berlin, Germany). RT-PCR was performed using the LightCycler apparatus (Roche, software ver­sion 3.5). Assays for quantification of uPAR-wt and uPAR-del5 were established, with primer sequences as follows: Ex 2F: GAC CTC TGC AGG ACC ACG AT; Ex 6,4R: CAG ATT TTC AAG CTC CAG GAC TT; Ex 5A: GGT GGC GGT CAT CCT TTG. RT-PCR was performed with a master mix with 3.0 µM MgCl2, 0.6 µM of the primers, 0.2 µM of each of the hybridization probes and 2 µl of reagent mix, in a total volume of 20 µl. The amplification program started with pre-de­naturation at 95 °C, followed by 45 cycles of amplification: denaturation 10 sec at 95 °C, annealing for 15 sec at 66 °C, and elongation at 72 °C for 15 sec. A standard curve was gen­erated for each run using eight glass capillar­ies (Roboscreen, Leipzig, Germany) coated with a defined number of molecules of uPAR (wild type or del5) plasmid (range from 100,000 to 10 copies). The generation of the plasmids pRcRSV-GPI-uPAR-wt (encoding uPAR-wt) and pRcRSV-GPI-uPAR-del5 have been described previously.11 A negative con­trol containing buffer only was included in each run. For normalization of the data h­G6PDH (human glucose-6-phosphate-dehy­drogenase) Housekeeping Gene Set (Roche) was used, according to the manufacturer´s protocol. All cDNA samples displaying less than 10,000 molecules of G6PDH were con­sidered to be of non-optimal quality and were excluded from further analyses (18 of 192 cDNA samples = 9.4%). Relative expression levels were determined calculating the ratio between absolute template molecule and Domains of uPAR antigen uPAR-wt (376 bp) Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 1 Ex 2F Ex 6,4R uPAR-del5 (372 bp) Figure 1. Detection of uPAR-wt and uPAR-del5 mRNA. uPAR consists of three homologous do­mains. Each individual domain is encoded by two exons: DI by exons 2+3, DII by exons 4+5, and DI­II by exons 6+7 (exon 1 encodes the signal se­quence of uPAR). For specific amplification of uPAR-wt mRNA a reverse-primer directed to exon 5 was designed. In order to specifically amplify uPAR-del5 a primer overlapping the unique boundary between exons 4 and 6 was chosen. Both RT-PCR assays use the same forward primer with­in exon 2. The amplicon length is 376 bp for uPAR-wt and 372 bp for uPAR-del5. G6PDH housekeeping molecule numbers. The ratios of target genes and reference gene were multiplied with factor 1,000. Statistics The association of uPAR mRNA levels with histomorphological and clinical parameters was analyzed using non-parametric tests (Mann-Whitney U test; Kruskal-Wallis test). Statistical analyses were performed using the SPSS 11.5 software. Results and discussion Development of RT-PCR assays for uPAR-del5 and uPAR-wt For quantification of uPAR-del5 mRNA, we established a highly sensitive real-time RT­PCR assay applying the LightCycler technol­ogy. In this assay, the 5’ amplification primer (Ex 2F) is identical to the 5’ primer used in the RT-PCR assays previously described 11 for detection of uPAR-2/3/4 mRNA (encom­passing exons 2, 3, and 4) and uPAR-del4/5 mRNA (encompassing exons 2, 3, and 6 and lacking exons 4 and 5, respectively). The 3’ amplification primer (Ex 6,4R) overlaps the alternative splicing site of exons 4 and 6 and, therefore, selectively binds to uPAR-del5 mRNA (and not to uPAR-wt or uPAR­del4/5). For generation of standard curves, glass capillaries coated with defined num­bers of uPAR-del5 plasmid -exactly deter­mined by HPLC calibration 19 - were used. In addition to the uPAR-del5 assay, a quantita­tive RT-PCR assay for uPAR-wt mRNA was established, which does neither amplify uPAR-del5 nor uPAR-del4/5 mRNA, since the 3’ amplification primer (Ex 5A) is direct­ed to exon 5 (Figure 1). Since in both assays the 5’ and 3’ amplification primers are di­rected to different exons, amplification of (possible contaminating) genomic uPAR-DNA is excluded due to the large amplicon size of > 10 kbp. With both assays, the de­tection limit was (at least) 10 copies of cDNA, which corresponds to the lowest glass capillary standard. Figure 2 depicts a plot of measured molecules versus theoretical mole­cule numbers of uPAR-del5 in 48 independ­ent PCR runs for the standards ranging from 10 to 100,000 copies. In case of uPAR-wt, similar results were obtained (data not shown). To test for specificity of the uPAR­del5 and uPAR-wt assays, we analyzed sam­ples containing large plasmid copy numbers (corresponding to about 1.5 pg DNA) of ei­ther uPAR-wt, -del5, and -del4/5 or included glass capillaries coated with 100,000 copies of the three different plasmids. In neither case, we observed an amplification signal above the buffer control for the control plas­mids (data not shown). Thus, the target se­quence (either uPAR-del5 or uPAR-wt) is se­lectively amplified with the two newly estab­lished RT-PCR assays. Quantification of uPAR mRNA variants and uPAR antigen in cell lines Seven different cell lines were selected and the uPAR antigen content determined apply­ing the uPAR HU/IIIF10-ELISA.20 As can be seen from Table 1, the uPAR antigen content ranged from undetectable (< 0.05 ng/mg, breast cancer cell line MDA-MB 435) to about 7 ng/mg total protein in the case of the ovari­an cancer cell line OVMZ-10. uPAR-wt mRNA concentration (normalized to the ex­pression of G6PDH) was determined and compared to the uPAR antigen values: the highest antigen values (OVMZ-10 and MDA­MB 231 BAG) corresponded to the highest mRNA levels, the cell line with undetectable uPAR protein levels displayed an extremely low uPAR-wt mRNA expression as well. The other cell lines displayed an intermediate ex­pression both at the protein and mRNA level (r = 0.786; P < 0.05) (Table 1). uPAR-del5 ex­pression was detected in all of the cell lines albeit to a low extent ranging from 0.23 to 1.53% of uPAR-wt expression. Table 1. Detection of uPAR antigen, uPAR-wt and uPAR-del5 mRNA in seven different cancer cell lines. Cellular uPAR antigen content (expressed as ng per mg total protein) was measured using the HU/IIIF10 ELISA.20 uPAR-wt and uPAR-del5 mRNA was quantified by the newly established RT-PCR assays and ex­pression normalized to G6PDH mRNA . Cell lines ELISA [ng/mg] RT-PCR measurements [relative to G6PDH] Name Origin HU/IIIF10 uPAR-wt uPAR-del5 uPAR-del5 / uPAR-wt [%] MDA 435 breast < 0.05 21 0.2 0.95 MDA 231 breast 1.99 747 .27 2.00LN 18 brain 2.07 137 2.1 1.53 OVMZ-6 ovary 2.18 648 5.5 0.85 aMCF-7 breast 3.19 671 3.9 0.58 MDA 231 BAG breast 5.28 1204 2.8 0.23 OVMZ-10 ovary 6.86 3588 .78 28.00 A 40 Mean ± SE 339.0 ± 20.5 20 10 0 30 0 300 600 900 1200 1500 1800 Frequency Frequency Frequency uPAR-wt / G6PDH B 40 Mean ± SE 2.2 ± 0.1 30 20 10 0 0 1.5 3.0 4.5 6.0 7.5 9.0 10.5 12.0 uPAR-del5 / G6PDH 40 Mean ± SE 0.7 ± 0.04 30 20 10 0 0 0.5 1.0 1.5 2.0 2.5 3.0 uPAR-del5 / uPAR-wt [%] Figure 3. uPAR mRNA expression in human breast cancer tissue. 174 primary breast cancer samples were measured for uPAR-wt and uPAR-del5 mRNA with the newly established quantitative RT­PCR assays. The histograms depict the frequency distribution of (A) uPAR-wt expression (normal­ized to G6PDH), (B) uPAR-del5 expression (nor­malized to G6PDH), and (C) relative uPAR-del5 ex­pression compared to uPAR-wt [%]. Quantification of uPAR mRNA variants in breast cancer tissue In order to select an appropriate housekeep­ing gene for normalization of mRNA concen­trations, we first analyzed expression of three different housekeeping genes (PBDG, G6PDH, and GAPDH) in a representative set (n=46) of breast cancer cDNA samples. The absolute mRNA concentrations of PBGD, G6PDH, and GAPDH strongly correlated with each other (ranging from r = 0.880 to 0.908, P < 0.0001) indicating that these genes are, in fact, expressed constitutively in breast cancer. As both genes, G6PDH and uPAR, are moderately high expressed, G6PDH was subsequently used for normalization. We assessed the level of expression of uPAR-del5 and uPAR-wt mRNA in 174 cases of breast cancer patients (Figure 3). uPAR-wt expression was found in all cases (median rel­ative expression level: 263). uPAR-del5 was detected in the breast cancer samples with high frequency but with a significantly lower expression level (median: 1.57). In one case, there was no uPAR-del5-amplification signal at all, in further 40 of the 174 cases (23.6%), the determined copy number was below the lowest standard included in the assay. The relative expression rate of uPAR-del5 to uPAR-wt was between 0 and 3.1% (median uPAR-del5rel/uPAR-wtrel: 0.53%). The mRNA concentrations of uPAR-wt and uPAR-del5 significantly correlated with each other (r = 0.779; P < 0.001). We observed no statistical­ly significant association of uPAR-wt or uPAR-del5 expression with clinical parame­ters such as nodal status, tumor size or grade (Table 2). A significantly higher uPAR-del5 expression was found in estrogen receptor negative tumors (P = 0.023). Conclusions In the present study, we developed a quanti­tative real-time RT-PCR method to specifical­ly quantify uPAR-wt mRNA (excluding both Table 2. uPAR-wt and uPAR-del5 mRNA expression and clinical parameters. Associations of uPAR-wt and uPAR-del5 expression with clinical parameters were analyzed by using non-parametric tests. All tests were performed at significance level of a < 0.05. 174 breast cancer patients uPAR-wt / G6PDH uP AR-del5 / G6PDH Variable Total No. o f patie nts ( %) Mean ± SE P value Mean ± SE P value Meno pausal status a pre/peri post 174 39 (22.4) 135 (77.6) 378 ± 53 327 ± 22 0.397 (ns) 2.2 ± 0.3 2.1 ± 0.2 0.785 (ns) Nodal status a negative positive x 149 79 (53.0) 70 (47.0) 25 360 ± 31 291 ± 30 0.058 (ns) 2.5 ± 0.3 1.9 ± 0.2 0.070 (ns) Size (pT) b 1 2 3 4 x 171 55 (32.2) 97 (56.7) 15 (8.8) 4 (2.3) 3 398 ± 38 286 ± 20 405 ± 118 369 ± 133 0.101 (ns) 2.6 ± 0.3 1.9 ± 0.2 2.1 ± 0.4 1.5 ± 0.6 0.240 (ns) Grade a I/II III x 123 65 (52.8) 58 (47.2) 51 275 ± 23 397 ± 47 0.116 (ns) 1.7 ± 0.2 2.4 ± 0.3 0.112 (ns) ER statusa negative positive 174 56 (32.2) 118 (67.8) 427 ± 48 297 ± 19 0.066 (ns) 2.8 ± 0.3 1.9 ± 0.1 0.023 PgR status a negative positive 174 67 (38.5) 107 (61.5) 390 ± 39 307 ± 22 0.274 (ns) 2.6 ± 0.3 1.9 ± 0.2 0.153 (ns) a Mann-Whitney-U Test b Kruskal-Wallis Test x Unknown status splice variants uPAR-del5 and uPAR-del4/5) and uPAR-del5 mRNA in breast cancer. The assays are rapid, robust, and sensitive. As these assays require only minute amounts of cDNA, they are well suited for studies when the amount of sample is limited. For valida­tion of the uPAR-wt assay, we initially meas­ured uPAR mRNA expression in cell lines of different origin and observed that the meas­ured mRNA expression levels corresponded well to the respective antigen levels deter­mined by a uPAR ELISA (HU/IIIF10). In the tumor cell lines, we also detected low expres­sion of uPAR-del5 mRNA. In vivo expression of this uPAR splice variant was subsequently proven by analyzing breast tumor samples. Similar to uPAR-wt, we found no significant association of uPAR-del5 expression in rela-tion to the nodal status, tumor size or grade in the analyzed patients cohort. The cDNA sequence of the uPAR-del5 variant has been originally published by Casey et al. 15 Recently, we verified expres­sion of uPAR-del5 in tumor cells by amplifi­cation of uPAR mRNA with primers directed to exon 1 and 6, respectively, followed by di­rect sequencing of the resulting PCR prod­ucts.11 Further evidence for the in vivo occur­rence of uPAR-del5 comes from searching available nucleotide databases, in which a se­ries of independent submissions of the cDNA sequence encoding uPAR-del5 obtained from various sources (e.g. accessions AX281707, AA481366, BM543893 or BM767461) is found. Thus, the uPAR-del5 splice variant seems to be often expressed in human cells. Previously, we have generated stably trans-fected Chinese hamster ovary cells, which harbor an expression plasmid encoding uPAR-del5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed syn­thesis, secretion and cell surface-association of uPAR-del5. These experiments indicate that uPAR-del5 mRNA is translated and processed in a similar manner as wild-type uPAR. Therefore, experiments are on the way to search for (new) functions of uPAR-del5 (and the other expressed splice variant uPAR­del4/5) which may play a role for tumor inva­sion and metastasis. Acknowledgements Part of this work was supported by grants of the Kapitel Klinische Forschung (KKF) of the Technical University Munich, the Graduiertenkolleg 333 (Deutsche Forschung­sgemeinschaft), and Wilex AG, Munich. We thank our collaborators N. Brünner, A. Krüger, V. Möbus, and J. Schlegel for provid­ing cell lines. References 1. Magdolen U, Krol J, Sato S, Mueller MM, Krüger A, Sperl S, Schmitt M, Magdolen V. Natural inhibitors of tumor-associated proteas­es. Radiol Oncol 2002; 36: 131-43. 2. Sidenius N, Blasi F. The urokinase plasminogen activator system in cancer: recent advantages and implication for prognosis and therapy. Cancer Metast Rev 2003; 22: 205-22. 3. Blasi F, Carmeliet P. uPAR: a versatile signaling orchestrator. Nat Rev Mol Cell Biol 2002; 3: 932­43. 4. Harbeck N, Kates R, Gauger K, Willems A, Kiechle M, Magdolen V, Schmitt M. Urokinase-type plasminogen activator (uPA) and its in­hibitor PAI-1: novel tumor-derived factors with a high prognostic and predictive impact in breast cancer. Thromb Haemost 2004; 91: 450-6. 5. Reuning U, Magdolen V, Wilhelm O, Fischer K, Lutz V, Graeff H, Schmitt M. Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis. Int J Oncol 1998; 13: 893-906. 6. Muehlenweg B, Sperl S, Magdolen V, Schmitt M, Harbeck N. Interference with the urokinase plasminogen activator system: a promising therapy concept for solid tumours. Expert Opin Biol Ther 2001; 1: 683-91. 7. Sperl S, Mueller MM, Wilhelm OG, Schmitt M, Magdolen V, Moroder L. The uPA/uPA recep­tor system as a target for tumor therapy. Drug News Perspect 2001; 14: 401-11. 8. Reuning U, Sperl S, Kopitz C, Kessler H, Krüger A, Schmitt M, Magdolen V. Urokinase-type plasminogen activator (uPA) and its recep­tor (uPAR): development of antagonists of uPA/uPAR interaction and their effects in vitro and in vivo. Curr Pharm Des 2003; 9: 1529-43. 9. Liu S, Aaronson H, Mitola DJ, Leppla SH, Bugge TH. Potent antitumor activity of a uroki­nase-activated engineered anthrax toxin. Proc Natl Acad Sci USA 2003; 100: 657-62. 10. Roldan AL, Cubellis MV, Masucci MT, Behrendt N, Lund LR, Danř K, Appella E, Blasi F. Cloning and expression of the receptor for human urokinase plasminogen activator, a cen­tral molecule in cell surface, plasmin depend­ent proteolysis. EMBO J 1990; 9: 467-74. 11. Luther T, Kotzsch M, Meye A, Langerholc T, Füssel S, Olbricht N, Albrecht S, Ockert D, Muehlenweg B, Friedrich K, Grosser M, Schmitt M, Baretton G, Magdolen V. Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer. Thromb Haemost 2003; 89: 705­17. 12. Montuori N, Carriero M, Salzano S, Rossi G, Ragno P. The cleavage of the urokinase recep­tor regulates its multiple functions. J Biol Chem 2002; 277: 46932-9. 13. Beaufort N, Leduc D, Rousselle JC, Magdolen V, Luther T, Namane A, Chignard M, Pidard D. Proteolytic regulation of the urokinase recep­tor/CD87 on monocytic cells by neutrophil elastase and cathepsin G. J Immunol 2004; 172: 540-9. 14. Mřller LB, Pollanen J, Rřnne E, Pedersen N, Blasi F. N-linked glycosylation of the ligand-binding domain of the human urokinase recep­tor contributes to the affinity for its ligand. J Biol Chem 1993; 268: 11152-9. 15. Casey JR, Petranka JG, Kottra J, Fleenor DE, Rosse WF. The structure of the urokinase-type plasminogen activator receptor gene. Blood 1994; 84: 1151-6. 16. Magklara A, Scorilas A, Katsaros D, Massobrio M, Yousef GM, Fracchioli S, Danese S, Diamandis EP. The human KLK8 (neuropsin/ovasin) gene: identification of two novel splice variants and its prognostic value in ovarian cancer. Clin Cancer Res 2001; 7: 806-11. 17. Sweep CG, Geurts-Moespot J. EORTC external quality assurance program for ER and PgR measurements: trial 1998/1999. European Organisation for Research and Treatment of Cancer. Int J Biol Markers 2000; 15: 62-9. 18. Span PN, Tjan-Heijnen VC, Manders P, Beex LV, Sweep CG. Cyclin-E is a strong predictor of endocrine therapy failure in human breast can­cer. Oncogene 2003; 22: 4898-904. 19. Köhler T, Lerche D, Meye A, Weisbrich C, Wagner O. Automated analysis of nucleic acids by quantitative PCR using coated ready-to-use reaction tubes. J Lab Med 1999; 23: 408-14. 20. Kotzsch M, Luther T, Harbeck N, Ockert D, Lutz V, Noack F, Grossmann D, Albrecht S, Kramer MD, Lossnitzer A, Grosser M, Schmitt M, Magdolen V. New ELISA for quantitation of human urokinase receptor (CD 87) in cancer. Int J Oncol 2000; 17: 827-34. Radiol Oncol 2004; 38(2): 121-9. Ex vivo flow mammalian cell electropulsation Marie Christine Vernhes1, Nathalie Eynard1, Marie Pierre Rols1, Valentina Ganeva2 and Justin Teissié1 M C Vernhes1, Nathalie Eynard1, Marie Pierre Rols1, V Ganeva2 and Justin Teissié1 1 IPBS CNRS (UMR 5089) Toulouse, France, 2 Faculty of Biology, University of Sofia, Bulgaria Cell electropulsation brings a local and reversible permeabilization of cell membranes. This gives the tech­nical possibility to introduce (load) exogenous compounds (drugs, proteins, DNA) into cells. Flow-through electropulsation allows to treat a large volume (number) of cells as requested for cell therapy. Cells are flow­ing through a pulsing chamber where they are submitted to a well-defined number of calibrated pulses. A proper setting of pulse frequency and flow rate controls the number of pulses. A large volume of cells can therefore be electrotreated in a small sized pulsing chamber. The viability of pulsed cells appears to be great­ly preserved. Key words: cell membrane permeability; electroporation Introduction Cell treatment with high intensity electric field pulses provokes a change in the membrane structure leading to a loss of its barrier function -— phenomenon indicated as electropermeabi­lization or “electroporation”.1,2 By a proper choice of the parameters of the applied electric field, this change in the membrane permeabili­ty can be reversible or irreversible leading to leakage of cytoplasmic content and cell death.3 Received 19 March 2004 Accepted 5 April 2004 Correspondence to: Justin Teissié, IPBS CNRC (UMR 5089), 205 Route de Narbonne, 31077 Toulouse, Cedex, France. Phone: +33 5 6117 58 12; Fax: +33 5 6117 59 94; E-mail: justin.teissie@ipbs.fr This paper was presented at the “3nd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. When a short (microsecond) electric field pulse is applied to a cell, the resulting change in mem­brane potential difference may result in a local­ized long lived but reversible change in the membrane organization. This new state of the membrane is called « electropermeabilized » and can support the transfer of hydrophilic compounds into the cytoplasm and their leak­age out of the cell. A key feature is that under controlled electrical conditions this membrane change is transient and the «normal» imperme­able state can be recovered. The cell viability can therefore be preserved. This is obtained by a proper choice of electrical parameters (field strength, pulse duration and number of pulses) and buffers (pH, osmotic pressure and addi­tives). This brought the technical possibility to introduce (load) exogenous compounds (drugs) into cells. A clinical development was proposed with big success (electrochimiotherapy).4 This transient membrane organization remains poorly characterized from a structural point of view. A peculiar associated property is fuso­genicity. When cells, which are in the electrop­ermeabilized state, are brought into contact, membrane coalescence occurs leading to the formation of viable polykaryons (electrofu-sion).5 This is indicative that the repulsive hy­dration forces have been abolished. This was supported by the observation of an alteration of the interfacial layer of electropermeabilized cells. Another observation is that it was possible to get protein expression by electropulsing cells in a solution containing the relevant plasmid (electrotransformation, electrotransfection).6 This again can be obtained in vivo in tumors (electrogenotherapy).7 Ex vivo treatment of cells appears as an in­teresting procedure for cell therapy. A present limit of electropulsation is that most protocols were designed for batch process. Only limited volumes can be treated due to the power limi­tations of most pulse generators. Furthermore safe conditions avoiding contamination (either microbial or electrochemical) need the use of rather expensive equipments (large laminar flow hood, sterilization of the cuvettes). Introduced in the 80’s electric field treatment using a flow system seems nowadays a very promising technique for ex vivo cell therapy.8­12 Our conclusions were reproduced on differ­ent cell systems by other groups 13 and very re­cently either on a large volume as we did in 1992 14 or on microdevices.15 Recently we showed that application of se­ries of electric pulses could provoke not only a drug loading but an important release of differ­ent cytoplasmic enzymes by a batch process. The efficiency of this process was dependent on the intensity, number and duration of puls­es, on the growth phase and postpulse incuba­tion media composition. This batch approach even very efficient was suitable only for treat­ment of micro volumes. Large volumes were successfully treated by the flow process.16 A new aspect of flow-through electropermeabi­lization -— the release of macromolecules from mammalian cells and its potential clinical ap­plication is validated in the present study. Up-sizing of laboratory scale processes was always limited by the amount of energy to be delivered by the power generators. We pre­sented a vast field of evidences that it is pos­sible to utilize electropulsation using a flow system to work on a large volume of cells. Power specifications for the pulse generator are mostly driven by the required pulse fre­quencies. Nevertheless kHz trains can be de­livered meaning that high flow rate (i.e. large volumes) can be treated (up to 1 l/min).17,18 All aspects of electropulsation can be ob­tained with the flow process: drug loading, protein and metabolite extraction, eradica­tion, gene transfer and expression and hydrid production. A key advantage for clinical ap­plications is that contamination can be avoid­ed by using closed loop circuits. The present paper described the systemat­ic investigation of exogeneous compound loading and protein extraction from chinese hamster ovary cells by a flow electropulsation method by emphasing the good preservation of the viability of the treated cells. Materials and methods Cells Chinese hamster ovary (CHO) cells were used as a model system. The WTT clone was selected for its ability to both grow in suspen­sion and plate easily. They were grown in sus­pension in MEM medium as previously de­scribed.11 Electropermeabilization Culture medium was removed and replaced by a pulsing buffer (10 mM phosphate, 1 mM MgCl2, 250 mM sucrose, pH 7.4) at a cell den­sity of 106 per ml. This low volume fraction of cells was chosen to avoid any viscosity effect on the flow. Penetration of propidium iodide PI (100 µM, in pulsing buffer) was used to moni­tor permeabilization. Analysis was performed with a cytofluorimeter (Facscan, BD) to evalu­ate both the percentage of fluorescent cells (i.e. percentage of PI positive cells) and the mean level of fluorescence of the cell population. The protein release was monitored as fol­lows. Cells are kept 10-20 min at room tem­perature after the electrical treatment and then on ice. The protein concentration in the supernatant of pulsed and control cells was assayed by the Biorad kit. Cell viability was assayed 24h after the electrical treatment by the crystal violet method by taking advantage of the selectivity of viable cells to plate on a culture dish. Electropulsation Electric field treatment was performed on a CNRS high power cell electropulsator gener­ating rectangular pulses with adjustable volt­age up to 1.5 kV. A flow through pulsing chamber with a 0.3 mL volume was used. Two stainless steel flat parallel electrodes at a distance of 0.3 cm were used to apply repetitive pulses. Pulse duration (T) and frequency (Ff) were triggered by a TTL pulse generator. All pulsing parameters were monitored on line with an oscilloscope con­nected to a PC computer when storage was needed. An ohmic behaviour was observed. Cells were treated at room temperature with series of pulses with controlled duration and frequencies. The flow rate was in the range from 1.2 to 60 mL/min and freely adjustable with a peristaltic pump (Gilson, France). Flow electropulsation The basic concept was to apply calibrated pulses at a delivery frequency which was linked to the flow rate (Figure 1). The desired number of pulses was actually delivered on each cell during its residency in the pulsing chamber. The geometry of the chamber (flat parallel electrodes) was chosen to give a ho­mogeneous field distribution on a laminar flow. Therefore, the residency time Tof a res given cell in the chamber was: T= Vol / Q (Eq. 1) res where Vol was the volume of the pulsing flow chamber and Q , the flow rate. The number of pulses delivered per cell was: N = Vol F f / Q (Eq. 2) F f being the frequency of the pulses, there­fore N was under experimental control. Nevertheless one should take into account that a parabolic distribution of the flow rate was present in the chamber. More pulses were applied on cells close to the walls than in the middle of the chamber The field strength was taken as the voltage to electrode distance ratio E = U/d (Eq. 3) d being the width between the two elec­trodes, U, the voltage. The field distribution Figure 2. Control by the field on cell permeabilization. CHO cells (106 per ml) were treated by a train of 10 pulses lasting 1 ms at a frequency of 1 Hz with a flow rate of 1.2 ml/min. The pulsing buffer contains the hydrophilic dye PI. Permeabilization is assayed by the number of fluorescent cells (A) and by the mean fluorescence emission of the population (B) by use of a flow fluocytometer. was homogeneous when taking into account the geometry of the chamber (parallel flat electrodes). This average power associated to the train of pulses was:

= U I f T (Eq. 4) T being the single pulse duration. As the chamber resistance, when filled by the sample, could be approximated by R = d /( . S) (Eq. 5) where L is the conductance of the sample and S the section of the electrodes, then

= f T E2 . Vol (Eq. 6). From Eq. 2, an increase in the flow rate Q while keeping the number of applied pulses N con­stant needs to increase FVolfVol, i.e. either F f or Vol (or both). Only a limited energy and cur­rent are delivered with each pulse. The power and current specifications are not requiring so­phisticated designs. The only difficulty is to have a main power supply able to maintain the interpulse recharging of the internal capacitors when working at high frequencies. Another important parameter is the nature of flow which is given by the Reynolds num­ber Re Re = (v d . )/µ (Eq. 7) Where v is the flow velocity, r, the volumic mass of the liquid, m its dynamic viscosity In our experimental conditions, Re is al­ways smaller than 2000, i.e. the flow is under a laminar condition. No tumbling is affecting the cell population. Results Effect of the field strength A peculiar observation was that a rather high percentage of cells were PI positive just by flowing across the flow through chamber with­out any electrical treatment. This was much higher than the basal level in the native CHO cell population. This was associated with a de­crease in the cell viability of about 30%. A train of 10 pulses lasting each 1 ms was applied on the flow of cells at a frequency of 1 Hz (flow rate 1.2 ml/min). An increase in the number of PI positive cells and in PI stain- Figure 3. Viability of pulsed cells. CHO cells (106 per ml) were treated by a train of 10 pulses lasting 1 ms at a frequency of 1 Hz with a flow rate of 1.2 ml/min. Their viability was assayed 24 h after the treatment by the crystal violet test. The data were corrected from the effect of the flow on the con­trol cells, which brought a 30% loss. ing was clearly present as soon as the field strength of the applied pulses was a large as 0.5 kV/cm (Figure 2 A,B). A maximal effect was present around 1 kV/cm and a small de­crease was observed for larger intensities. The viability of the pulsed cells was affect­ed in a field dependent way (all other param­eters being kept constant). Cells in the flow process were apparently very sensitive to the field as shown by the effect of 0.25 kV/cm pulses. But cells were observed to be some­how resistant to very strong electric field (Figure 3). The loss in viability apparently lev­eled off above 1.5 kV/cm. Effect of pulse duration Cells were submitted to a train of 10 pulses at a frequency of 10 Hz and a magnitude of 1 kV/cm with a flow rate of 1.2 ml/min. These conditions were chosen by taking into ac­count that when the pulse duration was 1 ms, all cells were permeabilized. We observed that even with a pulse duration as short as 0.1 ms the electrical treatment brought a perme­abilization of all cells (Figure 4A). Increasing the pulse duration above this value did not in­duce any further change. PI staining was ob­served to increase continuously with the pulse duration (Figure 4B). Cell viability was strongly dependent on the pulse duration. A sharp decrease was ob­served up to 0.2 ms followed by a slow de­crease with a further increase of the pulse du­ration (Figure 4A). Interestingly if the pulse duration was in­creased up to 5 ms, a much more limited lev- Figure 4. Effect of the pulse duration on cell permeabilization. CHO cells (106 per ml) were treated by a train of 10 pulses of 1 kV/cm at a frequency of 1 Hz with a flow rate of 1.2 ml/min. The pulsing buffer contains the hydrophilic dye PI. Permeabilization is assayed by the number of fluorescent cells (A) and by the mean fluorescence emission of the population (B) by use of a flow fluocytometer. Their viability was assayed 24 h after the treatment by the crystal violet test (A). The data were corrected from the effect of the flow on the control cells, which brought a 30% loss. Figure 5. Effect of the flow rate on cell permeabilization. CHO cells (106 per ml) were treated by a train of 10 pulses of 1 kV/cm lasting 1 ms at a frequency of 1 Hz with a flow rate of 1.2 ml/min and 10 Hz with a flow rate of 12 ml/min. Permeabilization is assayed by the number of fluorescent cells (A). Their viability was assayed 24 h after the treatment by the crystal violet test (B). The data were corrected from the effect of the flow on the control cells, which brought a 30% loss. el of permeabilization was observed and was observed to be associated to an irreversible process (cell death). No reversible permeabi­lization was observed. Effect of the flow rate Treatment of a large volume would take advan­tage of a high flow rate. We compared the be­havior of cells when submitted to a train of 10 pulses of 1 ms. Their frequencies were adjust­ed to the flow rate, being 1 Hz at 1.2 ml/min and 10 Hz at 12 ml/min. The on-line monitor­ing of the signal on the oscilloscope showed that even under the 10 Hz procedure, the shape of the pulses remained square. The flow re­mained laminar under the two conditions. Permeabilization was detected as soon as the field strength was larger than a critical value of 0.5 kV/cm. Its increase was sharper with a further increase in the field strength under the low flow rate conditions. All cells were permeabilized under the two conditions when a field as large as 1.5 kV/cm was ap­plied (Figure 5 A). Cell viability was less affected for a given field strength at the high flow rate (Figure 5 B). As a conclusion, reversible permeabiliza­tion was obtained under higher field condi­tions with the high flow rate. Cytoplasmic protein release We previously showed that flow-through electropulsation was an efficient approach for protein extraction from yeasts.16 Clinical biotechnology is taking advantage of the bio-production of proteins in mammalian cells.19 CHO cells are one of the most successful cell factories. Results in the preceding part of this work was dealing with the loading of small (drug size like) molecules. In this part of the work we checked how effective was the flow-through electropulsation for the extraction of cytoplasmic proteins from CHO cells. Cells were submitted to a train of 10 puls­es lasting 1 ms at a frequency of 10 Hz with a flow rate of 1.2 ml/min. This pulse duration was chosen being shown in batch experi­ments to be needed to obtain macromolecule loading.20 No protein release was observed with field strength up to 1 kV/mscm. High level of extraction was observed between 1.2 and 1.5 kV/cm (Figure 6). Interestingly as re­ported above the viability while affected by the electrical treatment remained larger than Figure 6. Electrorelease of cytoplasmic proteins. CHO cells (106 per ml) were treated by a train of 10 pulses of 1 kV/cm lasting 1 ms at a frequency of 1 Hz with a flow rate of 1.2 ml/min. Their via­bility was assayed 24 h after the treatment by the crystal violet test. The data were corrected from the effect of the flow on the control cells, which brought a 30% loss. 40% under the field conditions where the high electroextraction was obtained. Discussion The present study shows the flexibility and the effectiveness of the flow through elec­tropulsation in the case of mammalian cells. A high level of loading evaluated both in the number of PI positive cells and in the number of internalized polar molecules (as quantified by the fluorescence of PI in the present experiments) was obtained while pre­serving the viability of most cells. By using the 1 ms and 1 Hz conditions, we observed that up to 60% of the population could be loaded (permeabilized) and remained viable. This takes into account the fact that more than 20% of the population was killed by the flow. This clearly should be optimized for a clinical development of the method for cell therapytreatment. This can be optimized by taking into account that 0.1 ms pulses were effective to obtain a high level of loading. Short pulses may be less detrimental for cells. Under laminar flow conditions (Re less than 2000), the maximal efficiency of loading was not affected by the flow rate up to 12 ml/min. Viability was sensitive to the flow rate. Higher flow rate can be obtained by an array of parallel chambers where all cells would be treated under the same conditions (electrical parameters, flow velocity). The current which was delivered during the pulses under the optimized conditions (0.75 kV/cm) was only 1 Amp. The average power was 2.5 W under the high flow rate (12 ml/min, 10 Hz). This condition brought a temperature increase of only 2°C of the cell suspension under the assumption that no heat dissipation occurred between the pulses. This conclusion further supports the safety of this approach for drug loading in cell therapy. We observed a shift in the permeabiliza­tion /field strength plots when the flow rate was increased. This could result from the vis­coelasticity of CHO cells. Their spherical shape observed under batch conditions would be altered by the drag of the flow. This drag increased with the flow rate. In simpler words, their shape turned in a more elongat­ed one. It was well established that the sensi­tivity of a cell to an electric field was con­trolled by its shape and its orientation relative to the field lines.21 Our observation that cell permeabilization needed higher field strength under the high flow rate where they were elongated in a direction perpendicular to the field was fully supported by the theoretical approach. This interpretation is further sup­ported by the shift of the viability plot: elon­gated cells in a direction perpendicular to the field are less sensitive to the field The protein extraction is clearly a very effi­cient new approach for clinical biotechnolo­gy. The observation that a large subpopula­tion was still viable after the train of 1 ms pulses suggested that it should be possible to recycle the pulsed population in a fermentor for a further growth of the cells. This is in­dicative that cell viability is not affected and that electrochemical contamination if present is not harmful and furthermore can be de­creased by a bipolar pulse. (22). As a final conclusion, flow electropulsa­tion offers a safe and powerful tool for the development of cell therapy Acknowledgements This work was made possible through finan­cial supports of Electricité de France and of NATO. References 1 Neumann E. The relaxation hysteresis of mem­brane electroporation. In: Sowers, Neumann E, Jordan, editors. Electroporation and electrofusion in cell biology. New York, Plenum Press; 1989. p. 61-82. 2. Rols MP, Teissié J. Electropermeabilization of mammalian cells: Quantitative analysis of the phenomenon. Biophys J 1990; 58: 1089-98. 3. Sale AJH, Hamilton WA. Effect of high intensi­ty electric fields on microorganisms. I. Killing of bacteria and yeast. Biochim Biophys Acta 1967; 148: 781-8. 4. Mir LM, Glass LF, Sersa G, Teissié J, Domenge C, Miklavcic D, Jaroszeski MJ, Orlowski S, Reintgen DS, Rudolf Z, Belehradek M, Gilbert R, Rols MP, Belehradek JR, Bachaud JM, Deconti R, Stabuc B, Cemazar M, Coninx P, Heller R. Effective treatment of cutaneous and subcutaneous malignant tumours by electro-chemotherapy. Br J Cancer 1998; 77: 2336-42. 5. Teissié J, Rols MP. Fusion of mammalian cells in culture is obtained by creating the contact between cells after their electropermeabiliza­tion. Biochem Biophys Res Comm 1986; 140: 258­66. 6. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982; 1: 841-5. 7. Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissié J. In vivo electrically mediated pro­tein and gene transfer in murine melanoma. Nat Biotechnol 1998; 16: 168-71. 8. Teissié J, Rols MP. Electrofusion of large vol­umes of cells in culture: 2- Cells growing in sus­pension. Bioelectrochem Bioenerg 1988; 19: 59­66. 9. Teissié J, Conte P. Electrofusion of large vol­umes of cells in culture: 1- Anchorage depend­ent strains. Bioelectrochem Bioenerg 1988; 19: 49­57. 10. Sixou S, Teissié J. Specific electropermeabiliza­tion of leucocytes in a blood sample and appli­cation to large volumes of cells. Biochim Biophys Acta 1990; 1028: 154-60. 11. Rols MP, Coulet D, Teissié J. Highly efficient transfection of mammalian cells by electric field pulses: application to large volumes of cell culture by using a flow system. Eur J Biochem 1992; 206: 115-21. 12. Teissié J. Electropulsation de cultures cellu­laires. Biofutur 1986; 45: 64-7. 13. Bruggemann U, Roux EC, Hannig J, Nicolau C. Low oxygen affinity red cells produced in a large volume, continuous flow electropration systems. Transfusion 1995; 35: 478-86. 14. Li LH, Shivakumar R, Feller S, Allen C, Weiss JM, Dzekunov S, Singh V, Holaday J, Fratantoni J, Liu LN. Highly efficient, large volume flow electroporation. Technol Cancer Res Treat 2002; 1: 341-50. 15. Lin YC, Jen CM, Huang MY, Wu CY, Lin XZ. Electroporation microchips for continuous gene transfection. Sensor Actuat B-Chem 2001; 79: 137-43. 16. Ganeva V, Galutzov B, Teissié J. High yield electroextraction of protein from yeast by a flow process. Anal Biochem 2003; 315: 77-84. 17. Barbosa-Canovas GV, Gongora-Nieto MM, Pothakamury UR, Swanson BG. Preservation of food with pulsed electric fields. New York: Academic Press, 1999. 18. Vernhes MC, Benichou A, Pernin P, Cabanes PA, Teissié J. Elimination of free - living amoe­bae in fresh water with pulsed electric fields. Water Res 2002; 36: 3429-38. 19. Andersen DC, Krummen L. Recombinant pro­tein expression for therapeutic applications. Curr Opin Biotechnol 2002; 13: 117-23. 20. Rols MP, Teissié J. Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 1998; 75: 1415-23. 21. Vali? B, Golzio M, Pavlin M, Schatz A, Gabriel B, Faurie C, Teissié J, Rols MP, Miklav?i? D. Electric field induced transmembrane poten­tial on spheroidal cell: theory and experiment. Eur J Biophys 2003; 32: 519-28. 22. Kotnik T, Miklavcic D, Mir LM . Cell mem­brane electropermeabilization by symmetrical bipolar rectangular pulses. Part II. Reduced electrolytic contamination. Bioelectrochemistry. 22001; 54: 91-5. Radiol Oncol 2004; 38(2): 131-5. Immunohistochemical expression of HER-2/neu in patients with lung carcinoma and its prognostic significance Gordana Petrusevska1, Biljana Ilievska-Poposka2, Sašo Banev1, Snežana Smickova3, Zoran Spirovski4 1Institute of Pathology, 2Institution for Lung Diseases and Tuberculosis, 3Institute for Oncology; 4Clinic for Thoracovascular Surgery, Medical Faculty, University of “Sv. Kiril and Metodij”, Skopje, Republic of Macedonia Background. The HER-2 protein or p185her2 is a membrane receptor with tyrosine kinase activity encod­ed by HER-2/neu gene. Overexpression of HER-2/neu has been observed in many human cancers, includ­ing lung cancer. In the study, the expression of HER-2 protein is determined in the spectrum of lung cancer (adenocarcinoma, squamous cell carcinoma and small cell carcinoma). Patients and methods. The study population consisted of two groups: 19 patients that had undergone sur­gical treatment and 10 patients that had undergone fiber-optic bronchoscopy and biopsy for primary diag­nosis only. Tissue specimens were neutral formaldehyde-fixed and paraffin-embedded. Standard histo­chemical and immunohistochemical staining were used for diagnosis. Expression of HER-2/neu protein was determined by immunohistochemical staining with Hercep TestTM (DAKO). The results were graded 0-1 as negative and 2-3 as positive. Results. Overall incidence of HER-2/neu overexpression was 34.4% (10 of 29). Higher incidence was found in the patients with adenocarcinoma 45.4% (5 of 11). In squamous cell carcinoma and small cell car­cinoma, the overexpression incidence was 30.7% (4 of 13) and 20% (1 of 5), respectively. No statistically significant difference was seen given the age and gender. HER-2/neu overexpression was more pronounced in the patients with advanced tumour: all patients with squamous cell carcinoma and HER-2/neu overex­pression had stage IIIB and stage IV disease, while 80 % of adenocarcinoma patients with HER-2/neu over-expression had stage IIIA and IIIB disease. Conclusions. These results are satisfactory and encourage us to continue this work in the follow-up study to evaluate HER-2/neu role as predictive and prognostic factor for the patients with lung cancer. Key words: lung neoplasms – pathology; receptors,, erb-2; prognosis; immunohistochemistry Received 26 February 2004 Accepted 15 March 2004 Correspondence to: Prof. Gordana Petrusevska, MD, PhD, Institute of Pathology, Medical Faculty, University of “Sv. Kiril and Metodij”, Skopje, Republic of Macedonia; Phone: +389 2 3163 806; Fax: +389 2 3229 166; E-mail: gordanap61@yahoo.com Introduction Lung cancer is the leading cause of cancer mortality worldwide. In Macedonia, lung can­cer is the cause of death in nearly 19.44% of all cancer deaths.1 Most of the patients had an advanced stage of the disease at the time of diagnosis. Even with early diagnosis and an adequate treatment, the 5-year survival rate of the patients with stage I disease is about 70%.2 We presume that there is an intrinsic fac­tor that determines the clinical course of the disease. Advances in molecular pathology showed that expression of p53, c-myc, c-fos, c-erbB-1,3 and Her-2/neu4,5 is associated with lung cancer. The HER-2 protein or p185-her 2 is a membrane receptor with tyrosine kinase activity encoded by HER-2/neu gene. HER-2/neu overexpression has been ob­served in many human cancers, including carcinoma of the breast, ovary, gastrointesti­nal tract, salivary gland and lung.6 There are different reports about the expression of this protein in different lung cancers. The aim of the study was to determine the expression of HER-2 protein in the adenocar­cinoma, squamous cell carcinoma (squamous CC) and small cell carcinoma (small CC) of the lung, and to assess the correlation be­tween HER-2/neu expression and clinical stage of the patients. The evaluation of the expression of HER-2/neu is important because of its prognostic significance and possibility to treat the pa­tients with trastuzumab (HerceptinTM). Material and methods The study population consisted of two groups: 19 patients that had undergone surgi­cal treatment and 10 patients that had under­gone fiber-optic bronchoscopy and biopsy for primary diagnosis only. The stage of the disease was classified ac­cording to the new international staging sys­tem for lung cancer, using the following clin­ical investigations: blood examination, bio­chemical studies, chest radiography, whole bone scan and computed topographic scan of the chest. Tissue specimens from surgical and biopsy material were neutral formaldehyde-fixed and paraffin-embedded. Standard histologi­cal stainings (Hemalaun-Eosin, PAS, Alcian blue –PAS and reticulin – Gomori) were used for diagnosis. HER-2/neu oncogene expression was de­termined by immunohistochemical staining with Hercep TestTM (DAKO). The slides were deparaffinized in xylene and rehydrated with graded ethanol. Following the incubation with the primary rabbit antibody to human HER-2 protein, ready-to-use Visualisation Reagent consisted of both secondary goat an-ti-rabbit immunoglobulin and horseradish peroxidase, was used. The enzymatic conver- Table 1. Distribution of the patients according to the histological type of the tumour and clinical stage Histological type Clinical stage Total Adenocarcinoma Squamous CC Small CC I II 05 05 0 2 IIIa 3 2 3 IIIb 3 4 0 IV 011 2 13 0 5 Total 012 8 7 2 29 Table 2. Overexpression of HER2 according to histological type and clinical stage of disease Histological type HER2+/total Clinical stage Total (%) I II IIIa IIIb IV Adenocarcinoma 0/0 1/5 2/3 2/3 0/0 5 (45.4%) Squamous CC 0/0 0/5 0/2 3/4 1/2 4 (30.7%) Small CC 0/0 0/2 1/3 0/0 0/0 1 (20%) Total of HER2+ 01 3 5 1 10(34.4%) sion of the subsequently added DAB chro­mogen resulted in formation of brown pre­cipitation at the antigen site. Hercep TestTM was interpreted as negative for HER-2 protein overexpression (0 and 1+ staining intensity), and positive for HER-2 protein overexpression (weakly positive 2+ staining intensity and strongly positive 3+ staining intensity). Results The mean age of the patients was 55.4 years, ranging from 41 to 72 years. Twenty-six pa­tients were males and 3 patients were fe­males. More tumours were found in the left lung (19 cases, 65.5%) than in the right lung (10 cases, 34.5%). In the patients who under­went surgical treatment, lobectomy and me-diastinal lymphadenectomy were performed. Other patients received radiotherapy and/or chemotherapy, depending on clinical stage. The follow-up of the patients was from 3 to 24 months. In two patients, the outcome of sur­gery was fatal. Two patients died of advanced disease, and one died of cardiac failure. Table 1 shows the patients distribution according to the histological type and clinical stage of lung cancer. Histological analysis revealed 11 cases of adenocarcinoma, 13 cases of squamous CC and 5 cases of small CC. The majority of pa­tients had a clinical stage II disease (12 cases), 8 patients had stage IIIA, 7 patients IIIB, and two stage IV. The overall incidence of HER-2 overex­pression was 34.4% (10 of 29). Higher inci­dence was found in the patients with adeno-carcinoma – 45.4% (5 of 11) (Figure 1), while in squamous CC patients, the incidence was 30.7% (4 of 13) (Figure 2), and in small CC pa­tients, the incidence was 20% (1 of 5) (Figure 3) (Table 2). There was no statistically significant dif­ference given the age and gender. Figure 3. Positive immunostaining for HER-2 in small cell lung carcinoma (DAKO Hercep Test TM). As shown in Table 2, HER-2 protein over-expression was more pronounced in the pa­tients with advanced clinical stage: 4 of 5 pa­tients (80%) with adenocarcinoma and HER-2 protein overexpression had stage IIIA and II­IB disease, and 3 of 4 patients (75%) with squamous CC and HER-2 protein overexpres­sion had stage IIIB disease, while of two pa­tients with stage IV squamous CC, only one (50%) showed HER-2 protein overexpression. One patient with small CC and positive for HER-2 had in stage IIIA disease. Discussion Immunohistochemistry is a frequently used method for evaluating gene expression in tu­mours. However, a great variation in the in­terpretation of results has been noted. In the case of lung carcinoma, the positive rate of HER-2 expression ranged from 14 to 87.5%.7 This variation is probably due to different methods, materials or subjective biases. There is also a possibility for heterogeneous expression of HER-2 in tumour cells. Hercep TestTM (DAKO) offers an objective and quan­titative evaluation of immunohistochemical results. This is especially important when this immunohistochemically based marker is em­ployed for assessing the therapy and progno­sis. In our study, the overall incidence of HER­2 overexpression was 34.4%, with a higher in­cidence found in the patients with adenocar­cinoma – 45.4%. The patients with squamous CC overexpressed HER-2 in 30.7 % and those with small CC in 20%. These results are slightly higher than those presented in the re­view of Agus et al.8 and could be due to the sensitivity of the antibody Hercep TestTM (DAKO). There were many previous reports which have described the p185neu expression in lung cancer.2-7 In some cases, the overexpres­sion of HER-2/neu was shown to correlate al­so with the survival.4,5 However, a limited number of cases, lack of a scoring system for HER-2/neu expression and conflicting results made the interpretation of these studies diffi­cult. Harpole et al9 suggested that, beside the other parameters (male, sex, presence of symptoms, tumour size, poor cell differentia­tion, vascular invasion, p53 expression and high Ki-67 index), the HER-2/neu expression could be an independent prognostic factor in the patients with stage I non-small cell lung cancer. They further propose that the out­come of the disease is the “dose response” of the additive effect of these parameters. It ap­peared that the disease progression could be dependent on several clinicopathologic pa­rameters. The cause of early cancer deaths as well as the mechanism for the early relapse and metastasis remain to be determined. Osaki et al10 have shown that the patients with stage IIIB or T4 lung adenocarcinoma had an increased serum level of HER-2 and that it was correlated with the overexpression in tissue sections. Diez et al.11, working on fresh samples of non-small cell lung cancer, did not find significant correlation between p185 level and TNM classification, but proved that shortened median time of tumour relapse was proportional to the rise of p185 in the tumour tissue. The results presented in this paper demon­strate that the overexpression of HER-2/neu in the patients with lung carcinoma correlat­ed with the advanced clinical stage. The fol­low-up of the patients in our study was short, so the determination of the relapse rate or disease-free period was limited. Two patients who died of the advanced disease had over-expression of HER-2 and short partial remis­sion of 5 and 8 months. In conclusion, our results are satisfactory and encourage us to continue this work in the follow-up study to evaluate HER-2/neu role as predictive and prognostic factor for the pa­tients with lung cancer. However, the deter­mination of this parameter is required in more patients and for longer period to con­firm the value of HER-2 overexpression. The correlation with other oncoproteins is needed for the elucidation of a cause of early cancer death as well as of a mechanism for an early relapse and metastasising. References 1. Cancer Registry in Macedonia. Skopje: Republic Institute for Health Protection; 1997. p. 83. 2. Pairolero PC, Williams DE, Bergstralh EJ, Piehler JM, Bernatz PE, Payne WS. Postsurgical stage I bronchogenic carcinoma: morbid impli­cations of recurrent disease. Ann Thorac Surg 1984; 38: 331-8. 3. Volm M, Effereth T, Mattern J. Oncoprotein (c-myc, c-erbB-1, c-erbB-2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung. Clinical and biological correlations. Anticancer Res 1992; 12: 11-20. 4. Weiner DB, Nordberg J, Robinson R, Nowell PC, Gazdar A, Greene MI, et al. Expression of the neu gene-encoded protein (P185neu) in hu­man non-small cell carcinomas of the lung. Cancer Res 1990; 50: 421-5. 5. Shi D, He G, Cao S, Pan W, Zhang HZ, Yu D, et al. Overexpression of the c-erbB-2/neu-en­coded p185 protein in primary lung cancer. Mol Carcinog 1992; 5: 213-8. 6. Hynes NE, Stern DF. The biology of erbB­2/neu/HER-2 and its role in cancer. Biochim Biophys Acta 1994; 1198: 165-84. 7. Hsieh CC, Chow KC, Fahn HJ, Tsai CM, Li WY, Huang MH, et al. Prognostic significance of HER-2/neu overexpression in stage I adenocar­cinoma of lung. Ann Thorac Surg 1998; 66: 1163­4. 8. Agus DB, Bunn PA Jr, Franklin W, Garcia M, Ozols RF. HER-2/neu as a therapeutic target in non-small cell lung cancer, prostate cancer, and ovarian cancer. Semin Oncol 2000; 27: 53-63. 9. Harpole DH, Herndon JE II, Wolfe WG, Iglehart JD, Marks JR. A prognostic model of re­current and death in stage I non-small cell lung cancer utilizing presentation, histopathology and oncoprotein expression. Caner Res 1995; 55: 51-6. 10. Osaki T, Mitsudomi T, Oyama T, Nakanishi R, Yasumoto K. Serum level and tissue expression of c-erbB-2 protein in lung adenocarcinoma. Ches. 1995;108: 157-62. 11. Diez M, Pollan M, Maestro M, Torres A, Ortega D, Gomez A, et al. Prediction of recurrence by quantification of p185-neu protein in non-small cell lung cancer tissue. Br J Cancer 1997; 75: 684-9. Radiol Oncol 2004; 38(2): 137-44. New saccharide derivatives of indolo[2,3-b]quinoline as cytotoxic compounds and topoisomerase II inhibitors Joanna Godlewska1,2, Katarzyna Badowska-Roslonek2, Jan Ramza2, Lukasz Kaczmarek2, Wanda Peczynska-Czoch3, Adam Opolski1 1 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland; 2 Pharmaceutical Research Institute, Warsaw, 3 Institute of Organic Chemistry, Biochemistry and Biotechnology, University of Technology, Wroclaw, Poland Some of alkyl- and alkylamino- derivatives of 6H-indolo[2,3-b]quinolines are known to be active antiprolif­erative and cell cycle modulating compounds. Their cytotoxic properties are, at least in part, due to DNA in­tercalation ability and topoisomerase II inhibition activity. To improve physicochemical and biological prop­erties of 6H-indolo[2,3-b]quinolines the series of new, saccharide (C-2, C-9 or N-6) derivatives were de­signed and synthesized. The influence of different carbohydrate units (D-glucose, D-lactose, L-rhamnose, L-acosamine, L-daunosamine), position of attachment and linker size on cytotoxic properties and topoiso­merase II inhibition activity were tested. Among compounds tested there were 2-deoxy-a-D-glucopyranoside (1-6), 2-deoxy-a-L-rhamnopyranoside (7-12) and 2-deoxy-a-D-lactopyranoside (13-18) derivatives in the group of saccharide moiety containing compounds and a-L-daunosaminide (19-24) and a-L-acosaminide (25­ 27) in the aminosaccharide derivatives series as well. Key words: DNA topoisomerases, type II – antagonists and inhibitors; indols; quinones Introduction Indolo[2,3-b]quinoline derivatives are a group of compounds being the synthetic analogs of a plant alkaloid neocryptolepine (5-methyl- Received 25 April 2004 Accepted Correspondence to: Adam Opolski, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 Weigla St., 53-114 Wroclaw, Poland. Phone: +48 (71) 3371172 ext.371; Fax: +48 (71) 3371382; E-mail: opolski@iitd.pan.wroc.pl This paper was presented at the “3rd Conference on Experimental and Translational Oncology”, Kranjska gora, Slovenia, March 18-21, 2004. 5H-indolo[2,3-b]quinoline), which (together with cryptolepine) is present in Cryptolepis sanguinolenta extracts used in natural medici­ne in Africa. Cryptolepine (5-methyl-5H-indolo[3,2­b]quinoline) -major Cryptolepsis sanguinolenta alkaloid - displays a plenty of pharmacologi­cal effects, such as antimuscarinic, noradre­nergic receptor antagonistic, antihypertensi­ve, vasodilative, antithrombotic, antipyretic and antiinflammatory properties. Neocryptolepine and cryptolepine deriva­tives reveal antiplasmodial and antitrypano­somal1 and, first of all, cytotoxic activities.2 Indolo[2,3-b]quinoline derivatives can also be considered as the analogs of the DNA in­tercalating anticancer drug, ellipticine, and its natural isomer, olivacine. The alkaloids of the pyrido[4,3-b]carbazole group are able to stabilize in vitro the topoisomerase II-DNA cleavable complexes. One of the cytotoxic oli­vacine derivatives - S 16020 -showing no im­munogenicity in clinical phase I trial was qu­alified for phase II studies.3 Previous studies revealed that topoisome­rase II is in fact a cellular target involved in the mechanism of cytotoxic action of 5,11-di­methyl-5H-indolo[2,3-b]quinoline (DiMIQ), first examined cytotoxic compound of the group.4 Further investigation into the impact of the substituents introduced into the indolo[2,3­b]quinoline was conducted. Consequently, the series of different derivatives (including vario­us types of substituents and position of their attachment) of 5H- and 6H-indolo[2,3-b]quino-lines were synthesized and their cytotoxic acti­vity and ability to induce topoisomerase II-de­pendent DNA cleavage in vitro were tested. It was initially stated that among methyl-substituted indolo[2,3-b]quinolines, only de­rivatives belonging to the 5H series (and none of the 6H series), display cytotoxicity against human cervix carcinoma KB cells -ID50 (inhi­bitory dose 50%) values were in the range of 2 to 9 µM - and against several human cancer cell lines of different origin (ID50 values va­ried from 0.6 to 1.4 µM), as well as stimulate the formation of calf thymus topoisomerase II-mediated DNA cleavage at concentrations between 0.2 and 10 µM.5,6 Further SAR (structure-activity relation­ship) studies conducted on 6H series showed that the introduction of an alkyl-amino-alkyl substituent at the N-6 position of indolo[2,3­b]quinoline accounts for the appearance of the cytotoxic properties against KB cell line. ID50 values obtained were in the range of 2.0 to 9.0 µM. These results indicate on a strong relation between 6H-indolo[2,3-b]quinoline derivatives structure and their cytotoxic acti­vity, corresponding well with the ability to bind DNA and to inhibit topoisomerase II ac­tivity.7 To avoid problems connected with poor solubility and resulting inefficient bioavaila­bility, another members of the cytotoxic indo­lo[2,3-b]quinoline family, well soluble in wa­ter in a non-pH-dependent manner, were syn­thesized and tested.8 Simultaneously, attempts at preparing li­posomally-formulated 5H-indolo[2,3-b]quino­lines9 and obtaining cytotoxic 6H sugar and aminosugar bearing indolo[2,3-b]quinoline derivatives were undertaken. The results of research on the 6H series are the subject of present publication. Material and methods Compounds All compounds tested were synthesized at Pharmaceutical Research Institute in War­saw.10 Human topoisomerase II activity inhibition assay Topoisomerase II Assay Kit (Catalog No. 1001-2) and Human Type II Topoisomerase (p170 Form) (Catalog No. 2000H-2); 2U/ml, used in the assays were purchased from TopoGEN, Inc. (Columbus, Ohio, USA). The start solutions of compounds tested (10 mM) were prepared by dissolving the substances in DMSO (dimethyl sulfoxide, Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) and diluting in reaction buffer to the concentrations of 0.0005, 0.0025, 0.005, 0.025, 0.05, 0.25 and 0.5 mM. Electrophoresis was run in 1% agarose gel (Roth, Karlsruhe, Germany) with ethidium bromide (0.5mg/ml, Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) at 150V. The sa­me amount of ethidium bromide was added Figure 1. The structures of saccharide and aminosaccharide derivatives studied. to electrophoresis buffer - 1x TBE (89mM Tris-borate, 1mM EDTA, pH 8.0). For results acquisition Typhoon 8600 Variable Mode Imager (Molecular Dynamics Inc, Sunnyvale, CA, USA) was applied. Inhibition of topoisomerase II activity by compounds tested was measured as a degree of inhibition of the kinetoplast DNA decate-nation. It was assumed that total enzyme ac­tivity inhibition resulted in the same out­comes as achieved for sample with the sub­strate kinetoplast DNA only.11,12 Cell lines Established in vitro, human cervix carcinoma (KB) and Jurkat (T-cell leukemia) cell lines were used. Both lines were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.) and maintained in the Cell Table 1. Cytotoxicity and inhibition of topoisomerase II activity revealed by D-glucose indolo[2,3-b]qu­inoline saccharide derivatives. Compound tested Cytotoxity -ID50 [µM] Total topoisomerase II inhibition [mM] (No. 1) R3 = 2-deoxy-a-D-glucopyranoside n = 2 . 98.30 ± 31.95 n.a. (No. 2) R1 = 2-deoxy-a-D-glucopyranoside n = 2 n.a. n.t. (No. 3) R2 = 2-deoxy-a-D-glucopyranoside n = 2 15.40 ± 5.05 n.a. (No. 4) R3 = 2-deoxy-a-D-glucopyranoside n = 5 68.20 ± 1.24 n.a. (No. 5) R1 = 2-deoxy-a-D-glucopyranoside n = 5 46.30 ± 2.05 n.a. (No. 6) R2 = 2-deoxy-a-D-glucopyranoside n = 5 57.60 ± 8.50 n.a. ID501) – compound concentration leading to 50% inhibition of cell proliferation; n – number of carbon atoms in linker moiety (as it is shown in Figure 1); n.a. -not active; n.t. -not tested. Culture Collection of the Institute of Immu­nology and Experimental Therapy, Wroclaw, Poland. The cells were cultured in the RPMI 1640 + Opti-MEM (KB) or RPMI 1640 (Jurkat) medium supplemented with 2mM glutamine (Gibco, Warsaw, Poland), streptomycin (100 mg/ml), penicillin (100U/ml) (both antibiotics from Polfa, Tarchomin, Poland) and 5% (KB) or 10% (Jurkat) fetal calf serum (Gibco, Grand Island, U.S.A.). The cell cultures were main­tained at 37°C in humid atmosphere satura­ted with 5% CO2. Anti-proliferative assay in vitro Test solutions of the compounds (1 mg/ml) were prepared by dissolving the substances in 100 µl of DMSO completed with 900 µl of tissue culture medium. Afterwards, the tested compounds were diluted in culture medium to reach the final concentrations of 100, 10, 1, and 0.1 µg/ml. Results were converted into µM concentrations. The details of the SRB assay were described by Skehan et al.13 Twenty-four hours before ad­dition of the tested agents, the cells were plated in 96-well plates (Sarstedt, U.S.A.) at a density of 1x104 cells per well. The cytotoxicity assay was performed after 72-hour exposure of the cultu­red cells to varying concentrations (from 0.1 to 100 µg/ml) of the tested agents. The cells atta­ched to the plastic were fixed by gently layering cold 50% TCA (trichloroacetic acid, Aldrich-Che­mie, Germany) on the top of the culture medium in each well. The plates were incubated at 4°C for 1 hour and then washed five times with tap water. The background optical density was me­asured in the wells filled with culture medium, without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (SRB, Sigma, Germany) and dissolved in 1% ace­tic acid (POCh, Gliwice, Poland) for 30 minutes. Unbound dye was removed by rinsing (4x) with 1% acetic acid. The protein-bound dye was extracted with 10mM unbuffered Tris base (POCh, Gliwice, Poland) for determination of Table 2. Cytotoxicity and inhibition of topoisomerase II activity revealed by L-rhamnose indolo[2,3-b]qu­inoline saccharide derivatives. Compound tested Cytotoxity -ID50 [µM] Total topoisomerase II inhibition [mM] (No. 7) R3 = 2-deoxy-a-L-rhamnopyranoside n = 2 n.a. n.t. (No. 8) R1 = 2-deoxy-a-L-rhamnopyranoside n = 2 n.a. n.t. (No. 9) R2 = 2-deoxy-a-L-rhamnopyranoside n = 2 43.70 ± 8.41 n.a. (No. 10) R3 = 2-deoxy-a-L-rhamnopyranoside n = 5 70.80 ± 0.90 n.a. (No. 11) R1 = 2-deoxy-a-L-rhamnopyranoside n = 5 n.a. n.t. (No. 12) R2 = 2-deoxy-a-L-rhamnopyranoside n = 5 39.60 ± 13.80 n.a. ID501) – compound concentration leading to 50% inhibition of cell proliferation; n- number of carbon atoms in linker moiety (as it is shown in Figure 1); n.a. -not active; n.t. - not tested. optical density (at 540 nm) in a computer-inter­faced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland). Each compound in given concentration was te­sted in triplicates in each experiment. Every experiment was repeated 3-5 times. Fluorescent microscopy studies The cultured cells were washed twice in PBS (phosphate buffer saline, Ca2+, Mg2+ - free) and centrifuged. Then the cells were resu­spended in the same buffer and incubated for 10, 20 and 60 seconds with 1µM of compound 24. Axioskop 20 (Zeiss, Germany) was used for results acquisition and imaging. Results and discussion The results of cytotoxic activity in vitro were expressed as ID50 – the dose of compound (in mM) that inhibits proliferation rate of the tu­mour cells by 50% as compared to control untreated cells. The results are presented in Tables 1-4. Inhibition of the topoisomerase II activity by compounds tested was measured as a de­gree of the inhibition of kinetoplast DNA de-catenation. Some of the compounds tested in­hibited the activity of topoisomerase II and the results are presented in Tables 1-4. As it is shown in Table 1, D-glucose deri­vatives (1-6) revealed moderate cytotoxic but no topoisomerase inhibitory activity. Among L-rhamnose containing compounds, only 3 out of 6 (9-11) revealed moderate an-tiproliferative activity - one with N-6 pentyl linker moiety and two (C-9 substituted) – con­taining pentyloxy and ethyloxy chains. No one compound from this group revealed any ability to inhibit activity of topoisomerase II. D-lactose derivatives are the least active cytotoxic compounds (Table 3). Only one compound - N-6 pentyl derivative - shows moderate anti-proliferative activity (16, ID50 = Table 3. Cytotoxicity and inhibition of topoisomerase II activity revealed by D-lactose indolo[2,3-b]quino-line saccharide derivatives. Compound tested Cytotoxity -ID50 [µM] Total topoisomerase II inhibition [mM] (No. 13) R3 = 2-deoxy-a-D-lactopyranoside n = 2 n.a. n.t. (No. 14) R1 = 2-deoxy-a-D-lactopyranoside n = 2 n.a. n.t. (No. 15) R2 = 2-deoxy-a-D-lactopyranoside n = 2 n.a. 0.5 (No. 16) R3 = 2-deoxy-a-D-lactopyranoside n = 5 45.90 ± 5.73 n.a. (No. 17) R1 = 2-deoxy-a-D-lactopyranoside n = 5 n.a. n.t. (No. 18) R2 = 2-deoxy-a-D-lactopyranoside n = 5 n.a. n.t. ID501) – compound concentration leading to 50% inhibition of cell proliferation; n - number of carbon atoms in linker moiety (as it is shown in Figure 1); n.a. -not active; n.t. -not tested. 45.90 ± 5.73 µM) but no topoisomerase inhi­bition is observed. Surprisingly, one of the compounds, which did not reveal antiprolif­erative activity, inhibited topoisomerase II ac­tivity in the highest concentration tested (compound 15, 0.5 mM). This derivative con­tains ethyloxy chain attached to indolo[2,3­b]quinoline C-9 carbon atom. It is evident that for the saccharide contain­ing compounds the crucial for cytotoxic and topoisomerase inhibition activities are C-9 and N-6 substitution positions, and that in the case of N-6 derivatives only five carbon units chain leads to the compounds with biological activity. Interestingly, substitutions with aminosac­charide moieties give an important increase of cytotoxic properties. All compounds are active and their ID50 values are few times lower com­pared to “non-amino” monosaccharide deriva­tives. These compounds reveal also strong topoisomerase II inhibitory activity what con­firms thesis that there is a third condition (apart from position of substituent and size of linker chain) essential for specific activities of indo-lo[2,3-b]quinolines derivatives. This condition concerns the type of sugar residue constituted. Figure 2. Jurkat cells after 10 seconds of incubation in 1.0µM of compound 24. The cultured cells were washed twice in PBS (phosphate buffer saline, Ca2+, Mg2+ - free) and centrifuged. Then the cells were re­suspended in the same buffer and incubated for 10, 20 and 60 seconds with 1µM of compound 24. This compound is a fluorophore, so lighting areas in the cells indicate places of its intake. Axioskop 20 (Ze­iss, Germany) was used for results acquisition and imaging (Zeiss Filterset 02 - max excitation: . = 365; emmission: . > 420 nm – was applied). Table 4. Cytotoxicity and inhibition of topoisomerase II activity revealed by L-daunosamine and L-acosa-mine indolo[2,3-b]quinoline saccharide derivatives. Compound tested Cytotoxity -ID50 Total topoisomerase II [µM] inhibition [mM] (No. 19) R3 = a-L-daunosaminide n = 2 69.42 ± 9.77 0.025 (No. 20) R1 = a-L-daunosaminide n = 2 6.41 ± 0.86 0.005 (No. 21) R2 = a-L-daunosaminide n = 2 10.26 ± 2.82 0.025 (No. 22) R3 = a-L-daunosaminide n = 5 12.80 ± 0.47 0.500 (No. 23) R1 = a-L-daunosaminide n = 5 7.05 ± 0.15 0.050 (No. 24) R2 = a-L-daunosaminide n = 5 7.05 ± 0.33 0.025 (No. 25) R3 = a-L-acosaminide n = 2 7.16 ± 0.002 0.025 (No. 26) R1 = a-L-acosaminide n = 2 12.20 ± 1.33 0.025 (No. 27) R2 = a-L-acosaminide n = 2 10.12 ± 2.25 0,050 DIMIQ* 1.14 ± 0.61 0.500 (No. 28) ID501) – compound concentration leading to 50% inhibition of cell proliferation; n - number of carbon atoms in linker moiety (as it is shown in Figure 1); n.a. -not active; n.t. - not tested; *DiMIQ -referential compound - 5,11-dimethyl-indolo[2,3-b]quinoline; Within range of aminosaccharide deriva­tives of indolo[2,3-b]quinolines, the most promising are compounds 20 and 21 – C-2 and C-9 substituted ethyloxy, as well as 23 and 24 - C-2 and C-9 substituted pentyloxy L-daunosamine derivatives (Table 4). In the se­ries of L-acosamine derivatives, the best properties were revealed by compound 25 with five carbon linker chain attached to N-6 position of indolo[2,3-b]quinoline. Cytotoxic activities observed for the active compounds in the series are comparable to values estimated previously for 5H and 6H derivatives.5-7 The most cytostatic against KB cells and inhibiting activity of topoisomerase II are aminosaccharide substituted indo-lo[2,3-b]quinolines. Since some of previously tested indolo[2,3­b]quinoline derivatives show poor solubility (data not shown) what causes the problems connected with bioavailability, next step of the studies was to examine the cell intake of the compound 24. These results are shown in Figure 2. It can be seen that the compound is well distributed in cytosol and accumulated in some subcellular structures. Although a great effort have been made to find an effective anticancer chemotherapeu­tics, the number of clinically active drugs re­mains quite small and their spectrum of anti-tumor activity is rather limited. Because of that, there are still many projects and research aiming to discover the new, more effective or more selective anticancer compounds.14 Beside the cytotoxic properties assessed in in vitro tests, the candidates for the new anti­cancer drugs should reveal some special fe­atures, as good solubility and bioavailability and of course low toxicity. Structure-activity relationship studies are one of the ways to se­lect the compound of the best properties. On the basis of presented here results, the most promising indolo[2,3-b]quinoline deri­vatives were chosen for further preclinical in vitro and in vivo studies. Acknowledgements This research was supported in part by a Pal-pharma grant No. 010/2002 References 1. Jonckers TH, van Miert S, Cimanga K, Bailly C, Colson P, De Pauw-Gillet MC, et al.Synthesis, cytotoxicity, and antiplasmodial and antitrypa­nosomal activity of new neocryptolepine deri­vatives. J Med Chem. 2002; 45(16): 3497-508. 2. Dassonneville L, Lansiaux A, Wattelet A, Wat-tez N, Mahieu C, Van Miert S, et al. Cytotoxici­ty and cell cycle effects of the plant alkaloids cryptolepine and neocryptolepine: relation to drug-induced apoptosis. Eur J Pharmacol. 2000; 409(1): 9-18. 3. Awada A, Giacchetti S, Gerard B, Eftekhary P, Lucas C, De Valeriola D, et al. Clinical phase I and pharmacokinetic study of S 16020, a new olivacine derivative: report on three infusion schedules. Ann Oncol. 2002; 13(12): 1925-34. 4. Pognan F, Saucier JM, Paoletti C, Kaczmarek L, Nantka-Namirski P, Mordarski M, et al. A car-boline derivative as a novel mammalian DNA topoisomerase II targeting agent. Biochem Phar­macol. 1992; 44(11): 2149-55. 5. Peczynska-Czoch W, Pognan F, Kaczmarek L, Boratynski J. Synthesis and structure-activity re­lationship of methyl-substituted indolo[2,3-b]qu­inolines: novel cytotoxic, DNA topoisomerase II inhibitors. J Med Chem. 1994; 37(21): 3503-10. 6. Kaczmarek L, Peczynska-Czoch W, Osiadacz J, Mordarski M, Sokalski WA, Boratynski J, et al. Synthesis, and cytotoxic activity of some novel indolo[2,3-b]quinoline derivatives: DNA topo­isomerase II inhibitors. Bioorg Med Chem 1999; 7(11): 2457-64. 7. Kaczmarek L, Luniewski W, Zagrodzki B, Go-dlewska J, Osiadacz J, Wietrzyk J, et al. Synthe­sis of 6-substituted 6H-indolo[2,3-b]quinolines as novel cytotoxic agents and topoisomerase II inhibitors. Acta Pol Pharm 2002; 59(3): 199-207. 8. Kaczmarek L, Peczynska-Czoch W, Opolski A, Wietrzyk J, Marcinkowska E, Boratynski J, et al. Methoxy- and methyl-, methoxy-5,6,11-tri­methyl-6H-indolo [2,3-b]quinolinium derivati­ves as novel cytotoxic agents and DNA topo­isomerase II inhibitors. Anticancer Res 1998; 18(4C): 3133-8. 9. Kociubinska A, Gubernator J, Godlewska J, Stasiuk M, Kozubek A, Peczynska-Czoch W, et al. A derivative of 5H-indolo[2,3-b]quinoline - a novel liposomally-formulated anticancer agent. Cell Mol Biol Lett 2002; 7(2):289. 10. K. Badowska-Roslonek, L. Kaczmarek, J. Ram-za, W. Szelejewski, J. Godlewska, W. Peczyn­ska-Czoch; Polish Patent Application P- 353811 Warsaw 2002. 11. Muller MT, Spitzner JR, DiDonato JA, Mehta VB, Tsutsui K, Tsutsui K. Single-strand DNA cleavages by eukaryotic topoisomerase II. Bio­chemistry 1988; 27(22): 8369-79. 12. Muller MT, Helal K, Soisson S, Spitzner JR. A rapid and quantitative microtiter assay for eu­karyotic topoisomerase II. Nucleic Acids Res 1989; 17(22): 9499. 13. Skehan P, Storeng R, Scudiero D, et al. New co-lorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990; 82: 1107-1112. 14. Boyd MR. Status of the NCI Preclinical Antitu­mor Agent Discovery Screen, Principles and Practice of Oncology Updates. 1989; 3(10): 1-12, Appendix XVI. Radiol Oncol 2004; 38(2): 145-51. Regional comparison of cancer incidence Nermina Obralic1, Faris Gavrankapetanovic1, Zehra Dizdarevic1, Osman Duric1, Fuad Šišic1, Ivan Selak2, Snježana Balta3, Bakir Nakaš4 1Clinical Center University Sarajevo, 2Institute of Pathology of Medical Faculty of University Sarajevo, 3Institute for Public Health of the Sarajevo Canton, 4General Hospital Sarajevo Background. Due to specific war and post-war situation in Balkan region, differences in the number, type, development, biological course, treatment of malignant tumours and its outcome are possible. In order to per­ceive the situation realistically, it is necessary to gather continuously exact data about malignant tumours and compare them with the data from other European and world countries.The aim of the study was to col­lect and analyse the data on cancer incidence in the region of Sarajevo city, which represents a symbol of difficult times in the recent past, and to compare it to the incidence in the neighbouring countries. Patients and methods. Data on all newly diagnosed cancer cases, permanent residents of Sarajevo Canton, in the years 1999 and 2000 were collected. Crude incidence rate has been calculated according to the years observed, gender and localizations of the disease The data were compared to the cancer registries of Slovenia and Croatia and were observed in the light of specific local situation. Results. The crude cancer incidence of all sites but skin was the highest in both years and by both genders in Croatia. The incidence of the most common tumours (lung and breast cancer) was similar in all three coun­tries. The differences in the incidence between both genders in the Sarajevo canton were registered in laryn­geal and urinary bladder cancer, as well as in bone and cartilage sarcoma. Cervical cancer had extremely high incidence and was high up on the incidence list in the Sarajevo canton, which correlates with the data in de­veloping countries. The incidence of other tumours in the post-war period is reaching expected numbers. Conclusions. It is difficult to identify whether the war and post-war stress, irregular and insufficient nu­trition during and after the siege of the city of Sarajevo or some other factor influenced the cancer incidence among exposed population. The prevalence of smoking in the whole region is extremely high, in Bosnia and Herzegovina almost complete, which can influence not only the incidence of lung cancer but also laryngeal and urinary bladder cancer. Key words: neoplasms- epidemiology; incidence; Bosnia - Hercegovina cancer incidence Received 15 March 2004 Introduction Accepted 15 April 2004 Correspondence to: Nermina Obralic, MD, Institute of Malignant tumours represent one of the ma- Oncology, Clinical Center University Sarajevo, jor health problems today; their occurrence Bolniška 25, Sarajevo, Bosnia and Herzegovina; Phone: +387 33 666 497; E-mail: nerminaobralic@gmx.net and severity are constantly increasing. According to the available data, malignant tu­mours are the leading cause of death in most countries in Europe and rank second, after cardiovascular diseases.1,2 The war in Balkan (1991-1996) had dire consequences leading to a great number of victims, destroyed infrastructure, industry, environment, health capacities and equip­ment. Health situation in several countries has significantly worsened.3,4 Due to specific war and post-war situation, the differences in the number, type, development, biological course, treatment of malignant tumours, and in the outcome are possible. Actual situation related to the use of ammunition containing depleted uranium has raised additional ques­tions about its influence on human health. Contamination with the debris from deplet­ed uranium shells could have increased the risk of developing cancer and kidney dam­age. The so-called Balkan-syndrome is often linked with depleted uranium contamina­tion.5-8 Consequently, eventual increase in cancer incidence in Bosnia and Herzegovina is possible. It has been very often publicly mentioned that the number of malignant tumours in Bosnia and Herzegovina is enormously in­creasing and that we are faced with cancer epidemic. But the information on cancers, cancer rates and trends is incomplete in Bosnia and Herzegovina. Bosnia and Herzegovina does not have a proper cancer registry for the population nor does it have precise data on its incidence. The claims ma­de about the increases in many types of can­cers were based on clinical observations, but were not substantiated by the information on cancer rates relating the number of cases to the population these cancers come from.9,10 In order to perceive the situation realisti­cally and to avoid panic and ignorant atti­tude, it is necessary to gather continuously exact data about malignant tumours, not only to detect risk factors, or follow them up and control them, but also to be able to compare them with the data from other European countries and countries worldwide. The above observations, strong fear from malignant tumours, and frequent disinforma­tion, motivated the authors to investigate and to define a true situation regarding malignant tumours at least in part of Bosnia and Herzegovina. We chose the canton of Sarajevo relying on the fact that it would be possible to obtain the complete data about malignant tumours there because patients had rarely been treated in other places or ci­ties (diagnostics and treatment for cancer are available in Sarajevo; there are no political or organizational obstacles in sending patients to oncology departments). Beside that, the number of citizens in the observed period of time was stable and known. Those facts hel­ped us, to conduct the study and to reach ba­sic indicators about the incidence and type of malignant tumours during the period of 1999 - 2000. Besides, Sarajevo went through difficult ti­mes. It was under siege that was the longest since World War II and suffered from ruth­less horrors of war. During the war, approxi­mately 3 tons of depleted uranium munitions were used against armoured targets, predo­minantly in the surroundings of Sarajevo.8,11 Its citizens’ health was jeopardized by nume­rous factors, some of which are: Stressful situations during the war and af­ter the war which could influence the appea­rance of certain diseases, including malignant tumours; Difficult communication due to the siege and blockades which prevented early detec­tion and treatment of diseases; Quantitatively and qualitatively insuffi­cient nutrition due to the siege, war conditi­ons and difficult economic situation after the war; Increasing trend of smoking and, after the war, increased consumption of alcoholic be­verages; Possible increase of radioactivity due to ammunition containing depleted uranium used in the wider region of the city and pos­sible effect of chemical weapon; A shift in the population’s attitude toward their own health. Material and methods We collected the data on newly diagnosed cancer cases in the Canton of Sarajevo during 1st the period from January , 1999 until December 31st, 2000. All data relate to per­manent residents of the 6 municipalities in Sarajevo at the time of diagnosis. Cases have been confirmed microscopical­ly or clinically. Intraepithelial tumours of cer­vix and urinary bladder have not been inclu­ded in this analysis. The main source of information was the re­ports filled in by doctors from hospitals or outpatient departments where disease had been diagnosed and/or treated. These re­ports, which were legally binding, were prin­ted on the form no. 3-35-86 as “The report of malignant tumours”. The collection of data was related to histological diagnoses of newly discovered tumours and was done at the Institute for Pathology at the Medical Faculty (Sarajevo University Hospital) and at the Department for Pathology at the Sarajevo General Hospital. The data on mortality (dea­ths caused by malignant tumours) were collected from obligatory reports written on DEM 2 death certificates. Institute for Statistics of the Federation of Bosnia and Herzegovina provided the data of the number of citizens in the Sarajevo can­ton.12 For the study, the population number was estimated in the middle of a certain year (Table 1). Crude incidence (per 100 000 population of a certain gender and of all ages) of malig­nant tumours in the years 1999 and 2000 was calculated, regarding site and gender. The da­ta were compared to those from the Cancer Registry of Slovenia 1999 and 2000, Cancer Registry of Croatia 1999 and 2000 and regio­nal indicators and predictions.13-17 Results During the period of 1999-2000, 2,897 new ca­ses of malignant tumours (1496 among males and 1401 among females) were registered in the Sarajevo Canton. Fifteen cases (0.5 %) were registered only on the basis of death certificate. Pathohistologically or cytologically proven tu­mours were found in 2,571 patients (88.75 %). The number of new cancer cases by year of diagnosis and sex is presented in Table 2. The crude incidence rate in males was 432/100 000 in 1999 and 384/100 000 in 2000 and in females 390/100 000 in 1999 and 354/100 000 in 2000. Compared to Slovenia, the incidence rate in our country was lower. If non-melanoma skin cancer is excluded, the crude incidence rate in 2000 in Canton Sarajevo was 339/100 000 males and 286/100 000 females, respectively. The estimated inci­dence from GLOBOCAN 2000 is 309.3 new cases per 100.000 men and 267.9 cases per 100 000 women in Bosnia and Herzegovina.13 Crude incidence rates of most important cancer sites in males and females in Southern Europe, Slovenia, Croatia and Canton Sarajevo are presented in Tables 3 and 4. Table 1. Estimated population by gender in the Sarajevo canton during the period 1999-2000 Year Males Females Total 1999 178 951 201 932 380 883 2000 183 161 207 373 390 534 Table 2. Number of malignant tumours in the Sarajevo canton in the years 1999-2000: all sites; by gender Table 3. Crude cancer incidence per 100,000 males of all ages in the years 1999 and 2000 according GLOBACON 2000,13 Cancer Registry of Slovenia,14,15 Cancer Registries of Croatia16,17 and the Sarajevo canton Years Males Females Total 1999 791 766 1557 2000 705 645 1350 Total 1496 1401 2897 Cancer Southern Slovenia Croatia Sarajevo canton Primary site Europe 1999 2000 1999 2000 1999 2000 2000 Oral cavity 13.7 11.8 13.0 17.1 15 7.3 9.27 Nasopharynx 1.2 0.4 0.4 0.8 1.0 0.6 0.0 Other pharynges 6.9 15 11.9 13.2 15.8 1.0 7.6 Oesophagus 7.3 8 7.3 9.1 8.2 1.1 1.1 Stomach 32.5 32.5 29.3 39.7 34.3 21 16.2 Colon / Rectum 55.1 59.2 60.5 71 73.0 40.8 41.5 Liver 16.5 7.8 7.4 12 11.0 9.5 12.6 Pancreas 11.5 11 12.1 15.3 14.2 2.8 4.9 Larynx 13.78 9.2 11.520.2 17.9 46.9 27.3 Lung 95.9 85.0 85.9 115.6 121.8 106.2 90.1 Melanoma of skin 5.2 11 12.3 9.9 11.1 18.6 14.5 Prostate 44.7 44,1 40.8 41.1 43.8 24.0 20.0 Testis 5.0 8.8 8.8 7.2 7.0 2.8 4.4 Bladder 41.6 15.0 14.9 31.1 33.0 27.8 38.8 Kidney 13.4 11 10.8 16.2 15.7 10.6 6.3 Brain/nervous system 8.9 8.4 5.4 15.1 15.4 10.4 13.9 Thyroid 1.5 3.0 2.8 3.5 3.7 2.8 1.1 Non-Hodgkin lymphoma 13.2 8 9.9 12.6 10.8 4.4 4.5 Hodgkin Disease 2.9 1.5 2.1 4.3 3.6 3.8 7.8 Multiple myeloma 4.7 4.3 3.3 5.3 4.4 1.1 2.8 Leukaemia 11.3 8.1 1.4 17.7 14.9 10.6 5.8 Bone/ cartilage 1 0.9 5.9 4.9 8.4 7.9 Soft tissue sarcoma 1.3 2.4 3.5 2.9 1.68 0.54 All sites but skin 443.4 402.7 393.9 533.3 523.9 390 354 Discussion The aim of our study was to analyse the can­cer incidence in Canton Sarajevo and compa­re it with neighbouring countries. When try­ing to explain the differences, it is important to emphasise, that they may be real, but also due to different practices in reporting. Compared to Slovenia, in Canton Sarajevo the incidence of all cancer sites was rather lo­wer. If melanoma skin cancer is excluded, the incidence in Croatia was the highest in both years and both genders. According to the Cancer Register of Croatia, the crude inci­dence per 100 000 population in 1999 was 533.3 for men, and 432.1 for women. The same proportion was reported in the year 2000, i.e. 523.9 for men and 424.3 for women, whereas in Slovenia, it was 448.3 for men and 420.4 for women, and in Sarajevo, 384 and 311, respectively.11,12,15,16 Therefore, the reg­istered crude incidence of cancer in the Sarajevo Canton correlates with the estima­tions for the whole region of South Europe rather than with the one estimated for Bosnia and Herzegovina. In both genders, the crude incidence is comparable to the one registered in the Republic of Slovenia for the year of 1999 (390 men and 339 women in the Sarajevo Canton; 402 men and 359 women in Slovenia). In Slovenia, the incidence of cancer of the upper respiratory and digestive organs in the years 1999 and 2000 was 36.4 and 36.8, res­pectively, in Croatia, 51.3 and 49.7 respec- Table 4. Crude cancer incidence per 100,000 females of all ages in the years 1999 and 2000 according GLOBACON 2000,13 Cancer Registry of Slovenia,14,15 Cancer Registries of Croatia16,17 and of the Sarajevo canton Cancer Southern Slovenia Croatia Sarajevo canton Primary site Europe 1999 2000 1999 2000 1999 2000 2000 Oral cavity 3.0 3,1 2.6 5.7 3.4 1.5 1.4 Nasopharynx 0.4 0,2 0.4 0.4 0.2 0.5 0.0 Other pharynges 0.7 0,8 1.2 1.52.1 0.5 0.5 Oesophagus 1.4 2.2 1.4 1.9 2.3 0.5 0.9 Stomach 20.3 16.3 18.0 21.0 23.2 8.9 5.8 Colon / Rectum 44.3 41.3 47.9 53.0 54.0 28.9 27.2 Liver 7.6 3.52.4 7.2 8.0 6.4 5.3 Pancreas 0.1 9.8 11.3 11.6 12.2 2.0 1.0 Larynx 0.9 0.7 1.3 2.0 1.5 8.4 6.3 Lung 15.2 21.2 23.523.528.0 18.3 22.4 Melanoma of skin 6.9 13.1 11.1 9.1 9.7 14.0 10.8 Brest 88.5 97.8 91.2 110.7 95.0 95.8 77.8 Cervix uteri 13.7 19.9 19.6 17.2 19.0 36.6 35.2 Corpus uteri 23.6 24.8 24.6 25.7 22.6 26.3 22.0 Ovary 13.8 17.0 19.4 22.9 24.6 12.5 14.5 Bladder 8.8 5.1 4.4 9.0 9.0 15.9 8.6 Kidney 6.4 8.1 6.9 9.0 10.7 6.0 5.9 Brain/ nervous system 6.8 3.7 5.4 13.0 12.0 8.9 5.3 Thyroid 6.7 6.7 5.8 12.1 12.0 6.9 6.7 Non-Hodgkin lymphoma 11.0 8.8 11.8 9.8 9.6 5.5 2.4 Hodgkin disease 2.5 1.9 1.6 3.4 3.2 4.5 1.9 Multiple myeloma 4.3 7.1 4.2 6.2 3.4 3.0 1.4 Leukaemia 8.4 3.7 7.9 13.3 11.3 4.9 5.8 Bone/ cartilage 1.3 0.6 3.2 4.4 8.4 3.8 Soft tissue sarcoma 2.2 3.2 2.6 2.52.4 2.8 All sites but skin 336.9 358.4 363.6 432.1 424.3 339 286 tively, and 56.6 and 44.73 in the respective years in the Sarajevo canton. The greatest dif­ference is registered in cases of laryngeal can­cer where incidence is 2-3 times greater in the Sarajevo Canton than in other countries in the region. The incidence of oral and pharyn­geal cancer was lower. Even if we presume that a certain number of cancers of upper re­spiratory organs were wrongly assigned to advanced laryngeal cancers, this still could not explain extremely and constantly high in­cidence of this tumour in the Sarajevo can­ton. Lung cancer is most common in all three countries, with the incidence comparable to the one predicted by GLOBCAN report. The lowest incidence was registered in Slovenia, where the prevalence of smokers is lowering. The incidence of all digestive tract tu­mours was the highest in Croatia. The inci­dences in Slovenia and Sarajevo are compa­rable to the ones predicted by GLOBCAN re­port. Somewhat lower incidences of stomach and pancreatic cancer were registered in Sarajevo. The incidence of urinary bladder cancer in Croatia and Sarajevo is 2 times higher than in Slovenia, but still correlates to GLOBCAN es­timation for Southern Europe. The prostate cancer incidence in Croatia and Slovenia is at the level of expected values that correlates to GLOBCAN estimation, but in the Sarajevo canton, it is lower. This can be explained by the fact that, in Bosnia and Herzegovina, no routine screening for PSA is being imple­mented, therefore only clinically apparent tu­mours are diagnosed. In clinical practice, an unusually high num­ber of histologically confirmed bone and carti­lage sarcomas from different parts of Bosnia and Herzegovina have been observed, which is illustrated and presented through the data from the Sarajevo canton and for which we don’t have an acceptable explanation. From the given data, it is evident that other tumours of haematopoietic system are not more common in the Sarajevo canton in comparison to what has been expected and to the number of cases registered in Slovenia and Croatia. In females, breast is the most common cancer site in all three countries. It is impor­tant to note that incidence of cervical cancer is the highest in Sarajevo Canton and is com­parable to the incidence in undeveloped countries. It certainly reflects the insufficient opportunistic screening. Among the women in the Sarajevo Canton, higher incidences of laryngeal and urinary bladder cancer were also registered as well as bone sarcomas. Conclusions Several factors may have contributed to can­cer incidence in Canton Sarajevo: the war and post-war circumstances with irregular nutri­tion during and after the siege of the city, heavy smoking and drinking. The incidence of smoking in the whole region, especially in Bosnia and Herzegovina is extremely high, which can influence not only the incidence of lung cancer but also of laryngeal cancer and urinary bladder cancer. Additional studies are needed to evaluate whether some other factors have been impli­cated in the aetiology of malignant tumours among exposed population. It is important to emphasize the need for establishing a national population registry of malignant tumours for the whole Bosnia and Herzegovina, not only to detect, follow-up and control the risk factors but also to be able to compare our data with the data from other European countries and the countries world­wide References 1. Bray F, Sankila R, Ferlay J, Parkin DM: Estimates of cancer incidence and mortality in Europe in 1995. Eur J Cancer 2002; 38: 99-166. 2. Boyle P, Autier P, Bartelink H, Baselga J, Boffetta P, Burn J, et al. European Code Against Cancer and scientific justification: third version (2003). Ann Oncol 2003; 14: 973-1005. 3. Institute for Public Health of Bosnia and Herzegovina. Health status of population and health care system in transition. Report for 1999. Sarajevo; 2000. 4. Institute for Public Health of Bosnia and Herzegovina. Health status of population and health care system in transition. Report for 2001. Sarajevo; 2002. 5. Royal Society Working Group on the Health Hazards of Depleted Uranium Munitions. The health effects of depleted uranium munitions: a summary. J Radiol Prot 2002; 22: 131-9. 6. UNDEP identifies depleted uranium risks in Bosnia and Herzegovina. 2002. 7. Papastefanon C. Depleted uranium in military conflicts and impact on the environment. Health Phys 2002; 83: 280-2. 8. Current News. CADU news issue 12. 2002. 9. Puvacic Z, Konjhodžic F, Puvacic S: Istraživanje mortaliteta od malignih bolesti u Bosni i Hercegovini. Posebna izdanja. Knjiga 30. Sarajevo: Akademija nauka Bosne i Hercegovine; 2002. p. 15-24. 10. Obralic N; Dizdarevic Z, Dizdarevic S; Duric O; Gavrankapetanovic F; Gribajcevic M; et al. Analiza broja oboljelih od malignih neoplazmi lijecenih u Klinickom centru Univerziteta u Sarajevu u predratnom i poslijeratnom perio­du. Medicinski žurnal 2001; 1: 50-8. 11. United Nations Environmental Program. Depleted uranium in Bosnia and Herzegovina: post-conflict environmental assessment. Switzerland 2003. 12. Institute for Statistics of Federation Bosnia and Herzegovina: Reports for years 1998, 1999, 2000, 2001 and 2002. 13. GLOBCAN 2000. Cancer incidence, mortality and prevalence worldwide. Version 1.0. 14. Cancer Registry of Slovenia. Cancer incidence in Slovenia 1999. Report No. 41. Ljubljana: Institute of Oncology Ljubljana; 2002. 15. Cancer Registry of Slovenia. Cancer incidence in Slovenia 2000. Report No. 42. Ljubljana: Institute of Oncology Ljubljana; 2003. 16. Croatian National Institute of Public Health. Cancer incidence in Croatia 1999. Bulletin 24. Zagreb; 2001. 17. Croatian National Institute of Public Health. Cancer Incidence in Croatia 2000. Bulletin 25. Zagreb; 2002. Radiol Oncol 2004; 38(2): 153-4. Letter to the Editor Chemotherapy for small-cell lung cancer with paraneoplastic nephrotic syndrome Katsunori Kagohashi, Gen Ohara, Hiroaki Satoh, Kiyohisa Sekizawa Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Japan Platinum-containing chemotherapy has been commonly used as standard therapy for small cell lung cancer (SCLC). However, platinum causes renal dysfunction.1,2 We report a SCLC patient with paraneoplastic nephrotic syndrome who was successfully treated with platinum-containing chemotherapy. A com­plete tumour response could be achieved; however, his proteinuria did not decrease and renal function got worse every time he re­ceived the chemotherapy. A 74-year-old male was admitted to our hospital with the oedema of the lower ex­tremities that developed during the last three months. On physical examination, oedema was still present. Laboratory results were as follows: haemoglobin 10.6 g/dl, potassium 4.3 mEq/l, serum creatinine 1.0 mg/dl, blood urea nitrogen 34.2 mg/dl. Creatinine clear­ance was 43.0 ml/min and the urine sediment was free of casts and erythrocytes. However, serum albumin was 2.1 g/dl, and the 24 h urine collection revealed proteinuria of 10 g Received 23 March 2004 Accepted 25 April 2004 Correspondence to, Hiroaki Satoh, MD, Division of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba-city, Ibaraki, 305 8575, Japan; Phone: +81 29 853 3210; Fax: +81 29 853 3320; E-mail: hirosato@md.tsukuba.ac.jp daily. Renal biopsy disclosed membranous glomerulonephritis. A chest X-ray on admis­sion revealed mediastinal widening and a right hilar mass. Chest CT scan showed a large mass in the right middle lobe with me-diastinal lymph node swellings. Small lung nodules up to 10 mm in both lungs were also observed. Transbronchial biopsies revealed SCLC in an advanced desease stage. Paraneoplastic nephrotic syndrome was diag­nosed to be associated with SCLC. Chemotherapy for SCLC was started with carboplatin (AUC 5 mg/ml per minute, Calvert formula, day 1) and etoposide (100 mg/m2, days 1, 2, and 3). After two courses complete remission was achieved, however, oedema of both extremities did not disap­peare. Serum creatinine and blood urea nitro­gen increased every time he received the chemotherapy, and proteinuria did not de­crease in spite of the complete tumour re­sponse. Because of the impaired renal func­tion, no additional chemotherapy was indi­cated. Thereafter, the patient received sup­portive care, and he died from brain metasta­sis 8 months after from the initial treatment. Since the introduction of platinum in the early 1980s, commonly used combination chemotherapy regimens for SCLC have been platinum analogy combined with etopo­side.3,4 Despite the advantage of the plat­inum-containing chemotherapy, the cy­clophosphamide, doxorubicin, and vin­cristine regimen has still been commonly used for SCLC patients with paraneoplastic nephritic syndrome.5-6 In our case, carbo­platin-contaning chemotherapy could achieve a complete tumour response; however, para-neoplastic nephrotic syndrome was not im­proved and the renal function was deteriorat­ed. Platinum is nehprotoxic and, therefore, platinum itself can induce nephrotic syn­drome.1,2 In comparison with cisplatin, the nephrotoxicity of carboplatin is reduced but not completely eliminated. We cannot con­clude by insisting on the disadvantage of plat-inum-containing regimens for SCLC patients with paraneoplastic nephrotic syndrome, but by recommending to be careful in indicating the platinum-containing regimens, especially for SCLC patients with paraneoplastic nephrotic syndrome. References 1. Metz-Kurschel U, Kurschel E, Niederle N, Aulbert E. Investigations on the acute and chronic nephrotoxicity of the new platinum analogue carboplatin. J Cancer Res Clin Oncol 1990; 116: 203-6. 2. Kintzel PE. Anticancer drug-induced kidney disorders. Drug Saf 2001; 24: 19-38. 3. Livingston RB. Combined modality therapy of lung cancer. Clin Cancer Res 1997; 3: 2638­1647. 4. Schiller JH. Current standards of care in small-cell and non-small-cell lung cancer. Oncology 2001; 61(Suppl 1): 3-13. 5. Jermanovich NB, Giammarco R, Ginsberg SJ, Tinsley RW, Jones DB. Small cell anaplastic carcinoma of the lung with mesangial prolifer­ative glomerulonephritis. Arch Intern Med 1982; 142: 397-9. 6. Boon ES, Vrij AA, Nieuwhof C, van Noord JA, Zeppenfeldt E. Small cell lung cancer with paraneoplastic nephrotic syndrome. Eur Respir J 1994; 7: 1192-3. 7. Tomoda K, Hamada K, Fukuoka K, Tsukaguchi K, Tsujimoto M, Mikasa K, et al. [Small cell lung cancer associated with nephrotic syndrome; remission after chemotherapy.] [Japanese with English ab­stract]. Nihon Kokyuki Gakkai Zasshi 1998; 36: 541- 4. Radio/ Oncol 2004; 38(2): 73-83. Tumorski oznacevalci v klinicni onkologiji Novakovic S Majhne razlike med normalnimi in tumorsko spremenjenimi celicami izkorišcamo v diagnostiki in zdravljenju malignomov. Te razlike oznacujemo z imenom tumorski oznacevalci in so lahko kvalitativne ali kvantitativne po svoji naravi. To pomeni, da lahko kot tumorski oznacevalci služijo tako molekule (snovi), ki jih tvorijo maligne celice kot tudi molekule (snovi), ki nastajajo v povecanih kolicinah v normalnih gostiteljevih tkivih pod vplivom malignih celic. Na splošno so tumorski oznacevalci znacilne molekule, ki se pojavijo v krvi ali tkivih v povezavi z maligno boleznijo. Glede na uporabo delimo tumorske oznacevalce v grobem na oznacevalce v klinicni onkologi­ji in oznacevalce v patologiji. V tem preglednem clanku bomo obravnavali le oznacevalce v klin­icni onkologiji. Sedanja razdelitev tumorskih oznacevalcev v klinicni onkologiji vkljucuje: onkofetalne antigene, placentalne proteine, hormone, encime, antigene, ki spremljajo tumor, posebne serumske proteine, kateholaminske metabolite ter skupino razlicnih oznacevalcev. Na kratko lahko povzamemo, da mora idealni tumorski oznacevalec izpolnjevati dolocene pogoje: (1) povišane koncentracije oznacevalca se morajo pojaviti že na zacetku razvoja malig­noma, (2) koncentracije oznacevalca morajo biti povišane le pri bolnikih z doloceno vrsto ma­lignoma, (3) povišane koncentracije se morajo pojaviti pri vseh bolnikih z enako vrsto maligno­ma, (4) izmerjene koncentracije morajo odražati velikost tumorske mase, (5) izmerjene koncen­tracije morajo odražati ucinek zdravljenja in (6) dolocanje oznacevalca mora biti enostavno. Marsikateri med tumorskimi oznacevalci, ki jih dolocamo vsakodnevno, izpolnjuje nekatere, ne pa vseh navedenih pogojev. Kot posledico tega so raziskovalci vpeljali nekaj pojmov, ki naj bi opredelili kvaliteto tumorskega oznacevalca in omogocili cim boljšo izbiro oznacevalca za spremljanje dolocene vrste malignoma. Ti pojmi vkljucujejo obcutljivost (senzitivnost) oznace­valca, njegovo zanesljivost (specificnost) in napovedno vrednost. Obcutljivost pove, kakšna je srednja verjetnost, da bomo pri bolniku z malignomom dolocili povišane vrednosti (nad razme­jitveno vrednostjo) tumorskega oznacevalca. Zanesljivost pove, kakšna je srednja verjetnost, da bomo pri preiskovancu, ki nima malignoma, ugotovili normalne vrednosti oznacevalca. Napovedna vrednost pa pove, kakšna je uporabna vrednost tumorskega oznacevalca v mešani skupini preiskovancev. Dolocitve tumorskih oznacevalcev lahko v klinicni onkologiji uporabljamo z razlicnim na­menom. Pomagajo lahko pri (i) zgodnjem odkrivanju malignomov, (ii) razlocevanju malignih in benignih procesov, (iii) ocenjevanju razširjenosti maligne bolezni, (iv) spremljanju odgovora na terapijo in (v) napovedovanju in dokazovanju ponovitve maligne bolezni. Ker žal ne poznamo idealnega oznacevalca, ki bi imel ustrezno visoko obcutljivost in zanesljivost, dolocitve tu­morskih oznacevalcev le izjemoma uporabljamo kot presejalne metode za odkrivanje maligno­ma (primer takšnega oznacevalca je prostaticni specificni antigen -PSA). Kljub vsemu pa tu­morski oznacevalci igrajo kljucno vlogo pri odkrivanju zgodnjih ponovitev bolezni in spreml­janju odgovora na terapijo pri izbranih bolnikih. Dolocanje tumorskih oznacevalcev z namenom odkrivanja ponovitev bolezni je smiselno le, kadar pri takšnem bolniku še obstaja možnost uspešnega zdravljenja. Radio/ Oncol 2004; 38(2): 155-60. Radio/ Oncol 2004; 38(2): 85-92. Klinicna uporabnost serinskih proteaz pri raku dojk Cufer T Serinska proteaza uPA in njen inhibitor PAI-1 sta udeležena pri razgradnji tumorske strome ter bazalne membrane. Neodvisni napovedni pomen serinske proteaze uPA in njenega inhibitorja PAI-1 pri raku dojke je bil enoznacno potrjen v številnih posameznih raziskavah kot tudi v metaanalizi, v katero je bilo vkljucenih 18 datotek s podatki vec kot 8000 bolnic. Glede na te izsledke je tveganje za ponovitev bolezni pri bolnicah brez prizadetih pazdušnih bezgavk in nizkimi vrednostmi uPA in PAI-1 v prvotnem tumorju manjše od 10%. Tem bolnicam lahko prihranimo dopolnilno sistemsko zdravljenje, katerega pogosto spremljajo neželeni ucinki. Za vsakodnevno klinicno delo pa je še pomembnejše, da prvi izsledki potrjujejo tudi možen napovedni pomen uPA in PAI-1 za odgovor na dopolnilno sistemsko zdravljenje zgodnjega raka dojk. Naša opažanja pri 460 bolnicah z operabilnim rakom dojk potrjujejo napovedni pomen uPA in PAI-1 za odgovor na hormonsko zdravljenje, ne pa za odgovor na citostatsko zdravljen­je. Bolnice z visokimi vrednostmi PAI-1 v prvotnem tumorju so imele vec kot dvakrat višje tve­ganje ponovitve bolezni, ce niso bile zdravljene z dopolnilno sistemsko terapijo (HR 2.14; C.I. 95%= 0.48-9.56; p=0.321) ali pa so bile zdravljene samo s kemoterapijo (RR 2.48; C.I. 95%= 1.35­4.57; p=0.003). Slab napovedni pomen visokih vrednosti uPA in PAI-1 pa se je povsem iznicil pri bolnicah, ki so prejele dopolnilno hormonsko zdravljenje, samo ali v kombinaciji z kemoterapi­jo. Še vec, pri bolnicah, ki so prejele hormonsko zdravljenje, je bilo tveganje za ponovitev bolezni celo manjše v primeru visokih vsebnosti uPA ali PAI-1 v prvotnem tumorju (HR 0.79; p=0.693 and HR 0.26 p= 0.204). Serinska proteaza uPA in njen inhibitor PAI-1 sta danes mocna napovedna dejavnika poteka raka dojk, ki omogocata ustreznejši izbor bolnic za dopolnilno sis­temsko zdravljenje. Kot kažejo prvi izsledki, pa sta tudi obetavna napovedna dejavnika odgov­ora na zdravljenje, na podlagi katerih bomo v prihodnosti lahko izbirali najucinkovitejše zdravl­jenje za vsako posamezno bolnico. Radio/ Onco/ 2004; 38(2): 93-100. Katepsini in njihovi inhibitorji kot tumorski oznacevalci pri raku glave in vratu Strojan P Invazija in zasevanje tumorskih celic sta tesno povezana z razgradnjo bazalnih membran in zu­najcelicnega matriksa. Nosilci teh procesov so razlicni proteoliticni encimi, med njimi tudi skupina ubikvitarnih lizosomskih encimov, tj cisteinske proteaze katepsini B, H in L, aspartat­na proteaza katepsin D in endogeni inhibitorji prvih, cistatini. Namen tega pregleda je bil zbrati dosedanje vedenje o napovedni in prognosticni vrednosti katepsinov in njihovih inhibitorjev pri plošcatocelicnem karcinomu glave in vratu. Pri tej vrsti raka so se UICC/ AJCC TNM-klasifikaci­jski sistem in histopatološke znacilnosti tumorjev izkazali kot nezanesljivi kazalci za napoved bodisi odgovora na zdravljenje bodisi preživetja bolnikov. Poleg tega do sedaj še noben dejavnik iz širokega spektra biokemicnih in histoloških dejavnikov ni bil spoznan kot zanesljiv napove­dovalec naravnega poteka bolezni ali odgovora na zdravljenje. Prav z namenom izdelati zanesljiv prognosticni profil tumorja potekajo intenzivne raziskave številnih novih oznacevalcev. Radio/ Onco/ 2004; 38(2): 101-9. Farmakogenetika tiopurinov: Ali so laboratorijski podatki lahko odlocilni v pozologiji? Stocco G, Martelossi S, Decorti G, Ventura A, Malusa N, Bartoli F, Giraldi T Izhodišce. Namen študije je bil ugotoviti odvisnost med mutacijami v genu TPMT, oslabljeno encimsko aktivnostjo zaradi teh mutacij in klinicno toksicnostjo zdravljenja s tiopurinskimi an­ timetaboliti. Material in metode. V študijo smo vkljucili 44 bolnikov z vnetnim obolenjem crevesja, ki so bili zdravljeni z azatioprinom. Iz vzorcev krvi vsakega bolnika smo izlocili DNK in dolocili genotip s polimerazno verižno reakcijo, da bi tako našli tri najpogostejše genske mutacije. Encimsko ak­tivnost smo merili z visokolocljivostno tekocinsko kromatografijo na razgrajenih celicnih vsebi­ nah rdecih krvnickah. Rezultati. Med izbranimi bolniki so bili 4 (9,0%) pri mutacijah gena TPMT heterozigotni, medtem ko med njimi pri teh mutacijah nihce ni bil homozigoten. Z raziskavo lahko potrdimo popolno soodvisnost med mutiranim genotipom TPMT in oslabljeno encimsko aktivnostjo. Pri primer­janju posameznikov z mutiranim genotipom in normalnim genotipom TPMT nismo opazili spre­memb v toksicnosti. Zakljucek. Z metodami genotipiziranja, ki temeljijo na strateški uporabnosti DNK, je mogoce enostavno in zanesljivo odkriti homozigote TPMT -posameznike, ki jih ne smemo zdraviti s tiopurini. Hkrati pa ugotavljamo, da obicajnejše in manj nevarne oblike toksicnosti tiopurinov povzrocajo drugi dejavniki, ki niso povezani z mutacijami omenjenega gena TPMT. Radio/ Oncol 2004; 38(2): 155-60. Radio/ Oncol 2004; 38(2): 111-9. Razvoj kvantitativnih RT-PCR testov za nemutirani urokinazni receptor (uP AR-wt) in njegovo izrezovalno razlicico uP AR-del5 * Farthmann J, Holzscheiter L, Biermann J, Meye A, Luther T, Kotzsch M, Sweep F, Schmitt M, Span P, Magdolen V Receptor serinske proteinaze plazminogenskega aktivatorja urokinaznega tipa, uP AR (CD 87) igra pomembno vlogo v invaziji tumorskih celic in pri metastaziranju cvrstih malignih tumorjev. uP AR je mocno glikoziliran membranski protein, vsajen v glikan-lipidne membrane in sestavl­jen iz treh homolognih domen. Posamezna domena je kodirana z dvema eksonoma: Dl z ek­sonoma 2 + 3, DII z eksonoma 4 + 5 in DIII z eksonoma 6 + 7. Poleg nemutirane uPAR-wt RNA, sta bili opisani tudi dve izrezovalni razlicici, ki bodisi nimata eksona 5 (uP AR-del5) bodisi obeh eksonov 4 in 5 (uPAR-del4/5). Predhodno smo z metodo RT-PCR v realnem casu za kvantita­tivno dolocevanje mRNA koncentracije proucevali izražanje mRNA razlicic uPAR-del4/5 in uPAR mRNA, ki vsebujeta eksone 2,3 in 4 (to je nemutirana uPAR-wt in uPAR-del5). V tem prispevku smo dodatno uveljavili dva specificna, robustna in zelo obcutljiva testa RT­PCR, ki temeljita na tehnologiji Light-Cyler, da bi specificno dolocevali vsebnosti uPAR-wt in njegovo izrezovalno razlicico uPAR-del5. Izražanje uPAR-wt in uPAR-del5 je bilo merjeno v ra­zlicnih cloveških malignih celicnih linijah (ovarijskih rakavih celicah OVMZ-6 in OVMZ-10; rakavih celic dojke MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435 in aMCF-7; možganskih celicnih linijah LN18) kakor tudi v zbiru 174 vzorcev tkiva raka dojke. uPAR-del5 mRNA je bi­la zelo pogosto izražena v razmeroma nizkih vsebnostih (znacilno manj kot 1 % glede na uP AR­wt mRNA). V tumorskem tkivu rakavih bolnikov je bila opažena statisticno znacilna korelacija med uPAR-del5 in uPAR-wt mRNA (r=0.779; P > 0.001). Povezave med izražanjem obeh vrst mRNA in klinicnimi parametri, kakor so status bezgavk velikostjo tumorja in gradusa ni bilo. Znacilno povišanje uPAR-del5 pa je bilo opaženo v mailgnih tumorjih z negativnim estrogen­skim receptorjem. Oba razvita kvantitativna testa RT-PCR, ki smo ju opisali tu, lahko v bodoce prispevata k funkcionalni analizi in klinicni pomembnosti uP AR-wt in ene od njegovih izrezovalnih razlicic uP AR-del5 v malignih tumorjih. Radio/ Onco/ 2004; 38(2): 121-9. Ex vivo pretocna elektroporacija Vernhes MC, Eynard N, Rols MP, Ganeva V, Teissie J Elektroporacija celic povzroci lokalno in reverzibilno permeabilizacijo celicne membrane. To omogoca vnašanje eksogenih snovi v celice, kot so zdravila proteini in DNA Pretocna elektropo­racija omogoca tretman velike kolicine celic, kar je potrebno v primeru celicne tarpije. Celice tece­jo skozi komoro za elektroporacijo, kjer so izpostavljene tocno dolecenim pogojem elektropo­racije. Potrebno je prilagoditi frekvenco elektricnih pulzov, hitrost pretoka celic in število dove­denih pulzov. Na ta nacin je mogoce z elektroporacijo vnesti v celice velik volumen celic v majh­ni komori za elektroporacijo. Tudi viabilnost celic po elektroporaciji je zelo dobro ohranjena. Radio/ Oncol 2004; 28(2): 131-5. Napovedni pomen imunohistokemicnega dolocevanja HER-2/neu pri bolnikih s pljucnim rakom Petrusevska G, Ilievska-Poposka B, Banev S, Smickova S, Spirovski Z Izhodišca. Protein HER-2 ali p185her2 je membranski receptor s tirozinsko kinazno aktivnostjo, ki je dolocena z genom HER-2/neu. Prekomerno aktivnost HER-2/neu lahko zasledimo pri bol­ nikih z razlicnimi onkološkimi obolenji, tako tudi pri raku pljuc. Ker obstajajo razlicna porocila o stopnji aktivnosti tega gena pri pljucnem raku, smo v raziskavi dolocali protein HER-2 pri bol­nikih z žleznim, skvamoznocelicnim in drobnocelicnim pljucnim rakom. Bolniki in metode. Protein HER-2 smo dolocali pri tumorjih 29 bolnikov s pljucnim rakom; 19 bol­nikov je bilo operiranih, pri 10 pa smo tumorsko tkivo dobili ob diagnosticnem postopku z up­ogljivim bronhoskopom preden smo jih zdravili s kemo/radioterapijo. Vzorce tkiva smo fiksirali v nevtralnem formalinu in naredili parafinske preparate. Za diagnosticiranje smo uporabljali stan­dardna histokemicna in imunohistokemicna barvanja. Prisotnost proteina HER-2/neu smo potrjevali z imunohistokemicnim barvilom s pomocjo Hercep Testa (DAKO). Rezultate testiranja smo oznacili s stopnjami od O do 3. Stopnjo O in 1 smo ocenili kot negativno, s stopnjo 2 ali 3 pa kot pozitivno. Rezultati. Prekomerno izraženost proteina HER-2/neu smo zasledili v 34,4% (pri 10 od 29 bol­nikov). V vecji meri smo ga ugotovili pri bolnikih z žleznim rakom in to v 45,4% (pri 5 od 11 bol­nikov), pri bolnikih s skvamoznocelicnim rakom pa v 30,7% (4 od 13 bolnikov) in pri bolnikih z drobnocelicnim rakom le v 20% (1 od 5 bolnikov). Glede na starost in spol nismo našli statisticno znacilnih razlik. Pri napredovalih oblikah bolezni smo ugotovili vecjo izraženost proteina HER-2/neu, saj so imeli vsi bolniki s skvamoznocelicnim rakom pljuc in stadijem bolezni IIIB in IV povišan protein, bolniki z žleznim karcinomom in stadijem IIIB in IV pa v 80%. Zakljucki. Obetajoci rezultati kažejo, da bi lahko imel HER-2/neu pri bolnikih s pljucnim rakom prediktivni in prognosticni pomen, kar bomo poskusili potrditi po daljšem spremljanju bol­nikov. Radio/ Oncol 2004; 38(2): 155-60. Radio/ Oncol 2004; 38(2): 13 7-44. Novi saharidovi derivati indolo[2,3-b]kinolina kot citotoksicne spojine in inhibitorji topoizomeraze II Godlewska J, Badowska-Roslonek K, Ramza J, Kaczrnarek L, Peczyriska-Czoch W, Opolski A Nekateri alkilni in alkilaminski derivati 6H-indolo[2,3-b ]kinolinov imajo antiproliferativen ucinek in modulirajo celicni cikel. Njihove citotoksicne lastnosti deloma izvirajo iz sposobnosti interkaliranja DNK in sposobnosti inhibiranja topoizomeraze II. Ker smo želeli doseci boljše fizikalno-kemicne in biološke lastnosti 6H-indolo[2,3-b]kinolinov, smo sintetizirali serijo novih saharidnih derivatov ((C­2, C-9 ali N-6). Testirali smo vpliv razlicnih karbohiratnih enot (D-glukoza, D-laktoza, L-ramnoza, L­akozamin, L-daunozamin), mesto vezave in velikosti linkerja na citotoksicne lastnosti in inhibicijo topoizomeraze II. Testirali smo spojine iz skupine saharidov, ki vsebujejo derivate 2-deoksi-?-D­glukopiranozida (1-6), 2-deoksi-?-L-ramnopiranozida (7-12) in 2-deoksi-?-D-laktopiranozida (13-18) ter tudi serijo aminosaharidnih derivatov ?-L-daunozaminid (19-24) in ?-L-akozaminid (25-27). Radio/ Oncol 2004; 38(2): 145-51. Primerjava incidence raka v Sarajevu z incidenco v Slovenijii in Hrvaški Obralic N, Gavrankapetanovic F, Dizdarevic Z, Duric O, Šišic F, Selak S, Balta S, Nakaš B Ozhodišca. Vojna in razmere v povojnem obdobju dela Balkana so lahko vplivali na incidenco razlicnih vrst raka pa tudi na potek bolezni, nacin in uspešnost zdravljenja. Zaradi tocne ocene pojava rakastih obolenj, je potrebno kontinuirano zbirati podatke in jih primerjati s podatki iz drugih držav Evrope in sveta. Namen naše raziskave je bil tako zbrati in analizirati podatke o malignih obolenjih v Sarajevu ter jih primerjati s podatki iz registrov raka drugih držav. Bolniki in metode. Zbarali smo podatke o rakastih obolenjih prebivalcev s stalnim prebivališcem v Sarajevu med leti 1999 in 2000. Grobo letno incidenco smo razvrstili po spolu in lokalizaciji bolezni ter plodatke primerjali s podatki iz Registra raka Slovenije in Registra raka Hrvaške. Rezultati. Najvišja skupna groba letna incidenca vseh vrst rakastih obolenj (brez raka kože) je bila na Hrvaškem .. Incidenca najbolj pogostih rakov (pljucni rak in rak dojke) je bila v vseh treh državah enaka. V Sarajevo pa je bila višja incidenca raka grla, raka mehurja ter raka kosti in hrustanca pri obeh spolih. V Sarajevu je tudi mocno izstopala visoka incidenca raka vratu mater­nice, ki je primerljiva z incidenco v razvijajocih deželah. Incidenca raka ostalih lokalizacij je bi­la enaka pricakovanim podatkom. Zakljucki. Iz zbranih podatkov je težko zakljuciti, da bi vojna, povojni stress, neredno in neza­dostno prehranjevanje med in po obleganju Sarajeva ali katerikoli drugi dejavniki okolja bistveno vplivali na opazovano incidenco raka. Incidenca kajenja pa je v celotni regiji izjemno visoka, kar bi lahko vplivalo ne samo na pogostnost raka pljuc, pac pa tudi na pogostnost raka grla in mehurja, ki sta zvišana v primerjavi s podatki iz Slovenije in Hrvaške. Notices Notices submitted far publication should contain a mailing address, phone and/or fax number and/ ar e-mail oj a Contact person ar department. Oncology June 24-27, 2004 The 16th international symposium "Supportive Care in Cancer" will take place in Miami Beach, Florida, USA Contact Ms. Cynthia N. Rittenberg, RN, MN, AOCN, Executive Director, MASCC, %00 Rue St. Ann, Suite 223, Matairie, LA 70005 USA; or call +1 504 828 2184; or fax +1 504 828 2180; or e-mail cyn­dyrit@bellsouth.net; or see http://www.mascc.org Oncology July 3-6, 2004 The 18th EACR (European Association for Cancer Research) Congress will be held in Innsbruck, Austria. See http:/ /WW\v .fecs. be/ conf erences/ eacrl 8 Gynaecological cancer August 26-28, 2004 The ESTRO advanced teaching course on "Brachyherapy for Gynaecological Cancer" will take place in Vienna, Austria. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Medica! physics August 29 -September 2, 2004 The ESTRO course "Physics for Clinical Radiotherapy" will take place in Leuven, Belgium. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Paediatric oncology September, 2004 The International Society of Paediatric Oncology ­SIOP Annual Meeting will be held in Oslo, Norway. See http://www.siop.nl Radiobiology September 19-23, 2004 The ESTRO course "Basic Clinical Radiobiology" will take place in Lausanne, Switzerland. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgium; or call +32 775 93 40; or fax +32 2 779 54 94; or e-mail info@estro.be; or see http://www.estro.be Lung cancer September 23-25, 2004 The "9th Central European Lung Cancer Conference" will be offered in Gdansk, Poland. Contact Conference Secretariat, "9th Central European Lung Cancer Conference", Via Medica, ul. Swietokrzyska 73, 80 180, Gdansk, Poland; or call/fax +48 58 349 2270; or e-mail celcc@amg.gda.pl; or see www.lungcancer.pl Radiation therapy October 3-7, 2004 ASTRO Annual meeting will be held in Atlanta, USA Contact American Society for Therapeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http:// www.astro.org 162 Notices Oncology October 7-9, 2004 The 3rd ASTRO Annual rneeting will be held in Atlanta, USA. Contact Arnerican Society for Therapeutic Radiology and Oncology Office, 1891 Preston White Drive, Reston, VA 20191, USA; or see http:// www.astro.org Therapeutic radiology and oncology October 24-28, 2004 The 23rd lnternational Chicago Syrnposium on "Malignancies of the hest and Head & Neck" will be held in Chicago, USA. Contact Center for Continuing Medica! Education, 950 East 61st Street, Suite 101, Chicago, IL, 60637, or fax +1 773 702 1736; or e-rnail rngoldber@uchicago.edu; or see http://www.uchicago.edu/bsd/crne/ Medica! oncology October 29 -November 2, 2004 The 28th ESMO Congress will be held in Vienna, Austria. See http://www.esrno.org Radiation oncology November 7-12, 2004 The ESTRO course "Evidence-Based Radiation Oncology: Methodological Basis and Clinical Application" will take place in Cyprus. Contact ESTRO office, Avenue E. Mounier, 83/12, B-1200 Brussels, Belgiurn; or call +32 775 93 40; or fax +32 2 779 54 94; or e-rnail info@estro.be; or see http://www.estro.be Radiation oncology November 25-28, 2004 The ISRO international teaching course on "Practical Radiation and Molecular Biology with Mayor Ernphasis on Clinical Application" will take place in Chiangmai Thailand. See http://www.isro.be Radiation oncology March, 2005 The ISRO international teaching course on "Palliative Care in Cancer Treatment" will take place in Dar es Salaam, Tanzania. See http://www.isro.be Lung cancer July 3-6, 2005 The "llth World Conference on Lung Cancer" will be offered in Barcelona, Spain. Contact Heather Drew, Irnedex, !ne., 70 Technology Drive, Alpharetta, GA 30005 USA; or call + 1 770 751 7332, or fax + 1 770 751 7334; or e-rnail h.drew@irnedex.com, or see www.irnedex.com/calenders/oncology /htm Rad.iation oncology September -October, 2005 The ISRO international teaching course on "Rational Developrnents frorn developing to developed Countries" will take place in Lombok, Indonesia. See http://www.isro.be Oncology October 30 -November 3, 2005 The ESTRO 24 / ECCO 13 Conference will take place in Paris, France. Contact FECS office, Av. E. Mounier, 83/4, B-1200 Brussels, Belgiurn; or call +32 7759340; or fax +32 2 7795494; or e-rnail info@estro.be; or see http://www.fecs.be As a service to our readers, notices of rneetings or courses will be inserted free of charge. Please send inforrnation to the Editorial office, Radiology and Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia. Sanolabor specialna laboratorijska plastika testi za aplikacijo v imunohistokemiji, za aplikacijo v imunologiji, mikro­biologiji-virologiji, ipd. aparati za pripravo histoloških preparatov mikro-inkriotomi, zalivalci, tkivni procesorji, barvalci, pokrivalci laminar flow tehnika, inkubatorji, sušilniki, suhi sterilizatorji in oprema za laboratorijsko vzrejo živali -kletke laboratorijska oprema za mikro­biologijo celic, molekularno biologijo in biotehnologijo diagnosticni kiti, reagenti za uporabo v mikrobiologiji, imunologiji, citogenetiki, molekularni biologiji patologiji, mikrobiologiji, virologiji, mono-in poliklonalna protitelesa laboratorijsko pohištvo, varnostne omare, ventilacijska tehnika in digestorji hladilna tehnika in aparati za laboratorije, transfuzijo, patologijo in sodno medicino .n hitri testi za diagnostiko, EIA /RIA testi kompletna oprema in pripomocki za delo v laboratoriju FONDACIJA "DOCENT DR. J. CHOLEWA" JE NEPROFITNO, NEINSTITUCIONALNO IN NESTRANKARSKO ZDRUŽENJE POSAMEZNIKOV, USTANOV IN ORGANIZACIJ, KI ŽELIJO MATERIALNO SPODBUJATI IN POGLABLJATI RAZISKOVALNO DEJAVNOST V ONKOLOGIJI. MESESNELOVA 9 1000 LJUBLJANA TEL 01 519 12 77 FAKS 01 251 81 13 ŽR: 501 00-620-1 33-05-1 0331 15-214779 Activity of »Dr. J. Cholewa« Foundation for Cancer Research and Education -A Report for the First Quarter Of 2004 In the course of the year 2004 the Dr. J. Cholewa Foundation for Cancer Research and Education Foundation and the Health continues to support various activities associat­ed with cancer research and education in Slovenia. Although it remains the fact that various public and privately owned enterprises find it more and more difficult to con­tribute financially to help running day to day operations of the Foundation and its many scopes of activity, severa! new initiatives and suggestions were discussed and evaluated during the recent meetings of the Foundation. The Supervising Board of the Dr. J. Cholewa Foundation for Cancer Research and Education Foundation at the last meeting in May of the year 2004 discussed severa! of the requests addressed to it. The requests included support for the publication of books of substantial professional in­terest, support for various research and educational activities and various study grants. Financial reports of the Foundation' s activity showed that despite severa! of the afore­mentioned constraints it achieved ali the anticipated and stated goals for the year 2003 and in the first five months of 2004. The main goal still remains to support and sustain research in cancer in Slovenia, and various pathways are to be undertaken to achieve this goal. The Foundation will continue to support the regular publication of "Radiology and Oncology" international scientific journal, which is edited, published and printed in Ljubljana, Slovenia. The Dr. J. Cholewa Foundation for Cancer Research and Education remains optimistic about the prospects in the year 2004 and notices for the competitive for research and education grants will soon appear in ali major dailies in Slovenia. On severa! meetings it was discussed that the Foundation will put considerable ef­forts in the plan to organise the first "Prof. dr. Vinko Kambi? Memorial Meeting". The "Memorial Meeting" is planned to be held in about one and a half year' s tirne and con­sequently to take place every two or three years. This opportunity may also help the Foundation to find the new ways to collaborate with institutions sharing the same goals ali over Europe and beyond. Tomaž Benulic, MD, MSc Borut Štabuc, MD, PhD Andrej Plesnicar, MD, MSc • sistemske kandidoze • mukozne kandidoze • vaginalna kandidoza • kriptokokoze • dermatomikoze • preprecevanje kandidoze Ucinkovit antimikotik, ki ga bolniki dobro prenašajo. · Podrobnejše informacije so na voljo pri proizvajalcu. Krka. d. d., Novo mesto ŠITlarješka cesta 6 8501 Novo mesto www.krka.sl kapsule raztopina za intravensko infundiranje Kontraindikacije: Preobcutljivost za flukonazol, pomožne sestavine zdravila in za druge azole. Socasno jemanje flukonazola s terfenadinom ali cisapridom. Stranski ucinki: Lahko se pojavijo slabost, napenjanje, bruhanje, bolecine v trebuhu, driska. Možni so glavobol, krci in alopecija. Zelo redke so preobcutljivostne reakcije. Pri bolnikih s hudimi glivicnimi obolenji lahko pride do levkopenije, trombocitopenije, povecane aktivnosti jetrnih encimov ter hujše motnje v delovanju jeter. Oprema in nacin izdajanja: 7 kapsul po 50 mg, 28 kapsul po 100 mg, 1 kapsula po 150 mg -samo na zdravniški recept. 1 viala s 100 ml raztopine za intravensko infundiranje (200 mg/100 ml) -uporaba samo v bolnišnicah. Datum priprave besedila: marec 2003 PE: Stritarjeva 5, 4000 Kranj, Slovenija tel.: (0)4/ 2015 050, fax: (0)4/ 2015 055 e-mail: kemomed@siol.net, KEMOMED www.kemomed.si o ag111m1111111 Promega IZDELKI ZA MOLEKULARNO BIOLOGIJO DOKUMENTACIJA IN ANALIZA GELOV CISTA VODA ZA LABORATORIJ ./-&178,, BIOHlT Biosciences ELEKTRONSKE IN MEHANSKE AVTOMATSKE PIPETE DIAGNOSTIKA SEKVENATORJI MIKOPLAZEIVl IN LEGIONEL AstraZeneca ->- Vaš partner pri zdravljenju raka dojke in prostate Zoladex®3.6mg goserelin Zoladex®LA 10.8mg Casodex goserelin AstraZeneca . AstraZeneca UK Limited, Podružnica v Sloveniji, Einspielerjeva 6, Ljubljana www.astrazeneca.com Radiologtj and Oncology Instructions f or authors Editorial policy of the journal Radiologi; and Oncologi; is to publish original scientific pa­pers, professional papers, review articles, case reports and varia (editorials, reviews, short communications, professional information, book reviews, letters, etc.) pertinent to diag­nostic and interventional radiology, computer­ized tomography, magnetic resonance, ultra­sound, nuclear medicine, radiotherapy, clini­cal and experimental oncology, radiobiology, radiophysics and radiation protection. The Editorial Board requires that the paper has not been published or submitted for publication elsewhere: the authors are responsible for all statements in their papers. Accepted articles become the property of the journal and there­fore cannot be published elsewhere without written permission from the editorial board. Papers concerning the work on humans, must comply with the principles of the declaration of Helsinki (1964). The approval of the ethical committee must then be stated on the manu­script. Papers with questionable justification will be rejected. Manuscript written in English should be submitted to the Editorial Office in triplicate (the original and two copies), including the il­lustrations: Radiology and Oncologi;, Institute of Oncology, Zaloška 2, SI-1000 Ljubljana, Slovenia; (Phone: +386 1 5879 369, Tel./Fax: +386 1 5879 434, E-mail: gsersa@onko-i.si). Authors are also asked to submit their manu­scripts on a 3.5" 1.44 Mb formatted diskette. The type of computer and word-processing package should be specified (W ord for Windows is preferred). All articles are subjected to editorial review and review by independent referee selected by the editorial board. Manuscripts which do not comply with the technical requirements stated herein will be returned to the authors for cor­ rection before peer-review. Rejected manu­ scripts are generally returned to authors, how­ ever, the journal cannot be held responsible for their loss. The editorial board reserves the right to ask authors to make appropriate changes in the contents as well as grammatical and stylistic corrections when necessary. The expenses of additional editorial work and re­ quests for reprints will be charged to the au­ thors. General instructions • Radiology and Onco­logy will consider manuscripts prepared accor­ding to the Vancouver Agreement (N Engl J Med 1991; 324: 424-8, BMJ 1991; 302: 6772; JA­MA 1997; 277: 927-34.). Type the manuscript double spaced on one side with a 4 cm margin at the top and left hand side of the sheet. Write the paper in grammatically and stylisti­cally correct language. Avoid abbreviations unless previously explained. The technical da­ta should conform to the SI system. The man­uscript, including the references may not ex­ceed 15 typewritten pages, and the number of figures and tables is limited to 4. If appropri­ate, organize the text so that it includes: Introduction, Material and methods, Results and Discussion. Exceptionally, the results and discussion can be combined in a single sec­tion. Start each section on a new page, and number each page consecutively with Arabic numerals. Title page should include a concise and in­formative title, followed by the full name(s) of the author(s); the institutional affiliation of each author; the name and address of the cor­responding author (including telephone, fax and e-mail), and an abbreviated title. This should be followed by the abstract page, sum­marising in less than 200 words the reasons for the study, experimental approach, the major findings (with specific data if possible), and the principal conclusions, and providing 3­6 key words for indexing purposes. Structured abstracts are preferred. If possible, the authors are requested to submit also slovenian version of the title and abstract. The text of the report should then proceed as follows: Introduction should state the purpose of the article and summarize the rationale for the study or observation, citing only the essential references and stating the aim of the study. Material and methods should provide enough information to enable experiments to be re­peated. New methods should be described in detail. Reports on human and animal subjects should include a statement that ethical ap­proval of the study was obtained. Results should be presented clearly and concisely without repeating the data in the ta­bles and figures. Emphasis should be on clear and predse presentation of results and their significance in relation to the aim of the inves­tigation. Discussion should explain the results rather than simply repeating them and interpret their significance and draw conclusions. It should review the results of the study in the light of previously published work. Illustrations and tables must be numbered and referred to in the text, with appropriate location indicated in the text margin. Illu­strations must be labelled on the back with the author's name, figure number and orien­tation, and should be accompanied by a de­scriptive legend on a separate page. Line drawings should be supplied in a form suit­able for high-quality reproduction. Photo­graphs should be glossy prints of high quality with as much contrast as the subject allows. They should be cropped as close as possible to the area of interest. In photographs mask the identities of the patients. Tables should be typed double spaced, with descriptive title and, if appropriate, units of numerical meas­urements included in column heading. References must be numbered in the order in which they appear in the text and their cor­responding numbers quoted in the text. Authors are responsible for the accuracy of their references. References to the Abstracts and Letters to the Editor must be identified as such. Citation of papers in preparation, or submitted for publication, unpublished obser­vations, and personal communications should not be included in the reference list. If essen­tial, such material may be incorporated in the appropriate place in the text. References fol­low the style of Index Medicus. All authors should be listed when their number does not exceed six; when there are seven or more au­thors, the first six listed are followed by "et al.". The following are some examples of refer­ences from articles, books and book chapters: Dent RAG, Cole P. In vitro maturation of monocytes in squamous carcinoma of the lung. Br J Cancer 1981; 43: 486-95. Chapman S, Nakielny R. A guide to radiolog­ical procedures. London: Bailliere Tindall; 1986. Evans R, Alexander P. Mechanisms of ex­tracellular killing of nucleated mammalian cells by macrophages. In: Nelson DS, editor. Immunobiology oj macrophage. New York: Academic Press; 1976. p. 45-74. Page proofs will be faxed to the correspon­ding author whenever possible. It is their re­sponsibility to check the proofs carefully and fax a list of essential corrections to the editori­al office within 48 hours of receipt. If correc­tions are not received by the stated deadline, proof-reading will be carried out by the edi­tors. Reprints: Fifty reprints are free of charge, for more contact editorial board. Far reprint information contact: International Reprint Corporation, 287 East "H" Street, Benicia, CA 94510, USA. Tei: (707) 746-8740; Fax: (707) 7 46-1643; E-mail: reprints@intlreprints.com Le. ,5 , ,e /0, ml, l.e./0.4 ml, 5000 /0,5 m , · /0,&ml, :ejg 7-ml, 8000 i.e./0, i.e./0 9 ml,-111 i.J!./\ • snovi: natrijev dihidrogen.t­ trijev klorid, palisolbat 80, i,ici in voila m injekcije. J ind, · Zdralijenje Me.[n .nje potreb po uaiisruzijf pri od@.ih bolnikih, pri kateri!. lieiiioterapijo'Zlf111'1imo soliane l!J. mo1e, mahg,,, hmfom ah mulaph mielom In pri tvegan1u zalransfuzi10, k, ga oremmo glede na bolnnca., 5!)1ošno zd stanje. Zdra,ijenje'&oemije, posledice kroi\'. od'. ledlic pri otrocih in odrasfill 2dravljenih s hemodializo in odraslih zdravljenih s pefitonealno dializo. Zdravljenje hude anemije zaradi bolezni ledvic pri bolnikih, ki še niso na dializi. Povecanje lzvodnje avtologne kNt. bolnikih v programu samciobdaLt7ianja krvi pred operacijo. Zmanjšanje izpostavljenosti alo­nim transfuzijam krvi pred vecjimi elektivnimi ortopedskimi kirurškimi posegi. Kontraindikacije: Nenadzorovana arterijska hiija, preobcutljivost za ka ro od sestavin zdravila, ter oolniki.e morejo dobiti ustrezne antitrombinske profila k.se. .·enju z epoetinom razvi· aplazija rdecih kmiih celic ustaVJttzdravfj. Eprexom ali drugim epoetinom. . Subkutano injiciranje zdravila je kontraindicirano samo pri bolnikih s kron. ledvicno odpovedjo. Pri bolnikih, pri katerih se po zd je bolnikih v programu avtolognega zbiranja krvi upoštevati . kontraindikacije povezane s programom za kaS11ejše avtotransfuzije. Z ·10 je kontraindicirano pn ikih pred vecjim e!ektivnim kirurškim posegonrs ktlronamo,...cerebn:JYastru1amo, karotidno ali periferno arterijsko boleznijo ali nedavnim miokardnim infarktom ali cerebrovaskularnim dogodkom. Posebna opomrila in nostnl ukrepi: Potrebno J no spremljati in po potrebi nadzorovati kivni tlak. Previdna uporaba epoe­ tina alfa je potrebna pri nezdravljeni, neustrezno zdravljeni ali slabo nadzorovani hipertenziji. Pri bolnikih, zdrav!jenih z epoetinom alfa, je redno meriti koncentracije ti obina, dokler ni dosežena stabilna vrednost, zatem pa se meritve "'2" opravljajo periodicno. Epoetin alfa je treba previdno uporabljati tudi pri bolnikih z epilepsjo in kronicno odpovedjo jeter. V posameznih z epoetinom alfa se lahko pojavi zmerno, prehodno, 1g od odmerkov odvisno povecanje števila trombocitov. Preuciti je treba še druge vzroke anemiije in jih zdraviti pred zacetkom zdravljenja z ....,;.\.,.-Pri..evanju primernosti zdravljenja z epoetinom M alfa (bolniki, k! jim grozi transfuzija), je treba upoštevati 2-3-tedenski zamik med dajanjem eritropoetina ter tvorbo rdecih kivnick, ki jo i zdravili: O vplivu zdravljenJa°Lepoetinom.alfa.na.m.e.tabolizem.drugih · zdravil ni dokazov. Ker pa se cik!osporin veže na rdece kivne celice, je možna interakcija med njim in zdravilom. Odmerjanje in nacinenpr. Hb 105 g/1), ki prejemajo kemoterapijo je uporabimo subkutani nacin dajanja. Zacetni odmerek je 150 i.e./kgsubkutano, trikrat na teden. ce vrednost Hb naraste za najmanj 10 g/1, ali se število retikulocitov poveca za 40.., 000 osnovno vrednost po 4 tednih zdravljenja, se naj še naprej uporablja §_. - odmerek 150 i.e.jkg. ce je zvecanje Hb < 10 g/1 in je število retikulocitov naraslo za <40.000 celic/ 1 nad osnovno vrednost, zvecajte odmerek na 300 i.e./kg. Ce je 4·tedoi!! zdravjenja s 300 i.e./kg. vrednost Hb narasla na 10 g/1, ali l0 se je število retikulocitov zvecalo na 40,000 celic/ 1, naj ostane odmerek 300 i.e./kg. ce se je vrednost Hb zvecala za <10 g/1, število retikulocitov pa je naraslo <40.000celic/ 1 nad osnovno-vr. odziv ni verjeten in je treba zdravljenje prekiniti. o Neželeni ucinki: Nespecificni kolni izpušcaji, "gripi podobni" simptomi (glavobol, bolecine v sklepih, slabost, vrtoglavica, utrujenost), trombocitoza (zelo redko), hipertenzja. Posebna navodila za shranJmnje:,Shraojyjte zašciteno pred svettobo„ pri--fu-­ itemperaturi od 2° do a0c. Zdravila ne zamrzujte ali stresajte. Nacin izdajanja zdravila: H/Rp. Podrobnejše informacije in navodila o pr.dpisovanju so vam na voljo pri: .A.. JANSSEN-CI LAG Johnson & Johnson J.E., Podružnica Ljubljana, Smartinska 140, 1000 Ljubljana ,.......,,.