Expression of the Free Beta Subunit of Human Chorionic Gonadotropin in Cancer of the Urinary Bladder and Kidney Kristina Hotakainen Department of Clinical Chemistry University of Helsinki Helsinki, Finland Academic Dissertation To be publicly discussed with the permission of the Medical Faculty of the University of Helsinki in Auditorium 2, Biomedicum Helsinki, on May 24, 2002, at 12 o’clock noon. Supervised by Professor Ulf-Håkan Stenman, MD, PhD University of Helsinki Finland Reviewed by Docent Martti Nurmi, MD, PhD University of Turku Finland and Professor Kim Pettersson, PhD University of Turku Finland Opponent Professor Mirja Ruutu, MD, PhD University of Helsinki Finland ISBN 952-91-4592-6 (Print) ISBN 952-10-0514-9 (PDF) Yliopistopaino Helsinki 2002 Table of contents List of original publications ..................................................................................... 5 Abbreviations .......................................................................................................... 6 Abstract .................................................................................................................. 7 Introduction ............................................................................................................ 8 Review of the literature ........................................................................................... 9 1. Chorionic gonadotropin and its subunits ............................................................. 9 1.1 Biochemistry and biology ..................................................................... 9 1.2 Biological function ............................................................................... 9 1.3 HCG in serum and urine of healthy subjects ......................................... 9 1.4 HCG in malignant disease .................................................................... 10 1.5 Clinical determinations ......................................................................... 10 1.5.1 Normal pregnancy ..................................................................... 10 1.5.2 Ectopic pregnancy ..................................................................... 10 1.5.3 Maternal screening for Down’s syndrome ................................... 11 1.5.4 Trophoblastic disease ................................................................. 11 1.5.5 Testicular cancer ........................................................................ 11 1.6 The beta subunit of chorionic gonadotropin .......................................... 11 1.6.1 Genes, mRNAs and proteins ..................................................... 11 1.6.2 HCGß expression in tissues and body fluids .............................. 12 1.6.3 HCGß expression in peripheral blood cells ................................ 12 1.6.4 HCGß in malignant disease ....................................................... 12 1.6.5 HCGß expression in bladder cancer ........................................... 13 1.6.6 HCGß expression in renal cell carcinoma (RCC) ........................ 14 2. Bladder cancer ..................................................................................................... 14 2.1 Epidemiology ....................................................................................... 14 2.2 Risk factors ........................................................................................... 14 2.3 Histology ............................................................................................. 14 2.4 Symptoms and signs ............................................................................. 14 2.5 Diagnostic procedures ........................................................................... 14 2.6 Staging and grading ............................................................................. 15 2.7 Treatment and prognosis ....................................................................... 15 2.8 Markers of bladder cancer ..................................................................... 16 2.8.1 Markers for screening and diagnosis ........................................... 16 2.8.2 Prognostic markers .................................................................... 17 3. Renal cell carcinoma ........................................................................................... 19 3.1 Epidemiology ....................................................................................... 19 3.2 Risk factors ........................................................................................... 19 3.3 Histology ............................................................................................. 19 Kristina Hotakainen 3.4 Symptoms and signs ............................................................................. 20 3.5 Diagnostic procedures ........................................................................... 20 3.6 Staging and grading ............................................................................. 20 3.7 Treatment and prognosis ....................................................................... 20 3.8 Markers of RCC .................................................................................... 21 3.8.1 Tissue markers ........................................................................... 21 3.8.2 Serum markers........................................................................... 22 Aims of the study .................................................................................................... 23 Material and methods .............................................................................................. 24 1. Subjects and samples (I-IV) ................................................................................ 24 2. Preparations and cultures of cells (I-III) .............................................................. 24 3. Leukocyte extracts (I) ......................................................................................... 25 4. Separation of mononuclear cells, granulocytes, monocytes, B- and T-cells (I) ...... 25 5. Isolation of RNA (I-III) ..................................................................................... 26 6. Removal of genomic DNA (I-III) ....................................................................... 26 7. Oligonucleotide primers (I-III) .......................................................................... 27 8. RT-PCR and gel electrophoresis (I-III) ............................................................... 27 9. Restriction enzyme analysis and sequencing of the PCR products (I-III) ............. 27 10.Determination of hCG, hCGß, hCGßcf, LH and LHß (I-IV) .............................. 28 11.Immunohistochemistry (III) ............................................................................... 28 12.Statistical methods (I-IV) ................................................................................... 29 Results and discussion ............................................................................................. 30 1. Expression of hCGß and LHß in peripheral blood cells (I) .................................. 30 1.1 Expression of hCGß- and LHß mRNA in peripheral blood cells and cell lines ....................................................................................... 30 1.2 LH- and LHß expression in cultured lymphocytes................................. 30 1.3 Expression of hCG protein and hCGß mRNA and protein in cultured lymphocytes ............................................................................. 30 1.4 Conclusions .......................................................................................... 32 2. Expression of hCGß in bladder cancer (II, III) .................................................... 33 2.1 Immunohistochemical expression of hCGß (III) .................................... 33 2.2 HCGß and hCG in serum of bladder cancer patients (II, III) ................. 34 2.3 HCGß, hCG and hCGßcf in urine of bladder cancer patients (II, III) .... 35 2.4 HCGß mRNA in urinary cells (II, III) .................................................. 36 3. HCGß expression in serum of RCC patients (IV) ............................................... 37 Summary ........................................................................................................... 39 Conclusions ........................................................................................................... 41 Acknowledgements ................................................................................................. 42 References .......................................................................................................... 44 ........................................................................................................... Original publications .............................................................................................. 55 4 List of original publications I. Hotakainen K, Serlachius M, Lintula S, Alfthan H, Schröder J, Stenman U-H. Expression of luteinising hormone and chorionic gonadotropin beta-subunit messenger-RNA and protein in human peripheral blood leukocytes. Mol Cell Endocrinol 2000; 162:7985. II. Hotakainen K, Lintula S, Stenman J, Rintala E, Lindell O, Stenman U-H. Detection of messenger RNA for the ß-subunit of chorionic gonadotropin in urinary cells from patients with transitional cell carcinoma of the bladder by reverse transcription-polymerase chain reaction. Int J Cancer 1999; 84: 304-308. III. Hotakainen K, Haglund C, Paju A, Nordling S, Alfthan H, Rintala E and Stenman U-H. Chorionic gonadotropin beta-subunit and core fragment in bladder cancer: mRNA and protein expression in urine, serum and tissue (Submitted). IV. Hotakainen K, Ljungberg B, Rasmuson T, Alfthan H, Stenman U-H. The free ßsubunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma. Br J Cancer, 2002; 86: 185-189. 5 Kristina Hotakainen Abbreviations ATCC cDNA ConA CRCC CRP EGF FCM FDP FGF FISH FRC FSH hCG hCGα hCGß hCGßcf IA IFMA IL IRP IS LH LHß LSCM MAb MLC mRNA NMP NSE PCNA PDGF PHA PRL PWM RCC RT-PCR TATI TCC TGF Tis TSH US UTI VEGF 6 American Type Culture Collection complementary DNA Concanavalin A Conventional RCC C-reactive protein Epidermal growth factor Flow cytometry Fibrinogen degradation products Fibroblast growth factor Fluorescence in situ hybridization Finnish Red Cross Follicle stimulating hormone Human chorionic gonadotropin α subunit of hCG ß subunit of hCG Core fragment of hCGß Image analysis Immunofluorometric assay Interleukin International Reference Preparation International Standard Luteinizing hormone Beta subunit of LH Laser scanning cytometry Monoclonal antibody Mixed lymphocyte culture messenger RNA Nuclear matrix protein Neuron specific enolase Proliferating cell nuclear antigen Platelet derived growth factor Phytohemagglutinin Prolactin Pokeweed mitogen Renal cell carcinoma Reverse transcription-polymerase chain reaction Tumor-associated trypsin inhibitor Transitional cell carcinoma Transforming growth factor Tumor in situ Thyroid stimulating hormone Ultrasonography Urinary tract infection Vascular endothelial growth factor Abstract Human chorionic gonadotropin (hCG) is a glycoprotein hormone consisting of two dissimilar subunits, α and ß. HCG is produced by the placental trophoblasts and its function is to maintain pregnancy. The free subunits have no known biological function. The ß subunit (hCGß) is commonly produced at low concentrations by many cultured cells and malignant tumors of various origin, in which it frequently is a sign of aggressive disease. Clinically hCGß is a valuable marker for monitoring of trophoblastic tumors and testicular carcinoma. Reverse transcription-polymerase chain reaction (RT-PCR) of hCGß mRNA was used in this study to detect cells expressing hCGß in blood and urine. HCGß expression was induced by culture of peripheral blood cells, but no expression was observed in unstimulated blood cells. HCGß mRNA expression was detected in the urinary cells of approximately 50% of patients with bladder cancer but not in healthy controls. The expression was strongly associated with histologically proven carcinoma but not with stage and grade of the tumor. Immunohistochemical expression of hCGß protein was found in tumor tissue from one third of the cancer patients but equally often in benign epithelium. Current or previous instillation therapies did not affect the detection of hCGß mRNA in urinary cells or tissue expression detected by immunohistochemistry. The urine concentrations of the hCGß core fragment, a degradation product of hCGß, were higher in patients with hCGß mRNA in urine and the urine to serum ratio of hCGß was strongly associated with both stage and grade of the disease, and also with immunohistochemical detection of hCGß. Our findings show that hCGß expression is not cancer specific, but it may occur in benign conditions. The ratio of urine to serum hCGß demonstrates that local production of hCGß by the tumor correlates with stage and grade of the disease, supporting previous data on the association of hCGß with advanced disease. To study whether hCGß has prognostic significance in renal cell carcinoma (RCC) preoperative serum samples from patients with RCC were analyzed. The serum concentrations were elevated in 23% of the patients and hCGß was a prognostic marker independent of stage and grade of the tumor. Our results suggest that hCGß expression characterizes a potentially aggressive subgroup of tumors. 7 Kristina Hotakainen Introduction Bladder and kidney cancer are two fairly common malignancies, for which few reliable tumor markers are available. Markers that could be used for diagnosis, monitoring and evaluation of prognosis would be of clinical value. The free ß subunit of human chorionic gonadotropin (hCG) in serum has proved to be useful as a prognostic marker especially for ovarian and various gastrointestinal cancers. This study was undertaken to investigate whether hCGß expression could be used as a marker for bladder and kidney 8 cancer. We studied whether hCGß mRNA in urinary cells can be used to detect bladder cancer and if this finding correlates with serum and urine concentrations of hCGß and immunohistochemical staining of hCGß in tumor tissue, as well as with stage and grade of the tumor. The clinical course of renal cell carcinoma (RCC) is unpredictable, and no specific serum markers are available. We therefore studied whether hCGß in serum of RCC patients is of diagnostic and prognostic value for this disease. Review of the literature 1. CHORIONIC GONADOTROPIN AND ITS SUBUNITS 1.1 Biochemistry and biology Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family, which also comprises luteinizing hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH). These hormones are heterodimers consisting of an α and a ß subunit. The free subunits are devoid of hormonal activity (Catt et al., 1973; Rayford et al., 1972). The α subunit common to hCG, LH, FSH and TSH contains 92 amino acids. The ß chains are hormone specific and determine the biological activity. The ß subunit of hCG (hCGß) contains 145 amino acids including a 24 amino acid C-terminal extension lacking in LHß. The homology between the ß chains of hCG and LH is about 80% and hCG and LH exert their action through the same receptor (McFarland et al., 1989). The molecular weights of hCG, hCGß and hCGα are 36700, 22200 and 14500 Da, respectively (Birken, 1984). HCGα is encoded by a single gene on chromosome 12q21.123 and hCGß by a cluster of six nonallelic genes on chromosome 19q13.3 (Boothby et al., 1981; Talmadge et al., 1984b). 1.2 Biological function HCG is produced at high concentrations by placental syncytiotrophoblasts. HCG maintains pregnancy by stimulating progesterone production in the corpus luteum during the first trimester (Yoshimi et al., 1969). It also stimulates testosterone production by fetal testes (Huhtaniemi et al., 1977). Receptors for hCG/LH have been identified in the nonpregnant uterus (Reshef et al., 1990), prostate (Dirnhofer et al., 1998a) and several extragonadal sites such as leukocytes (Lin et al., 1995), thyroid (Frazier et al., 1990) and epidermal structures (Venencie et al., 1999). 1.3 HCG in serum and urine of healthy subjects Low concentrations of hCG are produced by the pituitary giving rise to measurable plasma levels. The serum concentrations are correlated with those of LH and thus they increase with age both in men and in women (Stenman et al., 1987). Age- and gender specific reference values in serum and urine are given in table 1. In healthy nonpregnant subjects the α subunit originates mostly from the pituitary, but some pituitary adenomas may secrete hCGß (Gil-delAlamo et al., 1995). HCGα can be detected at levels up to 3 ng/l in the serum of most healthy individuals (87%), patients with benign diseases (93%), and various nontrophoblastic cancers (96%) (Marcillac et al., 1992). HCG and hCGß are excreted into urine and the concentrations are comparable to those in plasma. The concentrations of hCGα in urine are 3-5-fold those in serum (Landy et al., 1990; Iles & Chard, 1991). Much of hCG and hCGß is degraded during excretion and a variable part of the hCG immunoreactivity in urine consists of a fragment of hCGß called the core fragment 9 Kristina Hotakainen Table 1. Reference values for hCG, hCGβ, hCGβcf and hCGα in serum and urine of women and men (Alfthan et al., 1992a). Upper reference limit (pmol/l) Serum Age (years) hCG hCGβ hCGβcf hCGα Women Men <50 ≥50 <50 ≥50 8.6 1.6 1.1 301 15.5 2.0 1.1 602 2.1 1.9 1.1 6.1 2.1 1.1 8.8 1.7 8.1 11.5 4.3 9.5 2.9 1.3 6.7 8.4 3.6 8.5 1.5 Clinical determinations Urine hCG hCGβ hCGβcf 1 In subjects with a serum concentration of FSH < 20; FSH ≥ 20 (Alfthan et al., unpublished data). 2 (hCGßcf) (Papapetrou & Nicopoulou, 1986). 1.4 HCG in malignant disease The expression of hCG in serum of cancer patients is a classic example of ectopic hormone production (Braunstein et al., 1973; Weintraub & Rosen, 1973; Hattori et al., 1978). Most cultured human fetal and cancer cells express hCGß, hCGα as well as intact hCG (Acevedo et al., 1992). However, detectable serum concentrations of hCGα and hCG occur in normal individuals also, whereas elevated levels of hCGß are frequently associated with malignant tumors (Marcillac et al., 1992; Alfthan et al., 1992b). Most nontrophoblastic tumors produce hCGß exclusively, but isolated production of hCGα has recently been described in an immunohistochemical study of neuroendocrine lung carcinoma (Dirnhofer et al., 2000). Carcinoid tumors of the pancreas frequently express hCGα (70%) in the absence of hCGß (Öberg & Wide, 1981), and immunocytochemical staining of hCGα can be observed equally often in endocrine pancreatic tumors (Heitz et al., 1983). Overexpression of hCGα in relation to hCGß at the mRNA level has been shown in breast cancer tissue, and these transcripts are also 10 translated into hCGß protein (Giovangrandi et al., 2001). In addition, some gastrointestinal and gynecological cancers may produce hCGα exclusively (Huang et al., 1989). Various minor molecular forms of hCG may occur in the urine of cancer patients, e.g., nicked and hyperglycosylated forms, and variants lacking the C-terminal extension of hCGß (Cole et al., 2001). 1.5.1 Normal pregnancy During pregnancy the concentrations of hCG become detectable 5-7 days after conception and increase exponentially with a doubling time of about 1.5 days. Peak values are reached at 7-10 weeks of pregnancy. After this the levels decrease slowly until the 15th week, after which there is a small gradual increase towards term. The concentrations of hCGß are about 0.5-1% of the total hCG concentration, whereas those of hCGα are less than 10% during the first trimester increasing to 30-60% at term. Low concentrations of hCGßcf occur in pregnancy serum (<1% of the total hCG concentration) (Alfthan & Stenman, 1990; Kardana & Cole, 1990). In urine hCGßcf is the major form of hCG immunoreactivity and the concentration is about 4000-fold that in serum (Wehmann et al., 1990). Pregnancy can be diagnosed by detecting elevated hCG concentrations in urine or serum by the time of the first missed menstrual period. 1.5.2 Ectopic pregnancy Ectopic pregnancy can be diagnosed when no intrauterine gestational sac is seen by ultrasonography and the hCG level is greater than 1000 IU/l (Cacciatore et al., 1995). The hCG concentration at day 44 after the last menstrual period can also be used to predict spontaneous resolution of an ectopic pregnancy (Korhonen et al., 1994). Review of the Literature 1.5.3 Maternal screening for Down’s syndrome HCG and hCGß in serum or urine can be used in the diagnosis of various pregnancyrelated disorders and chromosomal abnormalities of the fetus (Cuckle et al., 1999). Serum concentrations of hCG or hCGß in combination with other markers are used in screening of fetal trisomy 21. The risk increases with increasing concentration of hCG and hCGß and decreasing levels of alpha fetoprotein (AFP) (Phillips et al., 1992). mRNA expression has been demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral blood of patients with germ cell tumors, and more frequently in those with elevated serum concentrations of hCGß. However, the clinical significance of this phenomenon is unclear (Hautkappe et al., 2000). 1.6 The beta subunit of chorionic gonadotropin 1.6.1 Genes, mRNAs and proteins 1.5.4 Trophoblastic disease Elevated levels of both hCG and hCGß are encountered in virtually all cases of molar disease and choriocarcinoma. The proportion of hCGß to hCG may be used to differentiate between these. In benign disease the proportion is below 5% (on a molar basis) and in choriocarcinoma the proportion exceeds 6% (Ozturk et al., 1988; Stenman et al., 1995; Vartiainen et al., 1998). 1.5.5 Testicular cancer Germ cell tumors of the testis represent more than 95% of all testicular cancers. These are classified as seminomas (30-40%) and nonseminomatous germ cell tumors of the testis. HCG and hCGß are important markers for both tumor types. HCG is used both as a prognostic factor, for monitoring of the response to therapy and during follow up of the disease. Seminoma patients have elevated serum levels of hCG in 1030% of the cases. Of all hCG-producing seminomas 30- 40% have elevated serum levels of both hCG and hCGß, 25% of hCG only and 35% of only hCGß (Koshida et al., 1996; Weissbach et al., 1997). Elevated serum concentrations of hCG are associated with adverse prognosis (Bosl et al., 1981; Droz et al., 1988; von Eyben et al., 2001), and the risk increases with increasing concentrations (Vogelzang, 1987). HCGß HCGß is encoded by a cluster of genes numbered ß1 to ß9 on chromosome 19q13.3. The homology between the hCGß and LHß genes is 94% (Talmadge et al., 1983; Talmadge et al., 1984b), and the hCGß gene has probably arisen through duplication of an ancestral LHß gene (Boorstein et al., 1982). The gene cluster comprises six nonallelic hCGß genes and the gene encoding LHß. ß1 and ß2 are considered pseudogenes that are not expressed, while ß7 and ß9 are alleles to ß6 and ß3, respectively (Policastro et al., 1983; Policastro et al., 1986). However, it is possible that ß1 and ß2 can produce alternatively spliced transcripts giving rise to a hypothetical protein 132 amino acids in length (Dirnhofer et al., 1996). Genes ß6 and ß7 (type I genes) encode a protein with alanine at position 117 while genes ß3/9, ß5 and ß8 (type II genes) encode a protein containing aspartic acid at this position. The transcriptional activity varies among the various hCGß genes, and also among individuals. In the placenta, gene 5 is the one with the strongest expression, followed by genes 3 and 8 with gene 7 accounting for less than 2% of the total expression (Bo & Boime, 1992; MillerLindholm et al., 1997). A similar pattern is common also in nontrophoblastic tissues; Giovangrandi and coworkers recently showed that the increased hCGß mRNA expression observed in breast cancer tissue is mainly due to overexpression of genes 5, 11 Kristina Hotakainen 8 and 3 (Giovangrandi et al., 2001). Transcripts of these genes have been demonstrated in testicular tumors also (Madersbacher et al., 1994). The differential expression results from differences in the promoter regions of the genes (Otani et al., 1988). The mRNA of hCGß contains 879bp (Talmadge et al., 1984a). HCGß is 145 amino acids in length. It contains six carbohydrate chains and the molecular weight is 22200 Da. The threedimensional structure of hCGß resembles that of the so-called cysteine knot growth factors, e.g. nerve growth factor, platelet derived growth factor (PDGFß), transforming growth factor (TGFß) and vascular endothelial growth factor (VEGF) (Lapthorn et al., 1994; Muller et al., 1997). These growth factors bind to their receptors both as homo- and heterodimers. HCGß has been shown to exist as a homodimer (Butler et al., 1999), and thus it may also bind to a hypothetical receptor as a homodimer. However, no receptor for hCGß or hCGßß has been identified. 1.6.2 HCGß expression in tissues and body fluids HCGß occurs in serum of men and nonpregnant females at levels 5 to 10-fold lower than those of hCG. The serum levels are not related to those of hCG or LH and they are not age-dependent (Alfthan et al., 1992a). Expression of hCGß mRNA has been demonstrated in several tissues, i.e., bladder, adrenal, colon, testis, breast, thyroid and uterus, in which the expression is about 10,000-fold lower than in the placenta (Bellet et al., 1997). Expression of hCGß in normal nontrophoblastic tissues is mostly associated with type I gene expression but testicular tissue also expresses type II genes. The placenta, trophoblastic and other malignant tumors express predominantly type II genes (Bellet et al., 1997). Moderately elevated serum concentrations of hCGß protein occur in 30-70% of 12 most nontrophoblastic cancers (Alfthan et al., 1992b; Marcillac et al., 1992) and it has been suggested that activation of the hCGß/ LHß gene cluster is characteristic of malignant transformation (Acevedo et al., 1995; Bellet et al., 1997; Krichevsky et al., 1995). Membrane-bound hCGß has been found to be characteristic of a metastatic phenotype of cancer cells (Acevedo & Hartsock, 1996). HCGß stimulates the growth of certain cancer cell lines in culture, and it is proposed to act as an autocrine or paracrine growth factor (Gillott et al., 1996). The growth promoting effect of hCGß may partly result from inhibition of apoptosis (Butler et al., 2000). 1.6.3 HCGß expression in peripheral blood cells Low level expression of several genes thought to be specific for other organs can be observed in peripheral blood cells (Azad et al., 1993; Lintula & Stenman, 1997). Leukocytes have the potential of producing several peptide hormones (Blalock et al., 1985; Harbour-McMenamin et al., 1986; Smith et al., 1985), and receptors for LH/hCG have been identified on leukocytes (Lin et al., 1995). Cultured cells from leukemias and lymphomas express membrane-bound hCG and its subunits (Acevedo et al., 1992). HCG immunoreactivity has been induced in lymphocytes by mixed lymphocyte cultures, but not by other mitogenic stimuli (Harbour-McMenamin et al., 1986). During pregnancy, peripheral blood mononuclear cells are capable of secreting hCG (Alexander et al., 1998). 1.6.4 HCGß in malignant disease HCGß expression can be detected by immunohistochemistry in 20-52% of colorectal carcinomas and the expression is associated with poorly differentiated and advanced tumors (Kido et al., 1996; Yamaguchi et al., 1989; Campo et al., 1987). Review of the Literature In one study, only serum concentrations were found significantly correlated with the clinical outcome (Webb et al., 1995). The prognostic significance has later been confirmed both for serum concentrations and immunohistochemical detection of hCGß (Lundin et al., 2001; Carpelan-Holmström et al., 1996). HCG immunoreactivity has been described in squamous cell carcinoma cell lines (Cole et al., 1981; Cowley et al., 1985). In immunohistological studies of the esophagus (Burg-Kurland et al., 1986; Trias et al., 1991), oral cavity (Bhalang et al., 1999) and uterine cervix, hCG expression has been associated with poorly differentiated tumors. Preoperatively elevated serum levels of hCGß predict shorter recurrence free survival of patients with squamous cell carcinomas of the oral cavity and oropharynx (Hedström et al., 1999). Serum levels of hCGß are rarely elevated in vulvar carcinomas, but progressive disease is associated with rising levels (de Bruijn et al., 1997). The concentrations of hCGß in serum have a prognostic significance in ovarian carcinoma (Ind et al., 1997; Vartiainen et al., 2001). Urinary hCGßcf has been used as a marker of gynecological cancers (Cole et al., 1988; Wang et al., 1988). HCGß mRNA is expressed in several pancreatic cancer cell lines, and also in tissue samples from metastatic pancreatic cancer (Bilchik et al., 2000). Elevated serum concentrations of hCGß have been described in 40-70% of patients with pancreatic and biliary cancer. In most of these, urinay concentrations of hCGßcf are also elevated (Alfthan et al., 1992b; Motoo et al., 1996). Elevated hCGß in serum has been reported to correlate with adverse outcome in the cancer patients (Syrigos et al., 1998). RT-PCR of hCGß mRNA has been used to detect circulating melanoma cells (Hoon et al., 1995) and melanoma metastases in lymph nodes (Doi et al., 1996). Recently, Taback and coworkers showed that detection of hCGß mRNA expression in circu- lating cells from patients with breast cancer is significantly associated with tumor size (Taback et al., 2001). Hu and coworkers found an association between stage and detection of hCGß mRNA in blood of breast cancer patients (Hu & Chow, 2000). In both studies the combination of hCGß with another marker significantly improved the correlation (Hu & Chow, 2001). However, hCGß mRNA expression also occurs in benign breast tumors (Reimer et al., 2000). 1.6.5 HCGß expression in bladder cancer Approximately 70% of both benign and malignant urothelial cells in culture express hCG-like material, almost entirely consisting of hCGß (Iles et al., 1987; Iles & Chard, 1989; Iles et al., 1990b). Elevated levels of hCGß, and rarely intact hCG, can be detected in serum and urine from patients with transitional cell carcinomas (TCC) of the bladder. Both serum and urine concentrations of hCGß are elevated in up to 70% of the patients with metastatic disease (Iles et al., 1989; Iles et al., 1996) but in less than 10% of patients with local tumors (Iles et al., 1989; McLoughlin et al., 1991; Smith et al., 1994). The degradation of hCGß may lead to increased concentrations of hCGßcf in urine, but it is possible that some tumors synthesize and secrete hCGßcf into the urine (Iles et al., 1990a; Dirnhofer et al., 1998b). Approximately 30% of TCCs express hCGß and this expression seems to characterize an aggressive subgroup of tumors. Elevated serum and urine concentrations of hCGß in advanced disease have been shown to predict the development of metastasis and relapses as well as increased mortality (Iles et al., 1996; Marcillac et al., 1993). HCGß expression detected by immunohistochemistry is associated with a poor response to radiotherapy (Martin et al., 1989; Jenkins et al., 1990; Moutzouris et al., 1993) and the serum concentrations of hCGß correlate with the response to chemotherapy 13 Kristina Hotakainen (Dexeus et al., 1986; Mora, J. et al., 1996; Cook et al., 2000). 1.6.6 HCGß expression in renal cell carcinoma (RCC) Many earlier studies have failed to show any hCG immunoreactivity in serum or tumor tissue of patients with RCC (Sufrin et al., 1977; Kuida et al., 1988; Dunzendorfer et al., 1981). However, hCG immunoreactivity has been detected by radioimmunoassay in urinary concentrates from patients with advanced or poorly differentiated tumors (Fukutani et al., 1983), but the number of patients in the study was very small. Elevated serum concentrations of hCGß have been reported to occur in 10% of the patients, and the levels correlate with the clinical course (Dexeus et al., 1991). 2. BLADDER CANCER 2.1 Epidemiology Bladder cancer is one of the most common malignancies among men in the Western world (Parkin et al., 1999). Bladder cancer accounts for approximately 4% of all new cancer cases in Finland, being the 3rd most common malignancy in men and 16th in women. The age-adjusted incidence rate has increased from 10 to 19 cases per 100 000 inhabitants during the past 30 years (Finnish Cancer Registry, 2000), and it is still expected to grow. Bladder cancer is a disease of the aged; mean age at presentation in 1985-1994 was 69 years for males and 72 for females (Dickman et al., 1999). 2.2 Risk factors Smoking increases the risk of bladder cancer (Sorahan et al., 1994), and more so in women than in men (Castelao et al., 2001). Occupational and other exposure to aromatic amines is an established risk factor 14 (Boffetta et al., 1997; Steineck et al., 1990) and certain alkylating agents such as cyclophosphamide have also been reported to induce bladder cancer (Durkee & Benson, 1980). Chronic inflammation and regeneration processes caused by schistosomal infection of the bladder induce squamous metaplasia which may lead to development of cancer (Johansson & Cohen, 1997). 2.3 Histology TCC is by far the most common histological type of bladder cancer, accounting for 90-95% of the cases in industrialized countries (Dickman et al., 1999). The remainder are squamous cell carcinomas (3-6%), adenocarcinomas (1-3%), or undifferentiated carcinomas (1%) (Mostofi FK, 1973). Squamous cell carcinomas are frequently associated with schistosomal infection, and in endemic areas for schistosomiasis 75% of bladder cancers are squamous cell carcinomas, 6% adenocarcinomas and the remainder transitional (Johansson & Cohen, 1997). 2.4 Symptoms and signs The most frequently encountered sign of bladder cancer is painless and intermittent hematuria. This can be microscopic, but in 75% of the cases an episode of macrosopic hematuria can be verified (Varkarakis et al., 1974). Lower abdominal pain, frequency, urgency and dysuria occur in approximately one third of the patients with invasive disease (Utz et al., 1980). 2.5 Diagnostic procedures Urinary cytology is the primary tool for detection and follow up of bladder cancer (Lewis et al., 1976; Papanicolaou & Marshall, 1945). It is sensitive and specific in high-grade or invasive tumors, but less so in superficial and low-grade cancers (Esposti et al., 1978; Rubben et al., 1979; Review of the Literature Badalament et al., 1987b; Hudson & Herr, 1995; Koss et al., 1985). Cystoscopy is the gold standard for detecting bladder cancer. By diagnostic cystoscopy the extent and nature of the tumor can be evaluated in addition to the state of the urethral and bladder mucosae. Accurate staging requires biopsies including the muscular layer of the bladder wall (See & Fuller, 1992). Concomitant upper urinary tract disorders are excluded by intravenous urography (Hatch & Barry, 1986). Larger tumor masses in the bladder can be detected by transabdominal sonography. Possible muscle invasion can be evaluated by intravesical sonography. Computerized tomography and magnetic resonance imaging are used as supplements to clinical staging and evaluating metastatic spread of muscle-invasive tumors (Barentsz et al., 1996; See & Fuller, 1992). easier (Table 2). The tumors are graded into three grades according to the World Health Organization classification (Mostofi, 1973). More recently, new grading systems for papillary carcinomas have been suggested, in which the previous grades two and three are classified low and high grade, respectively. The previous grade 1 carcinoma is classified as a papillary tumor of low malignant potential (Table 3) (Epstein et al., 1998). Carcinoma in situ (Tis) is a distinct category of flat intraepithelial malignant proliferation (UICC, 1978). This carcinoma is an aggressive and potentially invasive high grade tumor (Wolf & Hojgaard, 1983). 2.7 Treatment and prognosis Superficial tumors (Ta and Tis) are treated by transurethral resection alone or in combination with intravesical chemo- or immunotherapy (Soloway, 1983; Soloway & Perito, 1992). Invasive and highly recurrent tumors are managed by cystectomy together with adjuvant treatments such as systemic chemotherapy and radiotherapy (Soloway, 1990). After initial therapy, all patients enter a follow up scheme consisting of urinary cytology and cystoscopy every 3-4 months during the first two years, and later 2.6 Staging and grading Stage classification of bladder cancer is performed according to the TNM classification of bladder cancer (Sobin & Wittekind, 1997). However, a previous classification of 1978 (UICC, 1978) is used in many studies to make comparison with earlier reports Table 2. TNM stage classification of bladder cancer. 3rd edition (UICC, 1978) TX T0 Tis Ta T1 T2 T3a T3b Primary tumor can not be assessed No evidence of primary tumor Carcinoma in situ (flat tumor) Papillary non-invasive carcinoma Tumor infiltrating the lamina propria Tumor infiltrating the superficial muscle Tumor infiltrating the deep muscle Tumor infiltrating through the bladder wall into the perivesical fat T4a Carcinoma involving the prostate, uterus or vagina T4b Carcinoma invading the abdominal or pelvic wall N Lymph node involvement (N0=without, N1-4=with) M Distant metastasis (M0=without, M1=with) 5th edition (Sobin & Wittekind, 1997) Tis Ta T1 T2 T2a T2b T3 T3a T3b T4 T4a Carcinoma in situ (flat tumor) Papillary non-invasive carcinoma Tumor infiltrating the lamina propria Tumor infiltrating the muscle Tumor infiltrating the superficial muscle Tumor infiltrating the deep muscle Tumor infiltrating perivesical tissues Microscopically Macroscopically Tumor infiltrating perivesical organs Carcinoma involving the prostate, uterus or vagina T4b Carcinoma invading the abdominal or pelvic wall N Lymph node involvement (N0=without, N1-3=with) M Distant metastasis (M0=without, M1=with) 15 Kristina Hotakainen Table 3. Histologic grading of papillary urothelial carcinoma. WHO 19731 WHO/ISUP 1998 2 Papilloma Grade 1 Grade 2 Grade 3 Papilloma LMP Low grade High grade Current proposal3 Papilloma Grade 1 Grade 2 Grade 3 1 Mostofi FK, 1973; 2Epstein et al., 1998; 3Cheng & Bostwick, 2000. every 6-12 months (Nurmi & Rintala, 2002). Most bladder cancers are superficial at diagnosis, but approximately 75% recur, and progression to more advanced stage occurs in up to 40% by ten years (Heney et al., 1983; Herr, 1997; Herr et al., 1995; Pagano et al., 1987). In Tis the long-term risk of progression to invasiveness or metastases is even higher, 60-70% (Cookson et al., 1997; Herr, 2000). High grade and tumor multiplicity are associated with a higher recurrence rate (Heney et al., 1983). However, 27% of patients with papillary tumors of “low malignant potential” will experience tumor recurrence and progression of grade occurs in 75% (Cheng et al., 1999). Stage is the most accurate prognostic indicator; the 5-year survival rate declines with increasing stage, being only 021% in stage T4 and as high as 80-94% in stage Ta (Malmström et al., 1987; Torti & Lum, 1984). Age is also consistently associated with survival, with a more favourable outlook in the younger age groups (Dickman et al., 1999). 2.8 Markers of bladder cancer The high recurrence rate and the risk of progression necessitates a close surveillance of bladder cancer. Furthermore, a significant number of recurrences occur more than five years after primary diagnosis, which justifies a lifelong follow up (Cheng et al., 1999; Cookson et al., 1997; Leblanc et al., 1999). An optimal marker would facilitate noninvasive diagnosis and follow up of blad16 der carcinoma. The test should be economic, well standardized and reproducible, easy to perform and most importantly, sensitive and specific. Furthermore, the ability to estimate stage and grade and to characterize the malignant potential and prognosis would be valuable. Urinary cytology is the reference standard to which potential new tests are compared. The sensitivity of cytology ranges from 70-100% in Tis and other high-grade carcinomas, with a specificity of more than 95% (Badalament et al., 1987a; Bastacky et al., 1999). However, in low-grade superficial tumors the sensitivity is at best no more than 40%. Urinary calculi, infection, instillation therapies and other treatments may cause cellular atypia leading to falsely positive cytology, but specificity is still better than 80% regardless of stage and grade (Murphy et al., 1984). A multitude of biomarkers for bladder cancer is being studied. These can roughly be classified into two categories; markers for screening and diagnosis of bladder cancer (Table 4) and potentially prognostic markers (Table 5). Some tests are being clinically used as complements to urinary cytology and cystoscopy and others are under clinical evaluation. The best new markers give higher sensitivity than urinary cytology, but specificity is generally lower (Brown, 2000; Burchardt et al., 2000). However, the cytologic methods, to which new tests are being compared, are not well standardized. This makes evaluation of their true performance difficult, and up to date no noninvasive test is sensitive and specific enough to replace cytology and cystoscopy. However, the intervals between follow up cystoscopies can be increased and the detection of relapse can be improved by using the new markers. 2.8.1 Markers for screening and diagnosis Assays for nuclear matrix protein 22 Review of the Literature (NMP22) and human complement factor H-related protein (BTA stat and TRAK tests) are the most widely used new markers for diagnosis and follow up of bladder cancer. NMP22 is a urothelial cancer associated nuclear matrix protein (Soloway et al., 1996). High NMP22 concentrations in urine are associated with active disease (Carpinito et al., 1996). The sensitivity is at least twice that of cytology (Sozen et al., 1999), but specifity (61-85%) (Serretta et al., 1998; Zippe et al., 1999) is compromized by benign urologic disorders (Miyanaga et al., 1997) and therapies (Öge et al., 2000). This limitation also applies to the various BTA-tests (Pode et al., 1999; Sanchez-Carbayo et al., 1999). Furthermore, elevated NMP22 concentrations in urine have also been observed in patients with renal cell carcinoma (Huang et al., 2000). Previous (Bacillus Calmette-Guérin, BCG) and current (any type) instillation treatments lower the specificity of the BTA stat significantly limiting its use in the follow up of bladder cancer (Raitanen et al., 2001). Part of the telomeres in somatic cells are degraded with every replication round (Harley et al., 1990). Telomerase is a ribonucleoprotein enzyme with reverse transcriptase activity, which maintains the telomeres in replicating germ cells thus making repeated rounds of replication possible without loss of parent DNA sequences (Blackburn, 1991). Telomerase activity is low or absent in normal cells but it is often detected in voided urine of bladder cancer patients regardless of grade (Kavaler et al., 1998). Telomerase tests have been reported to outperform both NMP22 and BTA assays as well as conventional cytology both in sensitivity and specificity (Ramakumar et al., 1999). However, the method is costly and laborious, making it less feasible for routine clinical use (Ross & Cohen, 2000). Increased urinary levels of fibrinogen degradation products (FDP) occur in patients with bladder cancer, and these can be detected by an immunoassay. Initially, Table 4. Markers for screening and diagnosis of bladder cancer. BTA (BTA TRAK; BTAstat) NMP22 FDP Telomerase Cell surface antigens (M344, 19A211, LDQ10) Hyaluronic acid/ hyaluronidase Cytokeratins the method was assigned a sensitivity of of 68-100% and a specificity of 75-96% (Johnston et al., 1997; Schmetter et al., 1997) in detecting bladder neoplasia, but later a sensitivity of merely 48% was reported (Ramakumar et al., 1999). 2.8.2 Prognostic markers An increasing list of protein and genetic alterations is under research as prognostic markers of bladder cancer (Table 5). Of these, assays for microsatellite instability, VEGF and some tumor-associated antigens can be performed on urinary cytology or bladder washing specimens. This also applies to flow cytometry (FCM), by which DNA-ploidy and S-phase fraction (SPF, fraction of cells in the population being in the DNA-synthesis phase) are measured. Aneuploid cell populations and those with a high SPF are typical of high grade and stage tumors (Klein et al., 1982; Koss et al., 1989; Schapers et al., 1993). The reported sensitivity of DNA-ploidy analyzed by FCM for detection of urothelial neoplasia ranges from 34% to 88% (Badalament et al., 1987a; Gregoire et al., 1997; Murphy et al., 1986) with a specificity exceeding 80% (Bakhos et al., 2000; Gregoire et al., 1997). However, the number of cells needed can rarely be obtained from voided urine, but a bladder washing sample is required. The test is compromised by large amounts of non-malignant cells in the sample. In addition, only gross genetic alterations leading to changes in DNA-ploidy can be detected by FCM. Thus, diploid tumors and those with bal17 Kristina Hotakainen anced translocations pass undetected, that is most of grade 1, stage 1 tumors and up to half of grade 2 lesions (Bucci et al., 1995; Kawamura et al., 2000; Liedl, 1995; Tribukait & Esposti, 1978). Furthermore, despite a higher overall sensitivity than for cytology (Badalament et al., 1987c; Giella et al., 1992; Song et al., 1995) FCM fails to detect most in situ carcinomas which are detected by urinary cytology. In summary, analyses for DNA-ploidy can be useful in differentiating benign atypia associated with instillation therapies from recurrent neoplasia (Bakhos et al., 2000), but the prognostic utility is limited (Bittard et al., 1996; Tetu et al., 1996) and the method is too insensitive for screening (Gourlay et al., 1995). Combined with conventional cytology, DNA-ploidy measured by static image analysis (IA) can detect up to 85% of recurrent tumors (Cajulis et al., 1995; de la Roza et al., 1996; Mora, L.B. et al., 1996; Richman et al., 1998). Laser scanning cytometry (LSCM) combines the advantages of FCM and image analysis and measures the DNA-content in individual cells. Small cell populations with aberrant DNA-content can be detected by these newer methods, and thus they may also be used for voided urine (Parry & Hemstreet, 1988; Wojcik et al., 1998). Aberrant cellular DNA-content can also be visualized by fluorescence in situ hybridization (FISH). This method has been reported to be capable of differentiating between Ta and T1 tumors and also of predicting response to immunotherapy (Pycha et al., 1997; Sauter et al., 1997). However, the sensitivity is highly dependent on the number of centromeric probes used, and due to the heterogenous nature of bladder cancer multiple probes are needed (Sauter et al., 1997). The possibility to also detect superficial tumors and to obtain prognostic information makes the method promising for the follow up of bladder cancer patients. Microsatellite analyses can detect tumor-associated alterations in 18 Review of the literature repetitive DNA sequences of the human genome (microsatellites). In bladder cancer, microsatellite instability and loss of heterozygosity is frequently observed in characteristic chromosomes. The analysis can be performed on voided urine with a high sensitivity (74-95%) in detecting recurrent tumors (Mao et al., 1996; Steiner et al., 1997; van Rhijn et al., 2001) even months before these are detected by cystoscopy. In addition, the test can also be used on frozen urine samples (Linn et al., 1997). However, microsatellite instability and loss of heterozygosity in urine are frequently found in benign urological disorders such as benign prostatic hyperplasia and cystitis also (Christensen et al., 2000). Thus the high specificity initially reported (Mao et al., 1996; Mourah et al., 1998; Steiner et al., 1997; van Rhijn et al., 2001) may be overestimated. The expression of many growth factors is altered in bladder cancers (Table 5) and the concentrations of these can be determined in urine. Determination of VEGF has been used to differentiate superficial (Ta) and low grade (G1) tumors from invasive (T1 or more) and high grade tumors (G23). High urinary concentrations of VEGF predict recurrence (Crew et al., 1997). Several monoclonal antibodies are being studied as tools for detection of bladder cancer. These identify antigens that are mostly absent in normal bladder epithelium. Some differentiation between various grades and stages as well as predicition of tumor recurrence can be achieved (Allard et al., 1995; Huland et al., 1987). Combinations of antibodies can be used as immunocytochemical tests on voided urine (Mian et al., 1999), and improve sensitivity and specificity when used together with cytology. P53 overexpression has been detected in bladder cancer, and this is associated with poor prognosis (Cordon-Cardo & Reuter, 1997; Sarkis et al., 1993; Tsuji et al., 1997). However, distinct mutations of p53 occur in bladder epithelium in smokers Review of the Literature Review of the literature Table 5. Potentially prognostic markers of bladder cancer. Microsatellite instability assays* DNA-ploidy/SPF *(FCM, IA, LSCM, FISH) Blood group-related antigens (ABO, Lewis-ag) Growth factors (EGF*, TGFβ, FGF, VEGF*) Cellular adhesion molecules (cadherins, integrins) Proliferation antigens (Ki-67, PCNA) Tumor-associated antigens* (486 P3/12, M344, 19A211, T138, DD23), Cell cycle regulatory proteins (p53, pRb, cyclins, p15, p16, p21) Oncogens (c-erb-B2, ras, c-myc, c-jun, mdm2) Components of fibrinolysis system (urokinase type I plasmin activator) * detection on urinary sediment or bladder wash specimens (Husgafvel-Pursiainen & Kannio, 1996), and p53 overexpression is related to the number of cigarettes smoked per day (Zhang et al., 1994). 3. RENAL CELL CARCINOMA 3.1 Epidemiology RCC or hypernephroma is the most common malignant tumor of the kidney accounting for more than 3% of the annual new cancer cases in Finland. It is the 7th most common cancer among men in Finland, and the 13th among women. The ageadjusted incidence rate has nearly doubled in the past 30 years (Dickman et al., 1999). 3.2 Risk factors Smoking doubles the risk for RCC in both men and women (McCredie & Stewart, 1992; McLaughlin et al., 1990). Obesity is another reported risk, and hypertension or medication for it have also been suspected (Chow et al., 1996; Messerli & Grossman, 1999). Certain dietary factors may play a role, but alcohol and coffee consumption do not (McLaughlin & Lipworth, 2000; Mellemgaard et al., 1996; Wolk et al., 1996). Occupational and other exposure to several carcinogens are suspected risk factors (Boffetta et al., 1997). Genetic predisposition to RCC has been suggested to be dependent on variations in metabolic pathways (Longuemaux et al., 1999). Nacetyltransferase 2 participates in the metabolism of drugs and carcinogens, of which arylamines are found in tobacco smoke (Hein et al., 1993). The so called slowacetylator genotype increases the risk associated with smoking. Toxification of nephrocarcinogenic chlorinated hydrocarbons proceeds by conjugation with glutathione. This is mediated by glutathione transferase, the activity of which may be altered in RCCs to promote carcinogenesis (Bruning & Bolt, 2000; Delbanco et al., 2001; Grignon et al., 1994). The cytochrome P450 system may similarly be involved in carcinogenesis (Murray et al., 1999). Familial RCC accounts for less than 5% of the cases, and most of these are associated with the von Hippel-Lindau syndrome. This autosomal dominant disorder is caused by errors in a tumor suppressor gene on chromosome 3p (Latif et al., 1993). In addition to several other tumors, approximately 70% of the patients develop RCC (Friedrich, 1999). 3.3 Histology RCCs are adenocarcinomas arising in the parenchymal epithelium. They are subclassified into five distinct types. Conventional (CRCC) and papillary renal cell carcinomas account for approximately 85-90% of RCCs, while the chromophobe and unclassified tumors account for up to 5% each. The remainder are collecting duct tumors. Transitional cell tumors may arise in the transitional epithelium of the renal pelvis; these account for approximately 5% of all kidney cancers. Oncocytoma is a benign renal tumor (Kovacs et al., 1997). 19 Kristina Hotakainen 3.4 Symptoms and signs The symptoms caused by RCC can be either systemic or local. The classic triad of local symptoms is hematuria, flank pain and a palpable tumor mass. A rapidly developing varicocele may be caused by a tumor invading the renal vein (left) or the inferior vena cava. The hematuria can cause obstruction of the ureter leading to painful attacks. Fatigue, fever and loss of weigth may occur (Nurmi & Rintala, 2002; Nurmi et al., 1985). 3.5 Diagnostic procedures Renal tumors are investigated by ultrasonography (US), urography, computerized tomography or magnetic resonance imaging, angiography and needle biopsies. The differentiation between renal cysts and tumors is mostly possible by US. Very small tumors can be detected by US and guided needle biopsies can be obtained. Computerized tomography is an aid in evaluating the extent of invasion into perirenal tissues and veins. Angiography is useful in localising and quantitating arteries in the planning of surgery. Bone scintigraphy and chest radiography are used to detect bone metastases (Hilton, 2000). 3.6 Staging and grading Staging of RCC is performed according to the TNM classification (Table 6) (Sobin & Wittekind, 1997) and nuclear grading according to Skinner and coworkers (Skinner et al., 1971). 3.7 Treatment and prognosis Curative treatment of RCC is usually possible only by surgical removal of the whole tumor. This is usually achieved by radical nephrectomy, but in selected cases nephronsparing surgery is an option. Response to hormonal and cytotoxic therapies is ob20 Table 6. TNM classification of renal cell carcinoma (Sobin & Wittekind, 1997). Tx T0 T1 Primary tumor can not be assessed No evidence of primary tumor Tumor 7 cm or less in greatest dimension, limited to the kidney T2 Tumor more than 7 cm in greatest dimension, limited to the kidney T3 Tumor extends into major veins, perinephric tissues or the adrenal gland, but not beyond Gerota fascia T3a Tumor infiltrates adrenal gland or perinephric tissues, but not beyond Gerota fascia T3b Tumor grossly invades the renal vein(s) or vena cava below the diaphragm T3c Tumor grossly extends into the vena cava above the diaphragm T4 Tumor invades beyond Gerota fascia N Lymph node involvement (N0=without, N1-2=with) M Distant metastasis (M0=without, M1=with) tained in no more than 10% of the patients (Motzer et al., 1997). Five to 20% of the patients respond to immunotherapy based on interferon-alpha or interleukin-2 (Malaguarnera et al., 2001; Minasian et al., 1993; Motzer et al., 2000; Nanus, 2000; Vogelzang et al., 1993). In approximately one third of the patients distant metastases are present at diagnosis (Dekernion et al., 1978). A solitary metastasis can be removed together with the primary tumor, and in cases with multiple metastases removal of the primary tumor may promote regression of the metastases (Golimbu et al., 1986a; Ljungberg et al., 2000; Wyczolkowski et al., 2001). Stage and nuclear grade are the most important prognostic factors (Fuhrman et al., 1982; Medeiros et al., 1988). The 5year survival rate in all stages is 50-60%, but patients with metastatic disease at first diagnosis survive only for approximately 12 months. Survival is consistently associated with age being lowest in the highest age groups (Dickman et al., 1999; Minasian et al., 1993; Nurmi, 1984). A third of initially nonmetastatic tumors recur after surgery, mostly incurably (Ljungberg et al., Review of the Literature 1999; Levy et al., 1998). Relapses can occur even decades after primary diagnosis and the 10-year relative survival rate is approximately 45% (Dickman et al., 1999). With the increasing use of imaging techniques such as US and computerized tomography the proportion of incidentally detected renal tumors has been growing (Konnak & Grossman, 1985; Smith et al., 1989). These tumors are smaller and of lower grade and stage. Thus, the 5-year survival rates for patients with such tumors are significantly higher (62-97%) than for those with symptomatic ones (42-83%)(Herr, 1994; Licht et al., 1994; Tsui et al., 2000). 3.8 Markers of RCC RCCs are a heterogenous group of tumors with a distinct genetic background and biology associated with the histological type (Takahashi et al., 2001; Young et al., 2001). The clinical outcome in the individual RCC patient is highly variable even within a given stage (Golimbu et al., 1986b; Kovacs et al., 1997). Several potential RCC markers have been described, but most of these can be studied only in surgically removed tissues (Table 7). No specific serum markers are available. Markers that could be used before surgery would be of value in planning of treatment and follow up. 3.8.1 Tissue markers Several genetic aberrations have been de- tected in RCC by restriction fragment length polymorphism analysis, comparative genomic hybridization, cDNA microarray screening, in situ hybridization and microsatellite analysis. A frequently involved region is located on chromosome 3p (Cohen et al., 1979), which also comprises the von Hippel Lindau tumor suppressor gene (Latif et al., 1993). Genetic losses of 3p occur commonly together with other alterations (Velickovic et al., 2001; Yamakawa et al., 1991). Some of these are associated with tumor histology (Velickovic et al., 2001), grade, stage (Morita et al., 1991; Wada et al., 1998) or recurrence (Moch et al., 1996; Thrash-Bingham et al., 1995). Multiple genes are up- or down regulated in RCC, and a distinction between histologic subtypes and clinical outcome is possible based on the expression profiles (Takahashi et al., 2001; Young et al., 2001). P53 mutations are fairly infrequent occurring in 2-33% of RCCs (Gelb et al., 1997; Ljungberg et al., 2001; Reiter et al., 1993; Vasavada et al., 1998). However, p53 expression has been found to have a prognostic significance (Girgin et al., 2001) in some papillary and chromophobe tumors, but not in CRCCs (Gelb et al., 1997; Kamel et al., 1994; Ljungberg et al., 2001). DNA-ploidy has been studied as a prognostic indicator in RCC, but its value is limited (Ljungberg et al., 1996; Shalev et al., 2001; Tannapfel et al., 1996). Vimentin expression can be detected in some RCCs (Beham et al., 1992; Dierick et al., 1991), and it may give addi- Table 7. Potential markers of RCC. Detected in Serum Tissue VEGF IL-10 CA-125 TATI NSE TNFβ Ferritin CRP Basic FGF Erythropoietin Ki-67 PCNA AgNOR P53 Growth factors (basic FGF) Adhesion molecules (CD44, cadherins, catenins) Cyclins (A) Vimentin Matrix metalloproteinases (2 and 9) Tissue inhibitors of MMPs (1 and 2) 21 Kristina Hotakainen tional prognostic information especially when analyzed together with tumor grade (Nativ et al., 1997; Sabo et al., 1997). Markers of cell proliferation such as nucleolar organizer regions analysis (AgNOR), Ki-67 index and proliferating cell nuclear antigen (PCNA) expression are promising as prognostic markers (de Riese et al., 1993; Delahunt et al., 1995; Hofmockel et al., 1995; Tannapfel et al., 1996). 3.8.2 Serum markers Of the many serum markers studied some are of potential prognostic value; neuron specific enolase (NSE), VEGF, interleukin10, CA-125, and tumor-associated trypsin inhibitor (TATI) (Yaman et al., 1996; Wittke et al., 1999; Grankvist et al., 1997; Jacobsen et al., 2000; Paju et al., 2001). Some of the general glycoprotein cancer markers are detected in serum of RCC patients. CA 125 in serum is an independent prognostic marker and CA 15-3 may also be of some value; both markers are associ- 22 ated with tumor stage and grade (Grankvist et al., 1997). Anemia or polycythaemia may be encountered in RCC, suggesting involvement of the erythropoietin processes. Elevated serum concentrations of erythropoietin have been described but although this is prognostically significant the sensitivity of this marker is too low for clinical use (Ljungberg et al., 1992). Ferritin appears to be produced by certain RCCs and can in some cases be used for monitoring of the disease (Essen et al., 1991; Kirkali et al., 1995; Özen et al., 1995). Acute phase reactants such as C-reactive protein (CRP) and inflammatory markers such as erythrocyte sedimentation rate may also be useful (Ljungberg et al., 2000; Nurmi et al., 1985). Several other general serum analytes such as calcium, lactate dehydrogenase and hemoglobin in combination with clinical features and other markers have been used in prognostic models (Motzer et al., 1999). However, none of these are specific for cancer in general and even less for renal cancer. Aims of the study The aims of the study were: 1. To study whether peripheral blood cells express hCGß and how the potential expression is regulated. Expression of hCGß mRNA in blood cells would limit the utility of hCGß mRNA as an indicator of tumor cells in circulation (I). 2. To study whether hCGß mRNA expressing cells can be detected in the cells of voided urine from bladder cancer patients (II). 3. To compare detection of hCGß mRNA in urinary cells with serum and urinary concentrations of hCGß and stage and grade of the disease, and with immunohistochemical detection of hCGß in tumor tissue (III). 4. To study whether the preoperative serum concentrations of hCGß are of prognostic significance in patients with RCC (IV). 23 Kristina Hotakainen Material and methods 1. SUBJECTS AND SAMPLES (I-IV) All studies were approved by the ethics committees of Helsinki University Central Hospital (HUCS) and the Finnish Red Cross (FRC), Helsinki, Finland, and the University Hospital in Umeå, Sweden. Patients as well as controls were recruited after informed consent. Whole blood buffy coats from healthy blood donors were obtained from the Blood Service of the FRC (I). Samples of venous peripheral blood (I) (Tables 8 and 9) control sera (III, IV) as well as urine (II, III) (Table 10) were collected from healthy laboratory personnel from HUCS. Placental tissue (I, II, III) was obtained after normal delivery from the Department of Obstetrics and Gynecology, HUCS. Urine and serum specimens of bladder cancer patients and control patients (II, III) were obtained from the Departments of Urology and Surgery, HUCS. All cases studied for this thesis were TCCs. TCC tissue specimens as well as samples of benign bladder tissue were obtained from the Department of Pathology, HUCS (Table 10). Staging of the tumors was performed according to the TNM classification of bladder cancer (UICC, 1978) and grading according to the WHO classification of bladder tumors (Mostofi FK, 1973) (II). In study III the more recent consensus classification of urothelial neoplasms was also used (WHO/ISUP 1998). The histological diagnosis was based on samples obtained within two weeks of urine sampling. Cytology of voided urine was classified according to Papanicolaou (Papanicolaou & Marshall, 1945). Sera of patients 24 with renal cell carcinoma (IV) were obtained from the serum bank of the Department of Urology and Andrology, University Hospital of Umeå (Table 10). Staging of RCC was performed according to the 1997 TNM classification (Sobin & Wittekind, 1997), nuclear grading according to Skinner and coworkers (Skinner et al., 1971), and DNAploidy according to Ljungberg and coworkers (Ljungberg et al., 1996). 2. PREPARATIONS AND CULTURES OF CELLS (I-III) Blood samples from five apparently healthy nonpregnant females and six males (Table 8) were drawn into 3 ml Vacutainer EDTA tubes (Becton Dickinson, Rutherford, NJ) (I). Erythrocytes in peripheral blood were hemolysed by incubating blood with 1.5 vol of diethylpyrocarbonate-treated water for 5 min. Mononuclear cells were isolated by Ficoll-Paque centrifugation according to the manufacturer’s instructions (Pharmacia, Uppsala, Sweden) and total RNA was isolated (Chomczynski & Sacchi, 1987). Urine samples (II, III) were stored at 4° C for a maximum of six h before processTable 8. Peripheral blood cell preparations (I). Leukocyte extracts Mononuclear cells B-cells T-cells Granulocytes Monocytes 1 Mononuclear cells 1 Lymphocytes 2 Peripheral blood (11) 3 Buffy coats Buffy coats Buffy coats Buffy coats from peripheral venous blood of a healthy male; 2 from buffy coats; five females and six males of healthy laboratory personnel. All buffy coats were obtained from the Blood Service of the FRC. 3 Table 9. Cultured peripheral blood lymphocytes and cell lines.All cell lines were obtained from the ATCC and buffy coats from the FRC. Cell type/ line Description Lymphocytes HL-60 Jurkat K-562 U-937 JAR UM-UC-3 TCC-SUP Used in Buffy coats Acute promyelocytic leukemia Acute T-cell leukemia Chronic myelogenous leukemia Histiocytic lymphoma Choriocarcinoma TCC cell lines TCC cell lines I I I I I I II II ing. The urinary cells were collected by centrifugation and washed once with phosphate buffered saline (PBS, 50 mM sodium phosphate, 150 mM NaCl, pH 7.4; HyClone Europe Ltd, Cramlington, UK). Total RNA was then isolated from the cell pellet. Peripheral blood lymphocytes from whole blood buffy coats were purified on a Ficoll-Paque gradient (I). 1 x 106 cells/ml were cultured in 15 ml Dulbecco’s Modified Eagle’s Medium (DMEM) (HyClone) supplemented with 5% fetal calf serum (FCS, Biological Industries, Kibbutz Beit Haemek, Israel), 100 IU/l penicillin, 100 mg/l streptomycin and 2 mM L-glutamine (GibcoBRL, Paisley, Scotland). The cells were stimulated with phytohemagglutinin (PHA) 10 mg/ml, concanavalin A (Con A) 2,5 mg/ml, pokeweed mitogen (PWM) at a final dilution of 1:100, and prolactin at different concentrations (14, 28, 70, 100, and 140 ng/ml) (all from Sigma Chemical Co., St. Louis, MO). Control cultures were performed without mitogens. Two-way mixed lymphocyte cultures (MLC, I) were performed in 25 cm2 tissue culture flasks with 5 x 105 lymphocytes/ml. The cultures were incubated at 37° C in a humified atmosphere of 8% CO2 in air. The cell number and the proliferative response to each stimulus was measured by counting the proportion of lymphoblasts in the culture by microscopy. All cell lines (Table 9) were obtained from Material and Methods the American Type Culture Collection (ATCC) and cultured according to instructions in RPMI 1640 medium (HyClone Europe Ltd, Cramlington, UK) supplemented with 10% FCS (Flow Laboratories, UK), 2 mM glutamine (GibcoBRL), 100 IU/l penicillin, 100 mg/l streptomycin (HyClone) and 2.5 mg/l amphotericin B (GibcoBRL). 3. LEUKOCYTE EXTRACTS (I) Eight ml of peripheral blood was taken in a Vacutainer Cell Preparation Tube (Becton Dickinson, Franklin Lakes, NJ) and mononuclear cells were isolated according to the manufacturer’s instructions (Table 8). Lymphocytes from whole blood buffy coats were purified by Ficoll-Paque centrifugation. The cells were washed twice with PBS and 108 cells were lysed in 100 µl of RIPA buffer containing 10 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulpahte, 1 mmol/l phenylmethanesulfonyl fluoride, 1 mM sodium vanadate, 10 mg/ml aprotinin (Sigma). The cell lysate was centrifuged for 15 min at 10 000 x g and the supernatant was stored at -20° C until analyzed. 4. SEPARATION OF MONONUCLEAR CELLS, GRANULOCYTES, MONOCYTES, B- AND T-CELLS (I) Mononuclear cells and granulocytes were isolated from buffy coats by density gradient centrifugation. Four ml of blood was mixed with 6 ml of PBS, layered over 10 ml Histopaque 1077 (Sigma) and spun at 600 x g for 30 min. The mononuclear cell layer was isolated and washed twice in PBS. Granulocytes were separated by a triple discontinuous gradient (Bhat et al., 1993) by layering 2 ml each of Histopaque 1119 at the bottom, Histopaque 1107 (two parts Histopaque 1119 and one part Histopaque 1083) in the middle and Histopaque 1077 on top. Two ml blood was diluted with 4 25 Kristina Hotakainen ml PBS and layered on top of the gradient. The tubes were centrifuged for 30 min at 600 x g. The granulocyte cell layer was then aspirated and washed twice with PBS. Remaining erythrocytes were hemolysed by incubating the cell pellet with 4 ml distilled water for 10 s, after which 1 ml 5 x PBS was added. The granulocytes were collected by centrifugation, lysed in 4 M guanidinium-thiocyanate buffer containing 0.1 M ß-mercaptoethanol and stored at -20° C. Magnetic cell sorting (MACS) was used to separate monocytes, B- and T-cells. Cells from the mononuclear cell layer (7,6 x 107) were resuspended at a concentration of 107 cells per 80 µl buffer (PBS, 2 mM EDTA, 0,5% BSA), and incubated with mouse monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec, Sunnyvale, CA) to human CD3, CD14 or CD19 (20 µl per 107 cells). CD3 antibodies were used for separation of T-lymphocytes, CD14 for monocytes and CD19 for B-lymphocytes. The labelled cells were washed twice in the buffer and immediately processed with a VarioMACS using an RS+ column (Miltenyi Biotec). The column was prewashed with the same buffer. Positive selection was done according to the manufacturer’s instruction. Cells from the positive fraction were eluted and washed in the buffer. The purity of the monocyte and Bcell fractions was determined on the basis of morphology and expression of MHC II antigens (Schröder et al., 1982). Table 10. Samples of patients and controls. The number of patients is given in parentheses when different from the number of samples used. Serum samples TCC patients (68) TCC patients (63) RCC patients Control patients with benign diseases Healthy controls Healthy controls n Used in 45 66 177 14 84 26 II III IV III IV III Tissue samples; source Placental tissue; normal delivery TCC tissue; TCC patients (66) Benign bladder Patients with hematuria dysuria pollacisuria recurrent lower UTI 1 I, II, III 96 III 6 4 5 6 III III III III 56 72 26 14 II III III III 68 84 14 15 8 3 1 1 1 1 1 1 1 1 1 1 3 II III II III II, III II III II, III II II II, III II, III II, III II, III II II, III II, III Urine samples Urine TCC patients (68) TCC patients (63) Healthy controls Control patients with benign diseases Urinary cells TCC patients (68) TCC patients (63) Healthy controls Healthy controls Patients with acute appendicitis urinary calculi Patient with urinary calculi BPH impotence epididymitis inflammatory pseudotumor chronic pyelonephritis cystitis hematuria cervical carcinoma renal carcinoma Patients with prostate carcinoma 5. ISOLATION OF RNA (I-III) 6. REMOVAL OF GENOMIC DNA (IIII) RNA was isolated from cells pelleted by centrifugation of urine (II, III) as well as from mononuclear blood cells and leukocyte subsets (monocytes, B- and T-lymphocytes, granulocytes) and cultured cells (I) according to Chomczynski and Sacchi (Chomczynski & Sacchi, 1987) with Heavy Phase Lock Gel tubes (5 Prime->3 Prime, Inc., Boulder, CO, USA). To diminish the content of genomic DNA in the RNA preparations each sample was subjected to DNase-treatment before RTPCR. Ten µg of total RNA was incubated at 37° C for 30 min in a 50-µl reaction mixture consisting of 1 x DNase buffer (40 mM Tris-HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl2, 10 mM CaCl2), 33 U of RNasin (Promega, Madison, WI) and 2 U of RQ1 26 Material and Methods A 1 3 E2 hCGβ mRNA E3 B LHβ mRNA E3 E2 2 4 1+2 3+4 Outer sense Outer antisense Nested sense Nested antisense 404 bp 292 bp 3 1 E1 Primer 1 2 3 4 2 4 1+2 3+4 Sequence CTG TTG CTG CTG CTG AG GCC TTT GAG GAA GAG GAG ACC CTG GCT GTG GAG AAG G TCA TCA CAG GTC AAG GGG TG 401 bp 292 bp Nucleotides 50-66 436-453 128-146 401-420 Figure 1. Schematic representation of the locations of the primers used for hCGβ (a). The primers are specific to the sequence of hCGβ mRNA, but they are also able to amplify LHβ mRNA (b). Primer 1 is homologous to both hCGβ- and LHβ mRNA, and the dashed primers (2, 3, and 4) contain mismatches with respect to LHβ mRNA. E; exon. RNase-free DNase (Promega, Madison, WI). The DNase was denatured for 10 min at 55° C. RNA was precipitated with 140 µl ethanol and 5µl 2 M sodium acetate and dissolved in diethylpyrocarbonate-treated water. 7. OLIGONUCLEOTIDE PRIMERS Oligonucleotide primers used for RT-PCR of hCGß mRNA were chosen from exon 2 of the hCGß5 gene and the antisense primers from exon 3 (Figure 1). 8. RT-PCR AND GEL ELECTROPHORESIS (I-III) For RT, 1 µg of total RNA and 1 pmol of the outer antisense primer were denatured for 10 min at 70° C in a 12-µl volume of diethylpyrocarbonate-treated water. The denatured RNA was incubated in a 20-µl reaction mixture (Lintula & Stenman, 1997) with 200 U SuperscriptTM reverse transcriptase (Gibco BRL) at 42° C for 50 min. The RT enzyme was denaturated for 15 min at 70° C. Contamination of RNA samples was excluded by subjecting each sample to the RT reaction without RT before PCR. In the PCR, 5 µl of the cDNA mixture was amplified in a 50-µl reaction volume in 1x PCR buffer as previously described (Lintula & Stenman, 1997) initially at 95° C for 5 min, 35 cycles at 95° C for 1 min, 50° C for 1 min. One µl of the first PCR product was further amplified with the nested primers in a 50-µl reaction volume for 35 cycles with an annealing temperature of 56° C but otherwise the same conditions as in the first PCR. Fifteen µl of the product was electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining. RNA isolated from placental tissue was used as a positive control for hCGß and water as a negative control in all experiments. The detection limit of the RTPCR was estimated by mixing the cultured cancer cells with PBS or urine from a healthy male to dilutions of 102-106 cells per liter PBS or urine and with blood to give dilutions up to 1 tumor cell per 107 peripheral blood leukocytes. 9. RESTRICTION ENZYME ANALYSIS AND SEQUENCING OF THE PCR PRODUCTS (I-III) The homology between the genes for LHß and hCGß is 94% (Talmadge et al., 1984b). There are three mismatches between the nucleotide sequences of the outer antisense primer used in reverse transcription and the 27 Kristina Hotakainen mRNA sequence of LHß. However, all mismatches are located in the 5'-end of the primer, and amplification of LHß cDNA occurs with these primers (Figure 1). The identity of the PCR products produced by the nested primers was therefore confirmed by Hae III restriction enzyme digestion and sequencing of the product. The 292 bp PCR product of hCGß, but not that of LHß mRNA, has a restriction site for Hae III producing fragments of 108 and 184 bp, respectively. Sequencing of the PCR products was performed using the nested sense primer with the ABI PrismTM Dye Terminator Cycle Sequencing Core Kit With AmpliTaq DNA Polymerase (Perkin Elmer, Foster City, CA). 10. DETERMINATION OF hCG, hCGß, hCGßcf, LH AND LHß (I-IV) Serum (II-IV), urine (II, III) and cell culture medium (I, II) were stored at -20° C before assay of the proteins. HCG (II, III) and hCGß (II-IV) in serum and urine, and hCGßcf in urine (II, III) were determined by ultra sensitive time-resolved immunofluorometric assays (IFMA) (Alfthan et al., 1992a). Cell culture media were collected at days 3, 5 and 7 of culture and concentrated 14-60-fold with Centricon-3 concentrators (Amicon, Beverly, MA). HCG and hCGß as well as LH and LHß in culture media and leukocyte extracts (I) were determined by IFMA. The detection limit of the hCG assay was 0.5 IU/l (WHO 3rd IS, 75/ 537), 0.5 pmol/l for hCGß, and 0.44 pmol/ l for hCGßcf (WHO 1st IRP, 75/551). To correct the results in urine for varying urine concentrations the results were divided by the concentration of urinary creatinine (II, III). The LHß assay (LHspec, Delfia, Wallac) detects LH, LHß, and a fragment of LHß whereas the LH assay detects only intact LH. The detection limit of the LH assay was 0.12 IU/l and that of the LHß assay was 0.05 IU/ l. The assays were calibrated against the 2nd WHO IS coded 80/552. 28 11. IMMUNOHISTOCHEMISTRY(III) HCGß immunoreactivity was analyzed using two monoclonal antibodies (MAbs) to hCGß; clones 9C11 (Alfthan et al., 1988) and 6G5 (Louhimo et al., 2001). MAb 9C11 is specific for hCGß, whereas MAb 6G5 also reacts with intact hCG (Louhimo et al., 2001). Immunoperoxidase staining was performed with the Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA). Four-µm thick, freshly cut sections of formalin-fixed, paraffin-embedded tissue specimens were deparaffinized in xylene and rehydrated by sequential incubation in graded ethanol/water solutions. The deparaffinized tissue sections were pretreated with 0.5% trypsin (DIFCO laboratories, Detroit, MI, USA) for 30 min at 37 °C, and washed with PBS. To quench endogenous peroxidase activity, the sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature and then blocked with normal rabbit serum (dilution 1:67) for 15 min to reduce non-specific binding. The primary antibodies diluted 1:10 000 in PBS containing 0.5% BSA (Sigma) and 0.1% sodium azide (Merck, Darmstadt, Germany) were added to the sections and incubated overnight at room temperature. After rinsing, biotinylated anti-mouse immunoglobulin was added (1:200) for 30 min to the sections followed by peroxidase labelled ABC reagent for 30 min. The peroxidase reaction was developed for 15 min with 3-amino-9ethyl-carbazole in acetate buffer, pH 5, containing 0.03% hydrogen peroxide. The sections were rinsed with water, counterstained with haematoxylin for 35 s and rinsed with water for 10 min. Paraffin sections of placental tissue were used as positive controls. As a negative control a placental section was stained by replacing the primary antibody with normal rabbit or mouse serum. Positive staining was graded 0-3 according to the estimated percentage of positive cells (negative <5%; +5-25%; ++ 25-50%; Material and Methods +++50-100%). Two persons (S. N. and K. H.) evaluated the staining independently. 12. STATISTICAL METHODS (I-IV) Student’s paired t-test, the chi-squared test for trend, Fisher’s exact test for correlation, Kruskal-Wallis test and the MannWhitney U test as well as receiver operat- ing characteristic (ROC) curve analysis were used in statistical analyses. Survival curves were plotted using the KaplanMeier method, and comparison of survival rates was performed with the log-rank test. The independence of hCGß as a predictor of survival was analysed by multivariate analysis using the Cox proportional hazard model. 29 Kristina Hotakainen Results and discussion 1. EXPRESSION OF hCGß- AND LHß mRNA IN PERIPHERAL BLOOD CELLS (I) 1.3 Expression of hCG protein and hCGß mRNA and protein in cultured lymphocytes 1.1 Expression of hCGß- and LHß mRNA in peripheral blood cells and cell lines Culture of lymphocytes alone or with all mitogens except PRL induced hCGß mRNA expression (Table 12). The PCR products were identified with Hae III, which digests the cDNA of hCGß but not that of LHß. However, a weak expression of hCGß may be obscured by an excess of LHß mRNA, potentially explaining why no hCGß mRNA was detected in cultured lymphocytes stimulated with PRL. No hCG protein was detected by IFMA in the control cultures without mitogens. PHA- and ConA stimulated cells secreted hCGß (Figure 3), but intact hCG protein was not detected in any of the cultures. Our results show that normal blood cells express LH and that the expression is stimulated by PRL. HCGß expression is not detectable in unstimulated cells but it is induced by culture and certain mitogens. LHß mRNA is expressed in all the nucleated blood cell fractions studied, and, when stimulated with PRL, leukocytes also produced intact LH protein at readily detectable levels. The protein production occurred LHß mRNA was detected by RT-PCR in the nucleated cells isolated from blood from nonpregnant females and healthy males and also in the isolated B- and T-cell fractions, monocytes and granulocytes. HCGß mRNA was not detected in any of these cell fractions (Table 11). LH, LHß, hCG or hCGß protein were not detected by IFMA in extracts from PBL or whole blood buffy coats. The Jurkat and K562 cell lines expressed mRNA for both LHß and hCGß (Table 12). 1.2 LH- and LHß expression in cultured lymphocytes The effect of mitogens on the expression of mRNA and protein was studied in lymphocyte cultures. LHß mRNA expression was detected by RT-PCR in lymphocytes cultured with and without mitogens (Table 12). PRL induced maximal mitogenic stimulation and LH-production at a concentration of 100 ng/ml (Figure 2). The LH concentrations were similar when measured by the assays detecting only LH or both LH and LHß indicating that the immunoreactivity consisted of intact LH. The other mitogens did not induce significant production of LH protein, and in control cultures without mitogen no LH immunoreactivity was detected. 30 Table 11. LHβ- and hCGβ mRNA detected by RTPCR in separated peripheral blood leukocytes without stimulation. Cell type mRNA expression LHβ B-cell T-cell Granulocyte Monocyte + + + + hCGβ - Results and Discussion Table 12. LHβ- and hCGβ mRNA detected by RT-PCR in cultured lymphocytes and cell lines Days of culture Mitogen PHA ConA PWM PRL MLC No mitogen 5 0 3 LHβ hCGβ + + + + + + - LHβ hCGβ + + + + + + + + + + (+) LHβ hCGβ + + + + + + + + + + + nd nd nd nd nd nd nd nd nd nd 7 LHβ hCGβ + + + + + + + + + + - Cell line nd nd nd nd nd HL-60 Jurkat JAR K562 U937 + (+) - (+) + + + (+)= weak positive, nd= not determined early (after 6 h, data not shown), i.e. before a mitogenic response was observed. This suggests that LH expression was directly induced by PRL and that it did not require a mitogenic effect. In contrast, production of hCGß protein was induced by the mitogens and the concentrations in the culture Concentration (mIU/l per 107 cells) LH , LHβ LH 120 * *** **** *** 100 *** ** 80 60 40 20 0 14 28 70 100 Prolactin (ng/ml) 140 Figure 2. The effect of PRL on the production of LH protein by cultured lymphocytes. LH, as well as LHβ and a fragment of LHβ in the culture medium were determined by IFMA. **** p<0.0001; *** p<0.001; ** p<0.01; * p>0.02 medium increased with time of stimulation. PRL has been regarded predominantly a Tcell mitogen (Clevenger et al., 1990; Pellegrini et al., 1992), but other T-cell specific mitogens such as Con A and PHA did not induce translation of the genes for the LH-subunits. The secretion of LH-protein was highest at a PRL-concentration of 100 ng/ml, which also induced the strongest mitogenic response. This concentration is similar to that in pregnant and lactating women and 5- to 10-fold that in males and nonpregnant females. However, the concentrations of LH in the culture medium are low in comparison with those of hCG occurring in blood during pregnancy. Thus the LH produced can be expected to play a physiological role only at a paracrine or autocrine level. PRL is produced by lymphocytes and it is thought to act as a growth factor for lymphoproliferation in a paracrine or autocrine way by inducing expression of genes associated with lymphocyte activation (Sabharwal et al., 1992a). PRL-receptors have been demonstrated in both B- and Tcells (Pellegrini et al., 1992; Dardenne et al., 1994; Russell et al., 1985) and both cell types respond to PRL-stimulation (Lahat et 31 Kristina Hotakainen 0.3 hCGβ (pmol/l per 107 cells) p=0.05 0.2 0.1 0 3 5 7 control 3 5 7 PHA 3 5 7 MLC 3 5 7 ConA 3 5 7 PWM Days Figure 3. The effect of mitogens on hCGβ production in cultured lymphocytes. The proteins were determined in the culture medium by IFMA at days 3, 5, and 7 of stimulation. In control cultures no mitogen was added. The mean concentrations and S.D. of parallel cultures are shown. al., 1993). Luteinizing hormone-releasing hormone (LHRH) is also thought to participate in lymphocyte activation (Batticane et al., 1991; Azad et al., 1993), and expression of its gene is regulated by PRL in lymphocytes (Wilson et al., 1995). Expression of the hCG/LH receptor gene has been demonstrated in T-lymphocytes from pregnant women (Lin et al., 1995). In line with these results a mitogenic LH-like protein has been observed in thymic extracts, suggesting that this glycoprotein would be secreted by thymocytes and act as an autocrine stimulator of lymphoproliferation (Sabharwal et al., 1992b). 1.4 Conclusions Together with the earlier described expression of the LH-receptor gene in T-lymphocytes (Lin et al., 1995) the expression of LH in leukocytes shown here suggests that LH may exert an autocrine function in blood cells, possibly as a result of an autocrine or paracrine effect of PRL. The expression of LHß mRNA appears to occur in all peripheral blood leukocytes. The concentrations of intact LH and LHß were similar when determined separately indicating that the 32 immunoreactivity detected consisted of intact LH. LH protein production occurred consistently and over a wide range of physiological PRL-concentrations, which supports the notion of a physiological role of this hormone in lymphocytes. The expression of hCGß was induced by mechanisms other than those stimulating LH expression, and only the free ß subunit, but not intact hCG protein, could be reliably detected. Expression of hCGß mRNA was detected only after culture of PBL. This is in accordance with results from other studies showing that culturing and certain other stimuli lead to production of hCG protein by blood cells (Goldstein et al., 1990; HarbourMcMenamin et al., 1986). It has been proposed that mRNA for genes transcribed in ectopic tissues at very low levels can be detected by RT-PCR if an excessive number of amplification rounds are used (Hu & Chow, 2000; de Graaf et al., 1997). We tested our RT-PCR with various numbers of cycles (20-45) and found the optimal result for hCGß mRNA at 35 cycles, whereas LHß mRNA could be detected also by 30 cycles. In addition, we showed translation of these transcripts in leukocytes, demonstrating that the mRNA expression is not only a non-functional background expression. During pregnancy, cultured monocytes have been reported to secrete hCG, and this has been suggested to be related to the immunotolerance associated with early pregnancy (Alexander et al., 1998). Although LH and hCGß have no known function in lymphocytes, the expression of LH appears to be a further example of the involvement of pituitary hormones in immune modulation. Because hCGß mRNA expression was induced by culture it is conceivable that disorders leading to lymphocyte activation may also induce hCGß expression. This may lead to positive results for hCGß mRNA in blood from patients without cancer. This is a potential limitation for the use of hCGß Results and Discussion expression for detection of cancer cells especially in blood and possibly also in urine. 2. EXPRESSION OF hCGß IN BLADDER CANCER (II, III) 2.1 Immunohistochemical expression of hCGß (III) We detected positive staining for hCGß equally often in sections from noninvasive (28%) and invasive tumors (35%), and in histologically benign samples (25%) from TCC patients (Table 13). There was no statistically significant association between tumor stage (p=0.6) or grade (p=0.75) and tissue staining for hCG antigen. Positive staining was observed in five of 26 (19%) cases with previous instillation therapy and in one of the 12 patients currently receiving instillations. The results were identical with both antibodies. Furthermore, 10 of 21 (48%) samples from control tissues from patients with benign conditions stained positive. Two of these samples were from control patients with hematuria and in both cases the epithelium was oedematous and inflamed; squamous metaplasia was observed in one case with dysuria, and in a patient with recurrent lower urinary tract infections (UTI). One of the positively staining samples was an inflammatory pseudotumor from a patient with recurrent UTI (Table 14). Two positive samples from patients with dysuria and pollacisuria were judged to be histologically normal. To our knowledge, immunohistochemical expression of hCGß in benign bladder epithelium has not been reported before, but mRNA expression has been observed in normal and malignant bladder epithelium (Lazar et al., 1995) and benign transitional cells in culture secrete hCGß into the culture medium (Iles et al., 1990b). Five of the ten positively staining control tissues showed signs of inflammation or squamous metaplasia of the epithelium. Thus, it is Table 13. Immunohistochemical expression of hCGβ-antigen in various stages and grades as well as nonmalignant samples from TCC-patients. Stage Tis Ta T1 T2 T3 T4 Not available Histologically benign tissue* n hCGβ-positive % 8 46 12 4 3 1 2 20 1 14 5 0 1 1 1 5 12 30 42 0 33 100 50 25 22 31 23 76 5 11 7 23 23 35 30 Grade 1 2 3 All 30 n= number of patients * Samples of histologically benign bladder epithelium from patients with cystoscopically verified TCC. possible that inflammation and hyperplasia may induce hCGß expression in benign transitional epithelium. Grade and stage of bladder cancer have been found to correlate with immunohistochemical staining of hCGß in most (Bacchi et al., 1993; Campo et al., 1989; Dirnhofer et al., 1998b; Jenkins et al., 1990; Martin et al., 1989; Moutzouris et al., 1993; Oliver et al., 1988; Özkardes et al., 1991), but not all earlier studies (Smith et al., 1994). We detected immunohistochemical expression of hCGß antigen in 30% of the TCC specimens but equally often in benign samples from TCC patients (25%) and in samples from patients with benign bladder diseases. The results were identical with both antibodies, one of which is specific for hCGß (Alfthan et al., 1988) whereas the other one also reacts with intact hCG. Therefore the staining most likely originates from hCGß expression. Expression of hCGß has been associated with malignant transformation, lack of differentiation of nontrophoblastic cells (Bellet et al., 1997) and aggressive disease (Bacchi et al., 1993; Marcillac et al., 1993; Martin et al., 1989; Moutzouris et al., 1993). Furthermore, the growth of bladder cancer cell 33 Kristina Hotakainen Table 14. Immunohistochemical expression of hCGβantigen in bladder epithelium of patients with benign conditions. Diagnosis Dysuria Hematuria Pollacisuria Recurrent lower UTI All n hCGβ-positive % 4 6 5 6 21 3 2 2 3 10 75 33 40 50 48 lines in vitro is stimulated by hCGß, and it has been proposed that hCGß acts as a paracrine or autocrine growth factor in epithelial bladder tumors (Dirnhofer et al., 1998b; Gillott et al., 1996). The stimulating action of hCGß in bladder cancer cell lines has been associated with an antiapoptotic effect interfering with normal cell turn over (Butler et al., 2000). While these findings suggest a role of hCGß in tumor progression, the frequently observed expression of hCGß in benign bladder tissue appears to need another explanation. Interestingly, tissue expression of hCGß has also been observed in some other non-malignant epithelial tissues (Iles et al., 1990b; Louhimo et al., 2001). Thus this phenomenon is less cancer specific than earlier thought. 2.2 HCGß and hCG in serum of bladder cancer patients (II, III) Serum hCGß was moderately elevated in 18 of 45 samples (40%) in study II, and in 19 of 66 samples (29%) in study III. The concentrations were not significantly associated with tumor stage or grade in either study, or with tissue expression of hCGß. Slightly elevated values in relation to the age and gender-specific reference values of hCG were observed in 4 of 43 patients (9%) in study II, and in 12 of 66 cases (18%) in study III. There was no difference in the serum levels between patients with and without instillation therapies. Values exceeding the upper reference limit of postmenopausal women (15.5 pmol/l) occurred in 8 patients. The concentrations of hCG, hCGß and 34 hCGßcf in serum and urine of bladder cancer patients are summarized in table 3, original publication III. Earlier studies have shown that elevated serum levels of hCGß occur in up to 50% of patients with metastasized bladder cancer (Iles et al., 1989; Dexeus et al., 1986), but only rarely in early stage disease. We determined hCGß by a highly sensitive assay and found elevated serum concentrations in one third of the cases including 30% (15 of 49) of those with superficial disease. This is probably explained by the fact that we specifically measured hCGß rather than “total hCGß”, i.e. hCG and hCGß together as has been done in most earlier studies (reviewed by Iles & Butler, 1998). The upper reference limit for hCGß is 2.1 pmol/l while that for hCG is 2.115.5 pmol/l depending on age and gender. A much higher cutoff was used in the studies of Iles et al., i. e. 25 IU/l roughly corresponding to 75 pmol/l. The sum of hCGß and hCG did not exceed this level in any of the sera in this study. Marcillac and coworkers determined hCGß in serum with a specific and sensitive technique. With a cutoff of 100 ng/l (4.5 pmol/l) they found elevated values in 18 of 38 patients (47%) with TCC (Marcillac et al., 1992) most of which had advanced disease (Marcillac et al., 1993). These results suggest that specific determination of hCGß provides better diagnostic accuracy than measurement of “total hCGß”. However, we observed elevated serum levels also of the intact hCG heterodimer in 18% of the cases. Although the levels were only moderately elevated, they were higher than in any case in our control material. This suggests that some tumors produce intact hCG, i.e. both α and ß subunits. This is supported by the finding of mRNA for both subunits in some TCC tumors (Lazar et al., 1995). Furthermore, Iles and coworkers showed that the urinary concentrations of hCGα in patients with hCGßpositive TCCs are higher than in controls Results and Discussion and patients with hCGß-negative TCCs (Iles et al., 1990) A p < 0.00001* Elevated levels of hCGß in urine were observed in only 5 of 55 cases (9%) in study II, and in 6 of 72 (8%) cases in study III. In 10 urine samples the creatinine concentration was below 4 mmol/l and in seven of these hCGß concentration was below detection limit. The median concentrations were higher in patients with invasive than with noninvasive tumors (p=0.03) and in cases with suspicious or malignant urinary cytology (Papa IV-V) (p=0.027). A slightly elevated value (4.6 pmol/l) was observed in one of the control patients, who had a benign inflammatory pseudotumor, which also stained positive in immunohistochemistry (Table 14). Urinary concentrations of hCGßcf were elevated in 4 TCC samples (II, III); two of which were from invasive grade 3 tumors. The median urinary hCGßcf concentrations were significantly higher in patients with invasive than noninvasive tumors (p=0.037), in cases with suspicious or malignant cytology (p=0.002), and in samples positive for hCGß mRNA (p=0.03). Urinary hCG was elevated in only one of 56 (2%) samples in study II and in two of 72 (3%) cases in study III. There was no difference in the urinary levels of hCGß and hCGßcf between patients with and without a history of instillation therapies, but urinary hCG was significantly higher in patients with previous or current instillation therapies (1.6 pmol/l) than in those without instillations (0.4 pmol/l, p=0.04). Theoretically, hCGß in urine might be expected to best reflect local secretion of hCGß from the tumor into urine. However, the urine concentrations of hCGß were less often elevated than those in serum. This could be explained by large variations in urinary flow rate leading to a wider refer- 1 0.1 0.01 Benign Noninvasive Invasive B p < 0.00001* U/S- hCGβ / mmol creatinine 2.3 HCGß, hCG and hCGßcf in urine of bladder cancer patients (II, III) U/S- hCGβ ratio 10 1 0.1 0.01 Benign Noninvasive Invasive Figure 4. Urine to serum-hCGβ ratio in TCC patients with benign histology, superficial and invasive tumors at the time of sampling (a). Panel b shows the ratio corrected for urinary creatinine concentration and expressed per mmol creatinine in urine. Samples with a urinary creatinine < 4 mmol/l were omitted. The horizontal lines indicate the medians in each category.*) Kruskal-Wallis test. ence range for hCGß in urine than in serum (Alfthan, 1994) This effect is often corrected for by dividing the value of urinary proteins with creatinine. We calculated this ratio for urinary hCGß but it did not improve the diagnostic accuracy. Similarly, correction for creatinine did not either improve the correlation between the serum and urine concentrations of luteinizing hormone in a study on prepubertal children (Demir et al., 1994). Although urinary flow rate affects its protein concentration, division by creatinine does not appear to be an optimal method of correction. We used morning urine, which usually is fairly concentrated, 35 Kristina Hotakainen but in spite of this ten samples had a low creatinine value (<4 mmol/l) and in seven of these hCGß was not measurable. Circulating hCG and hCGß are excreted into urine, and this is normally the source of hCGß urine concentrations. Therefore we studied whether the ratio of urine to serum hCGß would provide information on local secretion of hCGß in the urinary bladder. This ratio was significantly associated with histology (Kruskal-Wallis test for benign, noninvasive and invasive disease p<0.0001, Figure 4) and differentiated between high and low grade tumors (Kruskal-Wallis test for grades 1 to 3 p<0.00001; Mann – Whitney test for grade 1 versus grade 3 p=0.002 and grades 1 and 2 versus 3 p=0.009). In addition, the ratio correlated with tissue expression of hCGß detected by immunohistochemistry (p=0.019). This suggests that hCGß is directly released from the tumor into urine, whereas secretion from the tumor into the large plasma volume has a smaller impact on the concentrations in circulation, which are affected by secretion from various normal tissues. In accordance with earlier results (Baltaci et al., 1995; McLoughlin et al., 1991), the correlation between serum levels of hCGß and tissue expression was poor. Tissue expression did not either correlate with urinary hCGß. The ß-core fragment has been demonstrated by immunohistochemistry in TCC of the bladder (Dirnhofer et al., 1998b) and thus hCGßcf might be released directly from the tumor into urine. Urinary hCGßcf correlated with the invasiveness of the tumor and positivity for hCGß mRNA in urinary cells. However, elevated urine levels were rare and hCGßcf in urine was not useful as a marker. 2.4 HCGß mRNA in urinary cells (II, III) In study II, hCGß mRNA was detected in 29 of the 68 (43%) samples of urinary cells form TCC patients and in none of the 36 healthy controls (n=14). There was a highly significant association between histologically verified TCC and hCGß mRNA in urinary cells (p=0.0014). In study III 42 of 84 (50%) urinary cell samples were positive for hCGß mRNA. The positive cases tended to have a higher urine to serum ratio of hCGß (p=0.088) and hCGßcf concentrations in urine than the negative ones (p=0.03), but there was no correlation with tumor stage or grade (Table 15), urinary cytology, immunohistochemical hCGß expression and serum marker levels. Previous (more than 2 months earlier) or current instillation therapies did not either affect the result; 12 of 23 (52%) cases were positive. All healthy controls were negative, but in four of the 23 control patients (II, III) hCGß mRNA was present in the urinary cells. Three of these had a UTI and one had hematuria of unknown origin. HCGß mRNA in urinary cells is strongly associated with TCC. However, this was also detected in some patients with benign diseases, i. e. infection or hematuria. We found that hCGß mRNA expression may be incuced in leukocytes. Therefore hematuria and inflammation may be expected to cause a positive result in RT-PCR of hCGß mRNA in urinary cells. However, although hematuria occurred in several patients with TCC it was not consistently associated with Table 15. HCGβ mRNA in urinary cells from TCC patients according to tumor stage and grade. Stage n hCGβ mRNA % Tis Ta T1 T2 T3 T4 Not available 8 41 13 4 2 1 15 5 18 8 2 0 1 8 62 44 62 50 0 100 53 Grade 1 2 3 All 21 26 22 84 7 16 10 42 33 62 45 50 Results and Discussion 3. HCGß EXPRESSION IN SERUM OF RCC PATIENTS (IV) The concentration of hCGß in serum was elevated (>2 pmol/l) in 23% of the patients with RCC and 20 of these (11%) had values > 4pmol/l. The median concentration of hCGß in serum was 1.2 pmol/l (range 0.2 - 18 pmol/l), which was significantly higher (p<0.0001) than in controls (median 0.4 pmol/l, range 0.2 - 1.3 pmol/l). The serum concentrations of hCGß were not related to serum creatinine. There was no difference in hCGß levels between males and females, different age groups, different RCC types, aneuploid and diploid tumors, or tumors with and without venous invasion. Serum hCGß concentrations were not either significantly correlated with tumor stage or grade (Figure 5). Clinical stage and grade were highly predictive of disease specific survival (p<0.0001 each) in univariate analysis. Patients with serum hCGß concentrations above the median value (1.2 pmol/l) had significantly shorter survival than those with lower levels (p=0.0029) (Figure 6A). A difference in survival time was observed also among patients with metastasized tu- Table 16. Factors independently associated with decreased cumulative survival of patients with RCC (RR = Relative risk). Coefficient (β) Grade Stage hCGβ 1.15 2.34 0.48 RR p =0.032 <0.0001 =0.022 3.2 (1.1-9.0) 10.3 (4.8-22.3) 1.6 (1.1-2.5) mors (stage IV, Figure 6B) (p=0.06). When serum hCGß concentration was compared as quartiles there was no difference in disease specific survival between the two lowest quartiles or between quartiles 3 and 4 (p>0.6). In multivariate analysis using the Cox regression model with age, gender, serum hCGß, nuclear grade and stage as input variables, stage, grade, and serum hCGß concentration were independently associated with the disease specific survival (Table 16). RCC is known for its unpredictable clinical behavior and within stages and grades the clinical course is highly variable. Recurrence can occur many years after apparently successful surgery and metastases may spontaneously regress after removal of the primary tumor (Golimbu et al., 1986b). Several biomolecular markers have been studied as potential markers for RCC, but to date there is no specific clinically useful serum marker. Of the many serum markers studied, only a few, i. e. VEGF, interleukin-10, 12.8 6.4 (Serum hCGβ (pmol/l) a positive RT-PCR. It is possible that infections, instillation therapies, instrumentation and other conditions causing cellular atypia and inflammation also induce hCGß mRNA expression in transitional epithelium. The more frequent expression in patients with TCC could be explained by increased shedding of TCC cells from cancerous epithelium. However, half of the patients with a histologically verified tumor had a negative RT-PCR. This could be associated with a variable recovery of tumor cells in urine, or to selective expression of hCGß. Different hCGß genes are expressed in benign and malignant bladder epithelium. Our primers were designed according to the sequence of the hCGß5 gene which is expressed in TCC (Lazar et al., 1995). 3.2 1.6 0.8 0.4 0.2 0.1 I II III IV Stage Figure 5. Boxplot demonstrating preoperative serum hCGβ concentrations in RCC patients with various stages. The horizontal line indicates the upper reference limit for serum hCGβ (2.1 pmol/l). 37 Kristina Hotakainen A B 1 1 hCGβ ≥1.2 pmol/l 0.8 0.6 0.4 0.2 Cumulative survival Cumulative survival hCGβ <1.2 pmol/l hCGβ <1.2 pmol/l 0.8 hCGβ ≥1.2 pmol/l 0.6 0.4 Log-rank p-value =0.06 0.2 Log-rank p-value=0.0029 0 0 0 Cumulative survival A 25 50 75 100 125 Time (months) 0 150 175 200 24 48 72 96 120 Time (months) 144 168 1 0.8 hCGβ <1.2 pmol/l hCGβ ≥1.2 pmol/l 0.6 0.4 0.2 Log-rank p-value=0.0029 0 CA-125, and TATI are of potential prognostic value (Wittke et al., 1999; Grankvist et al., 1997; Jacobsen et al., 2000; Paju et al., 2001). There is little data on hCGß expression in renal tumours. Dexeus and coworkers observed increased hCGß concentrations in serum of 10% of the patients, and changes in the levels correlated with the clinical course (Dexeus et al., 1991). We found elevated concentrations in 23% of the patients, and the median concentrations were significantly higher than in controls (p<0.0001). Patients with serum hCGß levels above the median value (1.2 pmol/l) had significantly shorter survival than those with lower levels (p<0.0029), and in multivariate analysis serum hCGß, tumour stage and grade were independent prognostic variables. The fairly low frequency (23%) of elevated serum hCGß suggest that this marker is expressed by only part of the RCCs. However, because normal levels between 1.2 and 2.1 pmol/l were associated with adverse prognosis, it is possible that part of the hCGß in these patients is derived from the 0 38 25 50 tumor. The absence of a correlation with stage indicates that elevated levels are not only a result of tumor burden but that hCGß expression characterizes a subgroup of tumors. The lack of significant correlation with established prognostic variables such as grade and stage demonstrates the independent character of this marker. The clinical utility of hCGß in the management of RCC is limited by the infrequent expression. About one fourth of the patients had elevated levels and in half of these the elevation was substantial. However, a prognostic value was observed even with normal levels exceeding the median value. Furthermore, the prognostic significance of hCGß in serum was seen not only in patients with metastatic disease, but also in earlier stages (stages I-III). Thus it appears worthwhile to study whether hCGß could be used in identifying a subgroup of patients with increased risk of aggressive disease and as an aid in the selection of therapy, as well as in monitoring of the disease after primary therapy. 75 Time 100 125 (months) 150 175 200 Summary Methods for detection of cancer cells in blood and body fluids have been extensively studied in attempts to evaluate tumor spread. The presence of cancer cells in circulation reflects the metastatic potential of the tumor, although it is not necessarily a sign of metastatic disease. RT-PCR is a highly sensitive method of detecting cancer cells expressing a tumor- or tissue specific mRNA. With the best methods a single tumor cell may be detected among 107 leukocytes. However, a prerequisite is that peripheral blood cells do not express the mRNA used as a marker of cancer cells. Recently expression of several genes thought to be specific for other organs has been detected in blood cells, and leukocytes have the potential of producing many peptide hormones and receptors for these. HCG is a glycoprotein hormone consisting of two subunits, α and ß. It is produced at high concentrations by placental trophoblasts to maintain pregnancy. Isolated low level expression of hCGß commonly occurs in malignant tumors and this is associated with agressive disease. We studied whether hCGß mRNA could be used as a marker of cancer cells in blood and urine. To confirm the specificity of the method we first studied whether hCGß mRNA is expressed by peripheral blood cells. HCGß mRNA expression was detected by RT-PCR in cultured blood cells, but not in isolated peripheral blood leukocytes. HCGß mRNA expression occurred in the urinary cells from half of the patients with bladder cancer, and this was strongly associated with histological evidence of cancer. However, there was no association with stage or grade of the disease. Urine concentrations of hCGß were elevated in less than 10% of the cases, while the serum concentrations of hCGß and of intact hCG were elevated in 30-40% and 10-20%, respectively. However, the ratio of urine to serum concentration of hCGß protein was significantly associated with both stage and grade of the tumor, and also with immunohistochemical staining of hCGß in tumor tissue. Immunohistochemical expression of hCGß was observed in approximately one third of the tumors, and this was not associated with stage and grade. We also detected hCGß protein in benign transitional epithelium in 30% of the cases. In most of these the expression was associated with inflammation or squamous metaplasia. In previous studies, the serum concentrations of hCGß have been found to have prognostic value in bladder cancer, ovarian cancer, cancer of the oral cavity, oropharynx and colon. The prognostic significance of hCGß expression in bladder cancer was not studied, but the strong correlation between urine to serum ratio of hCGß and stage and grade of the disease suggest an association between hCGß expression and advanced and high grade tumors. Because hCGß appears to be a universal serum marker for aggressive cancers we studied the prognostic value of the preoperative serum concentrations of hCGß in patients with renal cell carcinoma (RCC). Serum hCGß was elevated in 23% of the patients. In addition to stage and grade, serum hCGß was an independent prognostic marker, and normal levels exceeding the median were 39 Kristina Hotakainen also of prognostic value. In preliminary studies we have found that hCGß can be detected by immunohistochemistry in approximately 15% of conventional RCCs and this is associated with survival. Our results add further support to pre- 40 vious studies linking hCGß to a potentially aggressive subgroup of tumors. A possible explanation for this association is the recently described anti-apoptotic effect of hCGß, but the detailed mechanism remains to be investigated. Conclusions 1. HCGß expression was detected in cultured peripheral blood lymphocytes but not in unstimulated cells. Because hCGß mRNA expression was induced by culture, disorders leading to lymphocyte activation may also induce hCGß expression giving positive results for hCGß mRNA in blood from patients without cancer. This is a potential limitation for the use of hCGß expression for detection of cancer cells in blood and possibly also in urine. 2. HCGß mRNA expression was detected in the urinary cells of 50% of the patients with bladder cancer but not in healthy controls. There was no association with stage or grade of the disease. The positive results in some benign diseases suggest that infections, instrumentation and other conditions causing cellular atypia and inflammation also induce hCGß mRNA expression in transitional epithelium. 3. Immunohistochemical expression of hCGß in bladder cancer was not associated with stage and grade of the tumor. The positivity in benign bladder tissues shows that the phenomenon is not restricted to malignant tumors but inflammation and metaplasia may also induce expression. While previous studies suggest a role of hCGß in tumor progression, these findings suggest that hCGß expression also is involved in growth stimulation. 4. Urinary hCGß and hCGßcf correlated with urinary cytology and the ratio of urine to serum concentration of hCGß protein was significantly associated with both stage, grade, and immunohistochemical staining. This suggests that hCGß is secreted into urine by many bladder tumors and supports the previously reported association between hCGß expression and advanced and high grade tumors. 5. Serum hCGß was elevated in 23% of the RCC patients and levels above the median concentration were independently associated with shorter survival. The absence of a correlation with stage indicates that elevated levels are not only a result of tumor burden. The fairly small proportion of RCC patients with elevated hCGß suggests that hCGß is not expressed by all tumors, but the expression identifies a subgroup of RCCs with unfavourable prognosis, supporting previous studies linking hCGß to aggressive tumors. 41 Kristina Hotakainen Acknowledgements This study was carried out at the Department of Clinical Chemistry in the University of Helsinki. I wish to express my sincere thanks to Professor Ulf-Håkan Stenman, Head of the Department, (who is fondly known as Uffen) for placing excellent working facilities at my disposal. I am also deeply grateful to Uffen for supervising this work. His enthusiasm for research and his profound knowledge of science are admirable. His never-ending encouragement and interest in my work have been invaluable. Despite numerous other ongoing projects and a tight schedule, Uffen has always been ready to comment on my latest findings and to give clues to help solve any problems. His kind personality, skill and positive attitude towards his students and life in general has made collaboration with him a pleasure. Docent Martti Nurmi and Professor Kim Pettersson were the official reviewers of this thesis. I wish to thank them for their thorough review and constructive criticism and comments. I am grateful for having had the opportunity to co-operate with Caj Haglund, MD, PhD, Professor Börje Ljungberg, Professor Jim Schröder, Stig Nordling, MD, PhD, Erkki Rintala, MD, PhD, and Ossi Lindell, MD, PhD. I wish to thank my coauthors Susanna Lintula, MSc, Henrik Alfthan, PhD, Annukka Paju, PhD, Jakob Stenman, MD, Martina Serlachius, MSc and Torgny Rasmuson, MD, PhD. Their participation has been most valuable. I also wish to thank Mrs Sanna Kihlberg, Mrs Anne Ahmanheimo, Mrs Taina Grönholm, Ms Maarit Leinimaa, Mrs 42 Kristiina Nokelainen, Mrs Elina Laitinen and Mrs Sari Nieminen for their friendship and technical assistance throughout the years. I also thank Mr Oso Rissanen for solving almost all the technical problems and Leena Vaara for her help in preparing slides. I am grateful to Jakob Stenman who took me to Uffen’s office during my years of medical studies to learn what research is all about; the consequences are now evident. I thank Susanna Lintula who introduced me to scientific work; her support and friendship have been most valuable, and Annukka Paju who has been an encouraging friend both inside and outside the laboratory. I am grateful to Doctors Jari Leinonen, Patrik Finne, and Henrik Alfthan for their patience with my questions about chemistry, statistics and everything else. I am indebted to Heini Lassus for her thorough guidance in the layout of this thesis. I also warmly thank all my friends and colleagues in the laboratory who have contributed to the good working atmosphere. Without you the work would have been lacking many of the most pleasant moments. I am grateful for the financial support from Finska Läkaresällskapet, the Foundation of K. Albin Johansson, the Helsinki University Central Hospital Research Funds and the Foundation of Else and Wilhelm Stockmann. Thanks go to all my friends and relatives outside the laboratory who have provided me with many joyful moments. I thank Ruut and Olli for their company and enthusiasm in sports activities, and their charming kids for being good examples and friends to our son. My parents-in-law Aila Acknowledgements and Mauri are thanked for always being available for baby-sitting and offering a helping hand when needed. My heartfelt thanks are directed to my mother Vuokko and my sister Johanna for their constant love and encouragement throughout the years. Without their support and company in daily matters from child-care to shopping, this work would not have been possible. I thank Toivo for his patience with our son, a frequent visitor in mother’s and Toivo’s home, and his interest in my work. I devote my warmest thoughts to my husband Markus. Your patience and encouragement towards my work and your love has carried me through these years. Together with our sunshine, Josia, you have brought much happiness to my life. Helsinki, April 2002 43 Kristina Hotakainen References Acevedo, H.F. & Hartsock, R.J. (1996). Metastatic phenotype correlates with high expression of membrane-associated complete beta-human chorionic gonadotropin in vivo. Cancer, 78, 2388-99. Acevedo, H.F., Krichevsky, A., Campbell-Acevedo, E.A., Galyon, J.C., Buffo, M.J. & Hartsock, R.J. (1992). Expression of membrane-associated human chorionic gonadotropin, its subunits, and fragments by cultured human cancer cells. Cancer, 69, 1829-42. Acevedo, H.F., Tong, J.Y. & Hartsock, R.J. (1995). Human chorionic gonadotropin-beta subunit gene expression in cultured human fetal and cancer cells of different types and origins. Cancer, 76, 1467-75. Alexander, H., Zimmermann, G., Lehmann, M., Pfeiffer, R., Schone, E., Leiblein, S. & Ziegert, M. (1998). HCG secretion by peripheral mononuclear cells during pregnancy. Domest Anim Endocrinol, 15, 377-87. Alfthan, H. (1994) Various molecular forms of human choriogonadotropin in serum and urine of nonpregnant and pregnant subjects and in patients with pancreatic disease. Thesis. Yliopistopaino, Helsinki. Alfthan, H., Haglund, C., Dabek, J. & Stenman, U.H. (1992a). Concentrations of human choriogonadotropin, its beta-subunit, and the core fragment of the beta-subunit in serum and urine of men and nonpregnant women. Clin Chem, 38, 1981-7. Alfthan, H., Haglund, C., Roberts, P. & Stenman, U.H. (1992b). Elevation of free beta subunit of human choriogonadotropin and core beta fragment of human choriogonadotropin in the serum and urine of patients with malignant pancreatic and biliary disease. Cancer Res, 52, 4628-33. Alfthan, H., Schröder, J., Fraser, R., Koskimies, A., Halila, H. & Stenman, U.H. (1988). Choriogonadotropin and its beta subunit separated by hydrophobic-interaction chromatography and quantified in serum during pregnancy by time-resolved immunofluorometric assays. Clin Chem, 34, 1758-62. Alfthan, H. & Stenman, U.H. (1990). Pregnancy serum contains the beta-core fragment of human choriogonadotropin. J Clin Endocrinol Metab, 70, 783-7. Allard, P., Fradet, Y., Tetu, B. & Bernard, P. (1995). Tumor-associated antigens as prognostic factors for recurrence in 382 patients with primary transitional cell carcinoma of the bladder. Clin Cancer Res, 1, 1195-202. Azad, N., La Paglia, N., Jurgens, K.A., Kirsteins, L., Emanuele, N.V., Kelley, M.R., Lawrence, A.M. & Mohagheghpour, N. (1993). Immunoactivation enhances the concentration of luteinizing hormone-releasing hormone peptide and its gene expression in human peripheral T-lymphocytes. Endocrinology, 133, 215-23. Bacchi, C.E., Coelho, K.I. & Goldberg, J. (1993). Expres- 44 sion of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: possible relationship with the prognosis. Rev Paul Med, 111, 412-6. Badalament, R.A., Gay, H., Cibas, E.S., Herr, H.W., Whitmore, W.F., Jr., Fair, W.R. & Melamed, M.R. (1987a). Monitoring endoscopic treatment of superficial bladder carcinoma by postoperative urinary cytology. J Urol, 138, 760-2. Badalament, R.A., Herr, H.W., Wong, G.Y., Gnecco, C., Pinsky, C.M., Whitmore, W.F., Jr., Fair, W.R. & Oettgen, H.F. (1987b). A prospective randomized trial of maintenance versus nonmaintenance intravesical bacillus Calmette-Guerin therapy of superficial bladder cancer. J Clin Oncol, 5, 441-9. Badalament, R.A., Kimmel, M., Gay, H., Cibas, E.S., Whitmore, W.F., Jr., Herr, H.W., Fair, W.R. & Melamed, M.R. (1987c). The sensitivity of flow cytometry compared with conventional cytology in the detection of superficial bladder carcinoma. Cancer, 59, 2078-85. Bakhos, R., Shankey, T.V., Flanigan, R.C., Fisher, S. & Wojcik, E.M. (2000). Comparative analysis of DNA flow cytometry and cytology of bladder washings: review of discordant cases. Diagn Cytopathol, 22, 65-9. Baltaci, S., Kupeli, S., Sak, S.D., Erden, E. & Sarica, K. (1995). Human chorionic gonadotropin in serum and neoplastic tissue from patients with bladder carcinoma. Int Urol Nephrol, 27, 289-95. Barentsz, J.O., Jager, G.J., Witjes, J.A. & Ruijs, J.H. (1996). Primary staging of urinary bladder carcinoma: the role of MRI and a comparison with CT. Eur Radiol, 6, 12933. Bastacky, S., Ibrahim, S., Wilczynski, S.P. & Murphy, W.M. (1999). The accuracy of urinary cytology in daily practice. Cancer, 87, 118-28. Batticane, N., Morale, M.C., Gallo, F., Farinella, Z. & Marchetti, B. (1991). Luteinizing hormone-releasing hormone signaling at the lymphocyte involves stimulation of interleukin-2 receptor expression. Endocrinology, 129, 277-86. Beham, A., Ratschek, M., Zatloukal, K., Schmid, C. & Denk, H. (1992). Distribution of cytokeratins, vimentin and desmoplakins in normal renal tissue, renal cell carcinomas and oncocytoma as revealed by immunofluorescence microscopy. Virchows Arch A Pathol Anat Histopathol, 421, 209-15. Bellet, D., Lazar, V., Bieche, I., Paradis, V., Giovangrandi, Y., Paterlini, P., Lidereau, R., Bedossa, P., Bidart, J.M. & Vidaud, M. (1997). Malignant transformation of nontrophoblastic cells is associated with the expression of chorionic gonadotropin beta genes normally transcribed in trophoblastic cells. Cancer Res, 57, 516-23. References Bhalang, K., Kafrawy, A.H. & Miles, D.A. (1999). Immunohistochemical study of the expression of human chorionic gonadotropin-beta in oral squamous cell carcinoma. Cancer, 85, 757-62. Bhat, N.M., Bieber, M.M. & Teng, N.N. (1993). One-step enrichment of nucleated red blood cells. A potential application in perinatal diagnosis. J Immunol Methods, 158, 277-80. Bilchik, A., Miyashiro, M., Kelley, M., Kuo, C., Fujiwara, Y., Nakamori, S., Monden, M. & Hoon, D.S. (2000). Molecular detection of metastatic pancreatic carcinoma cells using a multimarker reverse transcriptase-polymerase chain reaction assay. Cancer, 88, 1037-44. Birken, S. (1984). Chemistry of human choriogonadotropin. Ann Endocrinol (Paris), 45, 297-305. Bittard, H., Lamy, B. & Billery, C. (1996). Clinical evaluation of cell deoxyribonucleic acid content measured by flow cytometry in bladder cancer. J Urol, 155, 1887-91. Blackburn, E.H. (1991). Structure and function of telomeres. Nature, 350, 569-73. Blalock, J.E., Harbour-McMenamin, D. & Smith, E.M. (1985). Peptide hormones shared by the neuroendocrine and immunologic systems. J Immunol, 135, 858s-861s. Bo, M. & Boime, I. (1992). Identification of the transcriptionally active genes of the chorionic gonadotropin beta gene cluster in vivo. J Biol Chem, 267, 3179-84. Boffetta, P., Jourenkova, N. & Gustavsson, P. (1997). Cancer risk from occupational and environmental exposure to polycyclic aromatic hydrocarbons. Cancer Causes Control, 8, 444-72. Boorstein, W.R., Vamvakopoulos, N.C. & Fiddes, J.C. (1982). Human chorionic gonadotropin beta-subunit is encoded by at least eight genes arranged in tandem and inverted pairs. Nature, 300, 419-22. Boothby, M., Ruddon, R.W., Anderson, C., McWilliams, D. & Boime, I. (1981). A single gonadotropin alphasubunit gene in normal tissue and tumor-derived cell lines. J Biol Chem, 256, 5121-7. Bosl, G.J., Vogelzang, N.J., Goldman, A., Fraley, E.E., Lange, P.H., Levitt, S.H. & Kennedy, B.J. (1981). Impact of delay in diagnosis on clinical stage of testicular cancer. Lancet, 2, 970-3. Braunstein, G.D., Vaitukaitis, J.L., Carbone, P.P. & Ross, G.T. (1973). Ectopic production of human chorionic gonadotrophin by neoplasms. Ann Intern Med, 78, 3945. Brown, F.M. (2000). Urine cytology. It is still the gold standard for screening? Urol Clin North Am, 27, 25-37. Bruning, T. & Bolt, H.M. (2000). Renal toxicity and carcinogenicity of trichloroethylene: key results, mechanisms, and controversies. Crit Rev Toxicol, 30, 253-85. Bucci, B., Pansadoro, V., De Paula, F., Florio, A., Carico, E., Zupi, G. & Vecchione, A. (1995). Biologic characteristics of T1 papillary bladder cancer. Flow cytometric study of paraffin-embedded material. Anal Quant Cytol Histol, 17, 121-8. Burchardt, M., Burchardt, T., Shabsigh, A., De La Taille, A., Benson, M.C. & Sawczuk, I. (2000). Current concepts in biomarker technology for bladder cancers. Clin Chem, 46, 595-605. Burg-Kurland, C.L., Purnell, D.M., Combs, J.W., Hillman, E.A., Harris, C.C. & Trump, B.F. (1986). Immunocytochemical evaluation of human esophageal neoplasms and preneoplastic lesions for beta-chorionic gonadotro- pin, placental lactogen, alpha-fetoprotein, carcinoembryonic antigen, and nonspecific cross-reacting antigen. Cancer Res, 46, 2936-43. Butler, S.A., Ikram, M.S., Mathieu, S. & Iles, R.K. (2000). The increase in bladder carcinoma cell population induced by the free beta subunit of human chorionic gonadotrophin is a result of an anti-apoptosis effect and not cell proliferation. Br J Cancer, 82, 1553-6. Butler, S.A., Laidler, P., Porter, J.R., Kicman, A.T., Chard, T., Cowan, D.A. & Iles, R.K. (1999). The beta-subunit of human chorionic gonadotrophin exists as a homodimer. J Mol Endocrinol, 22, 185-92. Cacciatore, B., Korhonen, J., Stenman, U.H. & Ylostalo, P. (1995). Transvaginal sonography and serum hCG in monitoring of presumed ectopic pregnancies selected for expectant management. Ultrasound Obstet Gynecol, 5, 297300. Cajulis, R.S., Haines, G.K., 3rd, Frias-Hidvegi, D., McVary, K. & Bacus, J.W. (1995). Cytology, flow cytometry, image analysis, and interphase cytogenetics by fluorescence in situ hybridization in the diagnosis of transitional cell carcinoma in bladder washes: a comparative study. Diagn Cytopathol, 13, 214-23; discussion 224. Campo, E., Algaba, F., Palacin, A., Germa, R., Sole-Balcells, F.J. & Cardesa, A. (1989). Placental proteins in highgrade urothelial neoplasms. An immunohistochemical study of human chorionic gonadotropin, human placental lactogen, and pregnancy-specific beta-1-glycoprotein. Cancer, 63, 2497-504. Campo, E., Palacin, A., Benasco, C., Quesada, E. & Cardesa, A. (1987). Human chorionic gonadotropin in colorectal carcinoma. An immunohistochemical study. Cancer, 59, 1611-6. Carpelan-Holmstrom, M., Haglund, C., Lundin, J., Alfthan, H., Stenman, U.H. & Roberts, P.J. (1996). Independent prognostic value of preoperative serum markers CA 242, specific tissue polypeptide antigen and human chorionic gonadotrophin beta, but not of carcinoembryonic antigen or tissue polypeptide antigen in colorectal cancer. Br J Cancer, 74, 925-9. Carpinito, G.A., Stadler, W.M., Briggman, J.V., Chodak, G.W., Church, P.A., Lamm, D.L., Lange, P.H., Messing, E.M., Pasciak, R.M., Reservitz, G.B., Ross, R.N., Rukstalis, D.B., Sarosdy, M.F., Soloway, M.S., Thiel, R.P., Vogelzang, N. & Hayden, C.L. (1996). Urinary nuclear matrix protein as a marker for transitional cell carcinoma of the urinary tract. J Urol, 156, 1280-5. Castelao, J.E., Yuan, J.M., Skipper, P.L., Tannenbaum, S.R., Gago-Dominguez, M., Crowder, J.S., Ross, R.K. & Yu, M.C. (2001). Gender- and smoking-related bladder cancer risk. J Natl Cancer Inst, 93, 538-45. Catt, K.J., Dufau, M.L. & Tsuruhara, T. (1973). Absence of intrinsic biological activity in LH and hCG subunits. J Clin Endocrinol Metab, 36, 73-80. Cheng, L., Bostwick, D.G. (2000). World Health Organization and International Society of Urological Pathology classification and two-number grading system of bladder tumors: reply. Cancer, 88, 1513-6. Cheng, L., Neumann, R.M. & Bostwick, D.G. (1999). Papillary urothelial neoplasms of low malignant potential. Clinical and biologic implications. Cancer, 86, 2102-8. Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem, 162, 156-9. 45 Kristina Hotakainen Chow, W.H., McLaughlin, J.K., Mandel, J.S., Wacholder, S., Niwa, S. & Fraumeni, J.F., Jr. (1996). Obesity and risk of renal cell cancer. Cancer Epidemiol Biomarkers Prev, 5, 17-21. Christensen, M., Wolf, H. & Orntoft, T.F. (2000). Microsatellite alterations in urinary sediments from patients with cystitis and bladder cancer. Int J Cancer, 85, 614-7. Clevenger, C.V., Russell, D.H., Appasamy, P.M. & Prystowsky, M.B. (1990). Regulation of interleukin 2driven T-lymphocyte proliferation by prolactin. Proceedings Of The National Academy Of Sciences Of The United States Of America, 87, 6460-4. Cohen, A.J., Li, F.P., Berg, S., Marchetto, D.J., Tsai, S., Jacobs, S.C. & Brown, R.S. (1979). Hereditary renal-cell carcinoma associated with a chromosomal translocation. N Engl J Med, 301, 592-5. Cole, L.A., Hussa, R.O. & Rao, C.V. (1981). Discordant synthesis and secretion of human chorionic gonadotropin and subunits by cervical carcinoma cells. Cancer Res, 41, 1615-9. Cole, L.A., Shahabi, S., Butler, S.A., Mitchell, H., Newlands, E.S., Behrman, H.R. & Verrill, H.L. (2001). Utility of commonly used commercial human chorionic gonadotropin immunoassays in the diagnosis and management of trophoblastic diseases. Clin Chem, 47, 308-15. Cole, L.A., Wang, Y.X., Elliott, M., Latif, M., Chambers, J.T., Chambers, S.K. & Schwartz, P.E. (1988). Urinary human chorionic gonadotropin free beta-subunit and beta-core fragment: a new marker of gynecological cancers. Cancer Res, 48, 1356-60. Cook, A.M., Huddart, R.A., Jay, G., Norman, A., Dearnaley, D.P. & Horwich, A. (2000). The utility of tumour markers in assessing the response to chemotherapy in advanced bladder cancer. Br J Cancer, 82, 1952-7. Cookson, M.S., Herr, H.W., Zhang, Z.F., Soloway, S., Sogani, P.C. & Fair, W.R. (1997). The treated natural history of high risk superficial bladder cancer: 15-year outcome. J Urol, 158, 62-7. Cordon-Cardo, C. & Reuter, V.E. (1997). Alterations of tumor suppressor genes in bladder cancer. Semin Diagn Pathol, 14, 123-32. Cowley, G., Smith, J.A., Ellison, M. & Gusterson, B. (1985). Production of beta-human chorionic gonadotrophin by human squamous carcinoma cell lines. Int J Cancer, 35, 575-9. Crew, J.P., O’Brien, T., Bradburn, M., Fuggle, S., Bicknell, R., Cranston, D. & Harris, A.L. (1997). Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer. Cancer Res, 57, 5281-5. Cuckle, H.S., Shahabi, S., Sehmi, I.K., Jones, R. & Cole, L.A. (1999). Maternal urine hyperglycosylated hCG in pregnancies with Down syndrome. Prenat Diagn, 19, 91820. Dardenne, M., de Moraes M do, C., Kelly, P.A. & Gagnerault, M.C. (1994). Prolactin receptor expression in human hematopoietic tissues analyzed by flow cytofluorometry. Endocrinology, 134, 2108-14. de Bruijn, H.W., ten Hoor, K.A., Krans, M. & van der Zee, A.G. (1997). Rising serum values of beta-subunit human chorionic gonadotrophin (hCG) in patients with progressive vulvar carcinomas. Br J Cancer, 75, 1217-8. de Graaf, H., Maelandsmo, G.M., Ruud, P., Forus, A., 46 Oyjord, T., Fodstad, O. & Hovig, E. (1997). Ectopic expression of target genes may represent an inherent limitation of RT-PCR assays used for micrometastasis detection: studies on the epithelial glycoprotein gene EGP-2. Int J Cancer, 72, 191-6. de la Roza, G.L., Hopkovitz, A., Caraway, N.P., Kidd, L., Dinney, C.P., Johnston, D. & Katz, R.L. (1996). DNA image analysis of urinary cytology: prediction of recurrent transitional cell carcinoma. Mod Pathol, 9, 571-8. de Riese, W.T., Crabtree, W.N., Allhoff, E.P., Werner, M., Liedke, S., Lenis, G., Atzpodien, J. & Kirchner, H. (1993). Prognostic significance of Ki-67 immunostaining in nonmetastatic renal cell carcinoma. J Clin Oncol, 11, 18048. Dekernion, J.B., Ramming, K.P. & Smith, R.B. (1978). The natural history of metastatic renal cell carcinoma: a computer analysis. J Urol, 120, 148-52. Delahunt, B., Bethwaite, P.B., Thornton, A. & Ribas, J.L. (1995). Proliferation of renal cell carcinoma assessed by fixation-resistant polyclonal Ki-67 antibody labeling. Correlation with clinical outcome. Cancer, 75, 2714-9. Delbanco, E.H., Bolt, H.M., Huber, W.W., Beken, S., Geller, F., Philippou, S., Brands, F.H., Bruning, T. & Thier, R. (2001). Glutathione transferase activities in renal carcinomas and adjacent normal renal tissues: factors influencing renal carcinogenesis induced by xenobiotics. Arch Toxicol, 74, 688-94. Demir, A., Alfthan, H., Stenman, U.H. & Voutilainen, R. (1994). A clinically useful method for detecting gonadotropins in children: assessment of luteinizing hormone and follicle-stimulating hormone from urine as an alternative to serum by ultrasensitive time-resolved immunofluorometric assays. Pediatr Res, 36, 221-6. Dexeus, F., Logothetis, C., Hossan, E. & Samuels, M.L. (1986). Carcinoembryonic antigen and beta-human chorionic gonadotropin as serum markers for advanced urothelial malignancies. J Urol, 136, 403-7. Dexeus, F.H., Logothetis, C.J., Sella, A., Fitz, K., Striegel, A., Amato, R.J. & Liu, F.J. (1991). Serum biomarkers in metastatic renal cell carcinoma. Urology, 38, 6-10. Dickman, P.W., Hakulinen, T., Luostarinen, T., Pukkala, E., Sankila, R., Soderman, B. & Teppo, L. (1999). Survival of cancer patients in Finland 1955-1994. Acta Oncol, 38 Suppl 12, 1-103. Dierick, A.M., Praet, M., Roels, H., Verbeeck, P., Robyns, C. & Oosterlinck, W. (1991). Vimentin expression of renal cell carcinoma in relation to DNA content and histological grading: a combined light microscopic, immunocytochemical and cytophotometrical analysis. Histopathology, 18, 315-22. Dirnhofer, S., Berger, C., Hermann, M., Steiner, G., Madersbacher, S. & Berger, P. (1998a). Coexpression of gonadotropic hormones and their corresponding FSHand LH/CG-receptors in the human prostate. Prostate, 35, 212-20. Dirnhofer, S., Freund, M., Rogatsch, H., Krabichler, S. & Berger, P. (2000). Selective expression of trophoblastic hormones by lung carcinoma: neurendocrine tumors exclusively produce human chorionic gonadotropin alphasubunit (hCGalpha). Hum Pathol, 31, 966-72. Dirnhofer, S., Hermann, M., Hittmair, A., Hoermann, R., Kapelari, K. & Berger, P. (1996). Expression of the human chorionic gonadotropin-beta gene cluster in human pituitaries and alternate use of exon 1. J Clin Endocrinol References Metab, 81, 4212-7. Dirnhofer, S., Koessler, P., Ensinger, C., Feichtinger, H., Madersbacher, S. & Berger, P. (1998b). Production of trophoblastic hormones by transitional cell carcinoma of the bladder: association to tumor stage and grade. Hum Pathol, 29, 377-82. Doi, F., Chi, D.D., Charuworn, B.B., Conrad, A.J., Russell, J., Morton, D.L. & Hoon, D.S. (1996). Detection of betahuman chorionic gonadotropin mRNA as a marker for cutaneous malignant melanoma. Int J Cancer, 65, 454-9. Droz, J.P., Kramar, A., Ghosn, M., Piot, G., Rey, A., Theodore, C., Wibault, P., Court, B.H., Perrin, J.L., Travagli, J.P. & et al. (1988). Prognostic factors in advanced nonseminomatous testicular cancer. A multivariate logistic regression analysis. Cancer, 62, 564-8. Dunzendorfer, U., Drahovsky, D., Schmidt-Gayk, H. & Zahradnik, H.P. (1981). [Clinical significance of peptide hormones LH, FSH, TSH, prolactin, HCG, parathormone, calcitonin and prostaglandin F2 alpha in kidney neoplasms]. Z Urol Nephrol, 74, 13-9. Durkee, C. & Benson, R., Jr. (1980). Bladder cancer following administration of cyclophosphamide. Urology, 16, 145-8. Epstein, J.I., Amin, M.B., Reuter, V.R. & Mostofi, F.K. (1998). The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol, 22, 1435-48. Esposti, P.L., Edsmyr, F. & Tribukait, B. (1978). The role of exfoliative cytology in the management of bladder carcinoma. Urol Res, 6, 197-200. Essen, A., Ozen, H., Ayhan, A., Ergen, A., Tasar, C. & Remzi, F. (1991). Serum ferritin: a tumor marker for renal cell carcinoma. J Urol, 145, 1134-7. Finnish Cancer Registry (2000). Cancer incidence in Finland 1996 and 1997. Cancer Society of Finland: Helsinki. Frazier, A.L., Robbins, L.S., Stork, P.J., Sprengel, R., Segaloff, D.L. & Cone, R.D. (1990). Isolation of TSH and LH/CG receptor cDNAs from human thyroid: regulation by tissue specific splicing. Mol Endocrinol, 4, 126476. Friedrich, C.A. (1999). Von Hippel-Lindau syndrome. A pleomorphic condition. Cancer, 86, 2478-82. Fuhrman, S.A., Lasky, L.C. & Limas, C. (1982). Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol, 6, 655-63. Fukutani, K., Libby, J.M., Panko, W.B. & Scardino, P.T. (1983). Human chorionic gonadotropin detected in urinary concentrates from patients with malignant tumors of the testis, prostate, bladder, ureter and kidney. J Urol, 129, 74-7. Gelb, A.B., Sudilovsky, D., Wu, C.D., Weiss, L.M. & Medeiros, L.J. (1997). Appraisal of intratumoral microvessel density, MIB-1 score, DNA content, and p53 protein expression as prognostic indicators in patients with locally confined renal cell carcinoma. Cancer, 80, 1768-75. Giella, J.G., Ring, K., Olsson, C.A., Karp, F.S. & Benson, M.C. (1992). The predictive value of flow cytometry and urinary cytology in the followup of patients with transitional cell carcinoma of the bladder. J Urol, 148, 293-6. Gil-del-Alamo, P., Saccomanno, K., Lania, A., Pettersson, K.S., Beck-Peccoz, P. & Spada, A. (1995). Serum levels of beta-subunit of chorionic gonadotropin in patients with pituitary tumors. Eur J Endocrinol, 133, 33-7. Gillott, D.J., Iles, R.K. & Chard, T. (1996). The effects of beta-human chorionic gonadotrophin on the in vitro growth of bladder cancer cell lines. Br J Cancer, 73, 3236. Giovangrandi, Y., Parfait, B., Asheuer, M., Olivi, M., Lidereau, R., Vidaud, M. & Bieche, I. (2001). Analysis of the human CGB/LHB gene cluster in breast tumors by real-time quantitative RT-PCR assays. Cancer Lett, 168, 93-100. Girgin, C., Tarhan, H., Hekimgil, M., Sezer, A. & Gurel, G. (2001). P53 mutations and other prognostic factors of renal cell carcinoma. Urol Int, 66, 78-83. Goldstein, S., Jones, R.A., Hardin, J.W., Braunstein, G.D. & Shmookler Reis, R.J. (1990). Expression of alpha- and beta-human chorionic gonadotropin subunits in cultured human cells. In Vitro Cell Dev Biol, 26, 857-64. Golimbu, M., Al-Askari, S., Tessler, A. & Morales, P. (1986a). Aggressive treatment of metastatic renal cancer. J Urol, 136, 805-7. Golimbu, M., Joshi, P., Sperber, A., Tessler, A., Al-Askari, S. & Morales, P. (1986b). Renal cell carcinoma: survival and prognostic factors. Urology, 27, 291-301. Gourlay, W., Chan, V. & Gilks, C.B. (1995). Screening for urothelial malignancies by cytologic analysis and flow cytometry in a community urologic practice: a prospective study. Mod Pathol, 8, 394-7. Grankvist, K., Ljungberg, B. & Rasmuson, T. (1997). Evaluation of five glycoprotein tumour markers (CEA, CA50, CA-19-9, CA-125, CA-15-3) for the prognosis of renal-cell carcinoma. Int J Cancer, 74, 233-6. Gregoire, M., Fradet, Y., Meyer, F., Tetu, B., Bois, R., Bedard, G., Charrois, R. & Naud, A. (1997). Diagnostic accuracy of urinary cytology, and deoxyribonucleic acid flow cytometry and cytology on bladder washings during followup for bladder tumors. J Urol, 157, 1660-4. Grignon, D.J., Abdel-Malak, M., Mertens, W.C., Sakr, W.A. & Shepherd, R.R. (1994). Glutathione S-transferase expression in renal cell carcinoma: a new marker of differentiation. Mod Pathol, 7, 186-9. Harbour-McMenamin, D., Smith, E.M. & Blalock, J.E. (1986). Production of immunoreactive chorionic gonadotropin during mixed lymphocyte reactions: a possible selective mechanism for genetic diversity. Proc Natl Acad Sci U S A, 83, 6834-8. Harley, C.B., Futcher, A.B. & Greider, C.W. (1990). Telomeres shorten during ageing of human fibroblasts. Nature, 345, 458-60. Hatch, T.R. & Barry, J.M. (1986). The value of excretory urography in staging bladder cancer. J Urol, 135, 49. Hattori, M., Fukase, M., Yoshimi, H., Matsukura, S. & Imura, H. (1978). Ectopic production of human chorionic gonadotropin in malignant tumors. Cancer, 42, 232833. Hautkappe, A.L., Lu, M., Mueller, H., Bex, A., Harstrick, A., Roggendorf, M. & Ruebben, H. (2000). Detection of germ-cell tumor cells in the peripheral blood by nested reverse transcription-polymerase chain reaction for alphafetoprotein-messenger RNA and beta human chorionic gonadotropin-messenger RNA. Cancer Res, 60, 3170-4. Hedström, J., Grenman, R., Ramsay, H., Finne, P., Lundin, J., Haglund, C., Alfthan, H. & Stenman, U.H. (1999). Concentration of free hCGbeta subunit in serum as a prog- 47 Kristina Hotakainen nostic marker for squamous-cell carcinoma of the oral cavity and oropharynx. Int J Cancer, 84, 525-8. Hein, D.W., Doll, M.A., Rustan, T.D., Gray, K., Feng, Y., Ferguson, R.J. & Grant, D.M. (1993). Metabolic activation and deactivation of arylamine carcinogens by recombinant human NAT1 and polymorphic NAT2 acetyltransferases. Carcinogenesis, 14, 1633-8. Heitz, P.U., Kasper, M., Kloppel, G., Polak, J.M. & Vaitukaitis, J.L. (1983). Glycoprotein-hormone alphachain production by pancreatic endocrine tumors: a specific marker for malignancy. Immunocytochemical analysis of tumors of 155 patients. Cancer, 51, 277-82. Heney, N.M., Ahmed, S., Flanagan, M.J., Frable, W., Corder, M.P., Hafermann, M.D. & Hawkins, I.R. (1983). Superficial bladder cancer: progression and recurrence. J Urol, 130, 1083-6. Herr, H.W. (1994). Partial nephrectomy for incidental renal cell carcinoma. Br J Urol, 74, 431-3. Herr, H.W. (1997). Natural history of superficial bladder tumors: 10- to 20-year follow-up of treated patients. World J Urol, 15, 84-8. Herr, H.W. (2000). Tumor progression and survival of patients with high grade, noninvasive papillary (TaG3) bladder tumors: 15-year outcome. J Urol, 163, 60-1; discussion 61-2. Herr, H.W., Schwalb, D.M., Zhang, Z.F., Sogani, P.C., Fair, W.R., Whitmore, W.F., Jr. & Oettgen, H.F. (1995). Intravesical bacillus Calmette-Guerin therapy prevents tumor progression and death from superficial bladder cancer: ten-year follow-up of a prospective randomized trial. J Clin Oncol, 13, 1404-8. Hilton, S. (2000). Imaging of renal cell carcinoma. Semin Oncol, 27, 150-9. Hofmockel, G., Tsatalpas, P., Muller, H., Dammrich, J., Poot, M., Maurer-Schultze, B., Muller-Hermelink, H.K., Frohmuller, H.G. & Bassukas, I.D. (1995). Significance of conventional and new prognostic factors for locally confined renal cell carcinoma. Cancer, 76, 296-306. Hoon, D.S., Wang, Y., Dale, P.S., Conrad, A.J., Schmid, P., Garrison, D., Kuo, C., Foshag, L.J., Nizze, A.J. & Morton, D.L. (1995). Detection of occult melanoma cells in blood with a multiple-marker polymerase chain reaction assay. J Clin Oncol, 13, 2109-16. Hu, X.C. & Chow, L.W. (2000). Detection of circulating breast cancer cells by reverse transcriptase polymerase chain reaction (RT-PCR). Eur J Surg Oncol, 26, 530-5. Hu, X.C. & Chow, L.W. (2001). Detection of circulating breast cancer cells with multiple-marker RT-PCR assay. Anticancer Res, 21, 421-4. Huang, S., Rhee, E., Patel, H., Park, E. & Kaswick, J. (2000). Urinary NMP22 and renal cell carcinoma. Urology, 55, 227-30. Huang, S.C., Hsieh, C.Y., Hwang, J.L., Ouyang, P.C. & Chen, H.C. (1989). Free alpha subunit of human chorionic gonadotropin in women with non-trophoblastic tumors. Taiwan Yi Xue Hui Za Zhi, 88, 218-25. Hudson, M.A. & Herr, H.W. (1995). Carcinoma in situ of the bladder. J Urol, 153, 564-72. Huhtaniemi, I.T., Korenbrot, C.C. & Jaffe, R.B. (1977). HCG binding and stimulation of testosterone biosynthesis in the human fetal testis. J Clin Endocrinol Metab, 44, 963-7. Huland, H., Arndt, R., Huland, E., Loening, T. & Steffens, M. (1987). Monoclonal antibody 486 P 3/12: a valuable 48 bladder carcinoma marker for immunocytology. J Urol, 137, 654-9. Husgafvel-Pursiainen, K. & Kannio, A. (1996). Cigarette smoking and p53 mutations in lung cancer and bladder cancer. Environ Health Perspect, 104 Suppl 3, 553-6. Iles, R.K. & Butler, S.A. (1998). Human urothelial carcinomas—a typical disease of the aged: the clinical utility of human chorionic gonadotrophin in patient management and future therapy. Exp Gerontol, 33, 379-91. Iles, R.K. & Chard, T. (1989). Immunochemical analysis of the human chorionic gonadotrophin-like material secreted by ‘normal’ and neoplastic urothelial cells. J Mol Endocrinol, 2, 107-12. Iles, R.K. & Chard, T. (1991). Human chorionic gonadotropin expression by bladder cancers: biology and clinical potential. J Urol, 145, 453-8. Iles, R.K., Jenkins, B.J., Oliver, R.T., Blandy, J.P. & Chard, T. (1989). Beta human chorionic gonadotrophin in serum and urine. A marker for metastatic urothelial cancer. Br J Urol, 64, 241-4. Iles, R.K., Lee, C.L., Oliver, R.T. & Chard, T. (1990a). Composition of intact hormone and free subunits in the human chorionic gonadotrophin-like material found in serum and urine of patients with carcinoma of the bladder. Clin Endocrinol (Oxf), 33, 355-64. Iles, R.K., Oliver, R.T., Kitau, M., Walker, C. & Chard, T. (1987). In vitro secretion of human chorionic gonadotrophin by bladder tumour cells. Br J Cancer, 55, 623-6. Iles, R.K., Persad, R., Trivedi, M., Sharma, K.B., Dickinson, A., Smith, P. & Chard, T. (1996). Urinary concentration of human chorionic gonadotrophin and its fragments as a prognostic marker in bladder cancer. Br J Urol, 77, 619. Iles, R.K., Purkis, P.E., Whitehead, P.C., Oliver, R.T., Leigh, I. & Chard, T. (1990b). Expression of beta human chorionic gonadotrophin by non-trophoblastic non-endocrine ‘normal’ and malignant epithelial cells. Br J Cancer, 61, 663-6. Ind, T., Iles, R., Shepherd, J. & Chard, T. (1997). Serum concentrations of cancer antigen 125, placental alkaline phosphatase, cancer-associated serum antigen and free beta human chorionic gonadotrophin as prognostic markers for epithelial ovarian cancer. Br J Obstet Gynaecol, 104, 1024-9. Jacobsen, J., Rasmuson, T., Grankvist, K. & Ljungberg, B. (2000). Vascular endothelial growth factor as prognostic factor in renal cell carcinoma. J Urol, 163, 343-7. Jenkins, B.J., Martin, J.E., Baithun, S.I., Zuk, R.J., Oliver, R.T. & Blandy, J.P. (1990). Prediction of response to radiotherapy in invasive bladder cancer. Br J Urol, 65, 3458. Johansson, S.L. & Cohen, S.M. (1997). Epidemiology and etiology of bladder cancer. Semin Surg Oncol, 13, 291-8. Johnston, B., Morales, A., Emerson, L. & Lundie, M. (1997). Rapid detection of bladder cancer: a comparative study of point of care tests. J Urol, 158, 2098-101. Kamel, D., Turpeenniemi-Hujanen, T., Vahakangas, K., Paakko, P. & Soini, Y. (1994). Proliferating cell nuclear antigen but not p53 or human papillomavirus DNA correlates with advanced clinical stage in renal cell carcinoma. Histopathology, 25, 339-47. Kardana, A. & Cole, L.A. (1990). Serum HCG beta-core fragment is masked by associated macromolecules. J Clin Endocrinol Metab, 71, 1393-5. References Kavaler, E., Landman, J., Chang, Y., Droller, M.J. & Liu, B.C. (1998). Detecting human bladder carcinoma cells in voided urine samples by assaying for the presence of telomerase activity. Cancer, 82, 708-14. Kawamura, K., Tanaka, T., Ikeda, R., Fujikawa-Yamamoto, K. & Suzuki, K. (2000). DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry. Anal Quant Cytol Histol, 22, 26-30. Kido, A., Mori, M., Adachi, Y., Yukaya, H., Ishida, T. & Sugimachi, K. (1996). Immunohistochemical expression of beta-human chorionic gonadotropin in colorectal carcinoma. Surg Today, 26, 966-70. Kirkali, Z., Esen, A.A., Kirkali, G. & Guner, G. (1995). Ferritin: a tumor marker expressed by renal cell carcinoma. Eur Urol, 28, 131-4. Klein, F.A., Herr, H.W., Whitmore, W.F., Jr., Sogani, P.C. & Melamed, M.R. (1982). An evaluation of automated flow cytometry (FCM) in detection of carcinoma in situ of the urinary bladder. Cancer, 50, 1003-8. Konnak, J.W. & Grossman, H.B. (1985). Renal cell carcinoma as an incidental finding. J Urol, 134, 1094-6. Korhonen, J., Stenman, U.H. & Ylostalo, P. (1994). Serum human chorionic gonadotropin dynamics during spontaneous resolution of ectopic pregnancy. Fertil Steril, 61, 632-6. Koshida, K., Uchibayashi, T., Yamamoto, H. & Hirano, K. (1996). Significance of placental alkaline phosphatase (PLAP) in the monitoring of patients with seminoma. Br J Urol, 77, 138-42. Koss, L.G., Deitch, D., Ramanathan, R. & Sherman, A.B. (1985). Diagnostic value of cytology of voided urine. Acta Cytol, 29, 810-6. Koss, L.G., Wersto, R.P., Simmons, D.A., Deitch, D., Herz, F. & Freed, S.Z. (1989). Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors. Cancer, 64, 916-24. Kovacs, G., Akhtar, M., Beckwith, B.J., Bugert, P., Cooper, C.S., Delahunt, B., Eble, J.N., Fleming, S., Ljungberg, B., Medeiros, L.J., Moch, H., Reuter, V.E., Ritz, E., Roos, G., Schmidt, D., Srigley, J.R., Storkel, S., van den Berg, E. & Zbar, B. (1997). The Heidelberg classification of renal cell tumours. J Pathol, 183, 131-3. Krichevsky, A., Campbell-Acevedo, E.A., Tong, J.Y. & Acevedo, H.F. (1995). Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells. Endocrinology, 136, 1034-9. Kuida, C.A., Braunstein, G.D., Shintaku, P. & Said, J.W. (1988). Human chorionic gonadotropin expression in lung, breast, and renal carcinomas. Arch Pathol Lab Med, 112, 282-5. Lahat, N., Miller, A., Shtiller, R. & Touby, E. (1993). Differential effects of prolactin upon activation and differentiation of human B lymphocytes. Journal Of Neuroimmunology, 47, 35-40. Landy, H., Schneyer, A.L., Whitcomb, R.W. & Crowley, W.F., Jr. (1990). Validation of highly specific and sensitive radioimmunoassays for lutropin, follitropin, and free alpha subunit in unextracted urine. Clin Chem, 36, 3404. Lapthorn, A.J., Harris, D.C., Littlejohn, A., Lustbader, J.W., Canfield, R.E., Machin, K.J., Morgan, F.J. & Isaacs, N.W. (1994). Crystal structure of human chorionic gonadotropin. Nature, 369, 455-61. Latif, F., Tory, K., Gnarra, J., Yao, M., Duh, F.M., Orcutt, M.L., Stackhouse, T., Kuzmin, I., Modi, W., Geil, L. & et al. (1993). Identification of the von Hippel-Lindau disease tumor suppressor gene. Science, 260, 1317-20. Lazar, V., Diez, S.G., Laurent, A., Giovangrandi, Y., Radvanyi, F., Chopin, D., Bidart, J.M., Bellet, D. & Vidaud, M. (1995). Expression of human chorionic gonadotropin beta subunit genes in superficial and invasive bladder carcinomas. Cancer Res, 55, 3735-8. Leblanc, B., Duclos, A.J., Benard, F., Cote, J., Valiquette, L., Paquin, J.M., Mauffette, F., Faucher, R. & Perreault, J.P. (1999). Long-term followup of initial Ta grade 1 transitional cell carcinoma of the bladder. J Urol, 162, 194650. Levy, D.A., Slaton, J.W., Swanson, D.A. & Dinney, C.P. (1998). Stage specific guidelines for surveillance after radical nephrectomy for local renal cell carcinoma. J Urol, 159, 1163-7. Lewis, R.W., Jackson, A.C., Jr., Murphy, W.M., Leblanc, G.A. & Meehan, W.L. (1976). Cytology in the diagnosis and followup of transitional cell carcinoma of the urothelium: a review with a case series. J Urol, 116, 436. Licht, M.R., Novick, A.C. & Goormastic, M. (1994). Nephron sparing surgery in incidental versus suspected renal cell carcinoma. J Urol, 152, 39-42. Liedl, T. (1995). Flow cytometric DNA/cytokeratin analysis of bladder lavage: methodical aspects and clinical implications. Urol Int, 54, 22-47. Lin, J., Lojun, S., Lei, Z.M., Wu, W.X., Peiner, S.C. & Rao, C.V. (1995). Lymphocytes from pregnant women express human chorionic gonadotropin/luteinizing hormone receptor gene. Mol Cell Endocrinol, 111, R13-7. Linn, J.F., Lango, M., Halachmi, S., Schoenberg, M.P. & Sidransky, D. (1997). Microsatellite analysis and telomerase activity in archived tissue and urine samples of bladder cancer patients. Int J Cancer, 74, 625-9. Lintula, S. & Stenman, U.H. (1997). The expression of prostate-specific membrane antigen in peripheral blood leukocytes. J Urol, 157, 1969-72. Ljungberg, B., Alamdari, F.I., Rasmuson, T. & Roos, G. (1999). Follow-up guidelines for nonmetastatic renal cell carcinoma based on the occurrence of metastases after radical nephrectomy. BJU Int, 84, 405-11. Ljungberg, B., Bozoky, B., Kovacs, G., Stattin, P., Farrelly, E., Nylander, K. & Landberg, G. (2001). p53 expression in correlation to clinical outcome in patients with renal cell carcinoma. Scand J Urol Nephrol, 35, 15-20. Ljungberg, B., Landberg, G. & Alamdari, F.I. (2000). Factors of importance for prediction of survival in patients with metastatic renal cell carcinoma, treated with or without nephrectomy. Scand J Urol Nephrol, 34, 246-51. Ljungberg, B., Mehle, C., Stenling, R. & Roos, G. (1996). Heterogeneity in renal cell carcinoma and its impact no prognosis—a flow cytometric study. Br J Cancer, 74, 1237. Ljungberg, B., Rasmuson, T. & Grankvist, K. (1992). Erythropoietin in renal cell carcinoma: evaluation of its usefulness as a tumor marker. Eur Urol, 21, 160-3. Longuemaux, S., Delomenie, C., Gallou, C., Mejean, A., Vincent-Viry, M., Bouvier, R., Droz, D., Krishnamoorthy, R., Galteau, M.M., Junien, C., Beroud, C. & Dupret, 49 Kristina Hotakainen J.M. (1999). Candidate genetic modifiers of individual susceptibility to renal cell carcinoma: a study of polymorphic human xenobiotic-metabolizing enzymes. Cancer Res, 59, 2903-8. Louhimo, J., Nordling, S., Alfthan, H., von Boguslawski, K., Stenman, U.H. & Haglund, C. (2001). Specific staining of human chorionic gonadotropin beta in benign and malignant gastrointestinal tissues with monoclonal antibodies. Histopathology, 38, 418-24. Lundin, M., Nordling, S., Lundin, J., Alfthan, H., Stenman, U.H. & Haglund, C. (2001). Tissue expression of human chorionic gonadotropin beta predicts outcome in colorectal cancer: a comparison with serum expression. Int J Cancer, 95, 18-22. Madersbacher, S., Kratzik, C., Gerth, R., Dirnhofer, S. & Berger, P. (1994). Human chorionic gonadotropin (hCG) and its free subunits in hydrocele fluids and neoplastic tissue of testicular cancer patients: insights into the in vivo hCG-secretion pattern. Cancer Res, 54, 5096-100. Malaguarnera, M., Ferlito, L., Gulizia, G., Di Fazio, I. & Pistone, G. (2001). Use of interleukin-2 in advanced renal carcinoma: meta-analysis and review of the literature. Eur J Clin Pharmacol, 57, 267-73. Malmstrom, P.U., Busch, C. & Norlen, B.J. (1987). Recurrence, progression and survival in bladder cancer. A retrospective analysis of 232 patients with greater than or equal to 5-year follow-up. Scand J Urol Nephrol, 21, 18595. Mao, L., Schoenberg, M.P., Scicchitano, M., Erozan, Y.S., Merlo, A., Schwab, D. & Sidransky, D. (1996). Molecular detection of primary bladder cancer by microsatellite analysis. Science, 271, 659-62. Marcillac, I., Cottu, P., Theodore, C., Terrier-Lacombe, M.J., Bellet, D. & Droz, J.P. (1993). Free hCG-beta subunit as tumour marker in urothelial cancer. Lancet, 341, 13545. Marcillac, I., Troalen, F., Bidart, J.M., Ghillani, P., Ribrag, V., Escudier, B., Malassagne, B., Droz, J.P., Lhomme, C., Rougier, P. & et al. (1992). Free human chorionic gonadotropin beta subunit in gonadal and nongonadal neoplasms. Cancer Res, 52, 3901-7. Martin, J.E., Jenkins, B.J., Zuk, R.J., Oliver, R.T. & Baithun, S.I. (1989). Human chorionic gonadotrophin expression and histological findings as predictors of response to radiotherapy in carcinoma of the bladder. Virchows Arch A Pathol Anat Histopathol, 414, 273-7. McCredie, M. & Stewart, J.H. (1992). Risk factors for kidney cancer in New South Wales—I. Cigarette smoking. Eur J Cancer, 28A, 2050-4. McFarland, K.C., Sprengel, R., Phillips, H.S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D.L. & Seeburg, P.H. (1989). Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science, 245, 494-9. McLaughlin, J.K., Hrubec, Z., Heineman, E.F., Blot, W.J. & Fraumeni, J.F., Jr. (1990). Renal cancer and cigarette smoking in a 26-year followup of U.S. veterans. Public Health Rep, 105, 535-7. McLaughlin, J.K. & Lipworth, L. (2000). Epidemiologic aspects of renal cell cancer. Semin Oncol, 27, 115-23. McLoughlin, J., Pepera, T., Bridger, J. & Williams, G. (1991). Serum and urinary levels of beta human chorionic gonadotrophin in patients with transitional cell carcinoma. Br J Cancer, 63, 822-4. 50 Medeiros, L.J., Gelb, A.B. & Weiss, L.M. (1988). Renal cell carcinoma. Prognostic significance of morphologic parameters in 121 cases. Cancer, 61, 1639-51. Mellemgaard, A., McLaughlin, J.K., Overvad, K. & Olsen, J.H. (1996). Dietary risk factors for renal cell carcinoma in Denmark. Eur J Cancer, 32A, 673-82. Messerli, F.H. & Grossman, E. (1999). Beta-blockers and diuretics: to use or not to use. Am J Hypertens, 12, 157S163S. Mian, C., Pycha, A., Wiener, H., Haitel, A., Lodde, M. & Marberger, M. (1999). Immunocyt: a new tool for detecting transitional cell cancer of the urinary tract. J Urol, 161, 1486-9. Miller-Lindholm, A.K., LaBenz, C.J., Ramey, J., Bedows, E. & Ruddon, R.W. (1997). Human chorionic gonadotropin-beta gene expression in first trimester placenta. Endocrinology, 138, 5459-65. Minasian, L.M., Motzer, R.J., Gluck, L., Mazumdar, M., Vlamis, V. & Krown, S.E. (1993). Interferon alfa-2a in advanced renal cell carcinoma: treatment results and survival in 159 patients with long-term follow-up. J Clin Oncol, 11, 1368-75. Miyanaga, N., Akaza, H., Ishikawa, S., Ohtani, M., Noguchi, R., Kawai, K., Koiso, K., Kobayashi, M., Koyama, A. & Takahashi, T. (1997). Clinical evaluation of nuclear matrix protein 22 (NMP22) in urine as a novel marker for urothelial cancer. Eur Urol, 31, 163-8. Moch, H., Presti, J.C., Jr., Sauter, G., Buchholz, N., Jordan, P., Mihatsch, M.J. & Waldman, F.M. (1996). Genetic aberrations detected by comparative genomic hybridization are associated with clinical outcome in renal cell carcinoma. Cancer Res, 56, 27-30. Mora, J., Gascon, N., Tabernero, J.M., Rodriguez-Espinosa, J. & Gonzalez-Sastre, F. (1996). Different hCG assays to measure ectopic hCG secretion in bladder carcinoma patients. Br J Cancer, 74, 1081-4. Mora, L.B., Nicosia, S.V., Pow-Sang, J.M., Ku, N.K., Diaz, J.I., Lockhart, J. & Einstein, A. (1996). Ancillary techniques in the followup of transitional cell carcinoma: a comparison of cytology, histology and deoxyribonucleic acid image analysis cytometry in 91 patients. J Urol, 156, 49-54; discussion 54-5. Morita, R., Saito, S., Ishikawa, J., Ogawa, O., Yoshida, O., Yamakawa, K. & Nakamura, Y. (1991). Common regions of deletion on chromosomes 5q, 6q, and 10q in renal cell carcinoma. Cancer Res, 51, 5817-20. Mostofi FK, S.L., Torloni H. (1973). Histological typing of urinary bladder tumors. International Classification of Tumors. Vol. 19. WHO: Geneva. Motoo, Y., Watanabe, H., Yamaguchi, Y., Mouri, I., Fujii, T., Yamakawa, O., Satomura, Y., Okai, T. & Sawabu, N. (1996). Urinary gonadotropin peptide in patients with cancer of digestive organs. Anticancer Res, 16, 2041-8. Motzer, R.J., Mazumdar, M., Bacik, J., Berg, W., Amsterdam, A. & Ferrara, J. (1999). Survival and prognostic stratification of 670 patients with advanced renal cell carcinoma. J Clin Oncol, 17, 2530-40. Motzer, R.J., Mazumdar, M., Bacik, J., Russo, P., Berg, W.J. & Metz, E.M. (2000). Effect of cytokine therapy on survival for patients with advanced renal cell carcinoma. J Clin Oncol, 18, 1928-35. Motzer, R.J., Russo, P., Nanus, D.M. & Berg, W.J. (1997). Renal cell carcinoma. Curr Probl Cancer, 21, 185-232. Mourah, S., Cussenot, O., Vimont, V., Desgrandchamps, References F., Teillac, P., Cochant-Priollet, B., Le Duc, A., Fiet, J. & Soliman, H. (1998). Assessment of microsatellite instability in urine in the detection of transitional-cell carcinoma of the bladder. Int J Cancer, 79, 629-33. Moutzouris, G., Yannopoulos, D., Barbatis, C., Zaharof, A. & Theodorou, C. (1993). Is beta-human chorionic gonadotrophin production by transitional cell carcinoma of the bladder a marker of aggressive disease and resistance to radiotherapy? Br J Urol, 72, 907-9. Muller, Y.A., Li, B., Christinger, H.W., Wells, J.A., Cunningham, B.C. & de Vos, A.M. (1997). Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proc Natl Acad Sci U S A, 94, 7192-7. Murphy, W.M., Emerson, L.D., Chandler, R.W., Moinuddin, S.M. & Soloway, M.S. (1986). Flow cytometry versus urinary cytology in the evaluation of patients with bladder cancer. J Urol, 136, 815-9. Murphy, W.M., Soloway, M.S., Jukkola, A.F., Crabtree, W.N. & Ford, K.S. (1984). Urinary cytology and bladder cancer. The cellular features of transitional cell neoplasms. Cancer, 53, 1555-65. Murray, G.I., McFadyen, M.C., Mitchell, R.T., Cheung, Y.L., Kerr, A.C. & Melvin, W.T. (1999). Cytochrome P450 CYP3A in human renal cell cancer. Br J Cancer, 79, 183642. Nanus, D.M. (2000). New treatment approaches for metastatic renal cell carcinoma. Curr Oncol Rep, 2, 417-22. Nativ, O., Sabo, E., Raviv, G., Madjar, S., Halachmi, S. & Moskovitz, B. (1997). The impact of tumor size on clinical outcome in patients with localized renal cell carcinoma treated by radical nephrectomy. J Urol, 158, 72932. Nurmi, M. & Rintala E. (2002). Virtsateiden kasvaimet. In Urologia, Nurmi, M., Lukkarinen, O., Ruutu, M., Taari, K. & Tammela, T. (eds) pp. 91-107. Kustannus Oy Duodecim: Helsinki. Nurmi, M., Puntala, P., Tuominen, J. & Antila, L. (1985). Prognostic significance of symptoms and findings in renal adenocarcinoma. Strahlentherapie, 161, 632-5. Nurmi, M.J. (1984). Prognostic factors in renal carcinoma. An evaluation of operative findings. Br J Urol, 56, 2705. Oliver, R.T., Stephenson, C., Collino, C.E. & Parkinson, M.C. (1988). Clinicopathological significance of immunoreactive beta-hCG production by bladder cancer. Mol Biother, 1, 43-5. Otani, T., Otani, F., Krych, M., Chaplin, D.D. & Boime, I. (1988). Identification of a promoter region in the CG beta gene cluster. J Biol Chem, 263, 7322-9. Ozturk, M., Berkowitz, R., Goldstein, D., Bellet, D. & Wands, J.R. (1988). Differential production of human chorionic gonadotropin and free subunits in gestational trophoblastic disease. Am J Obstet Gynecol, 158, 193-8. Pagano, F., Garbeglio, A., Milani, C., Bassi, P. & Pegoraro, V. (1987). Prognosis of bladder cancer. I. Risk factors in superficial transitional cell carcinoma. Eur Urol, 13, 1459. Paju, A., Jacobsen, J., Rasmuson, T., Stenman, U.H. & Ljungberg, B. (2001). Tumor associated trypsin inhibitor as a prognostic factor in renal cell carcinoma. J Urol, 165, 959-62. Papanicolaou, G.N. & Marrshall, V.f. (1945). Urine sediment smears as a diagnostic procedure in cancers of the urinary tract. Science, 101, 519-520. Papapetrou, P.D. & Nicopoulou, S.C. (1986). The origin of a human chorionic gonadotropin beta-subunit-core fragment excreted in the urine of patients with cancer. Acta Endocrinol (Copenh), 112, 415-22. Parkin, D.M., Pisani, P. & Ferlay, J. (1999). Global cancer statistics. CA Cancer J Clin, 49, 33-64, 1. Parry, W.L. & Hemstreet, G.P., 3rd. (1988). Cancer detection by quantitative fluorescence image analysis. J Urol, 139, 270-4. Pellegrini, I., Lebrun, J.J., Ali, S. & Kelly, P.A. (1992). Expression of prolactin and its receptor in human lymphoid cells. Molecular Endocrinology, 6, 1023-31. Phillips, O.P., Elias, S., Shulman, L.P., Andersen, R.N., Morgan, C.D. & Simpson, J.L. (1992). Maternal serum screening for fetal Down syndrome in women less than 35 years of age using alpha-fetoprotein, hCG, and unconjugated estriol: a prospective 2-year study. Obstet Gynecol, 80, 353-8. Pode, D., Shapiro, A., Wald, M., Nativ, O., Laufer, M. & Kaver, I. (1999). Noninvasive detection of bladder cancer with the BTA stat test. J Urol, 161, 443-6. Policastro, P., Ovitt, C.E., Hoshina, M., Fukuoka, H., Boothby, M.R. & Boime, I. (1983). The beta subunit of human chorionic gonadotropin is encoded by multiple genes. J Biol Chem, 258, 11492-9. Policastro, P.F., Daniels-McQueen, S., Carle, G. & Boime, I. (1986). A map of the hCG beta-LH beta gene cluster. J Biol Chem, 261, 5907-16. Pycha, A., Mian, C., Haitel, A., Hofbauer, J., Wiener, H. & Marberger, M. (1997). Fluorescence in situ hybridization identifies more aggressive types of primarily noninvasive (stage pTa) bladder cancer. J Urol, 157, 21169. Raitanen, M.P., Hellstrom, P., Marttila, T., Korhonen, H., Talja, M., Ervasti, J. & Tammela, T.L. (2001). Effect of Intravesical Instillations on the Human Complement Factor H Related Protein (BTA stat) Test. Eur Urol, 40, 422-6. Ramakumar, S., Bhuiyan, J., Besse, J.A., Roberts, S.G., Wollan, P.C., Blute, M.L. & O’Kane, D.J. (1999). Comparison of screening methods in the detection of bladder cancer. J Urol, 161, 388-94. Rayford, P.L., Vaitukaitis, J.L., Ross, G.T., Morgan, F.J. & Canfield, R.E. (1972). Use of specific antisera to characterize biologic activity of hCG-beta subunit preparations. Endocrinology, 91, 144-6. Reimer, T., Koczan, D., Muller, H., Friese, K., Krause, A., Thiesen, H.J. & Gerber, B. (2000). Human chorionic gonadotrophin-beta transcripts correlate with progesterone receptor values in breast carcinomas. J Mol Endocrinol, 24, 33-41. Reiter, R.E., Anglard, P., Liu, S., Gnarra, J.R. & Linehan, W.M. (1993). Chromosome 17p deletions and p53 mutations in renal cell carcinoma. Cancer Res, 53, 3092-7. Reshef, E., Lei, Z.M., Rao, C.V., Pridham, D.D., Chegini, N. & Luborsky, J.L. (1990). The presence of gonadotropin receptors in nonpregnant human uterus, human placenta, fetal membranes, and decidua. J Clin Endocrinol Metab, 70, 421-30. Richman, A.M., Mayne, S.T., Jekel, J.F. & Albertsen, P. (1998). Image analysis combined with visual cytology in the early detection of recurrent bladder carcinoma. Cancer, 82, 1738-48. 51 Kristina Hotakainen Ross, J.S. & Cohen, M.B. (2000). Ancillary methods for the detection of recurrent urothelial neoplasia. Cancer, 90, 75-86. Rubben, H., Bubenzer, J., Bokenkamp, K., Lutzeyer, W. & Rathert, P. (1979). Grading of transitional cell tumours of the urinary tract by urinary cytology. Urol Res, 7, 8391. Russell, D.H., Kibler, R., Matrisian, L., Larson, D.F., Poulos, B. & Magun, B.E. (1985). Prolactin receptors on human T and B lymphocytes: antagonism of prolactin binding by cyclosporine. Journal Of Immunology, 134, 3027-31. Sabharwal, P., Glaser, R., Lafuse, W., Varma, S., Liu, Q., Arkins, S., Kooijman, R., Kutz, L., Kelley, K.W. & Malarkey, W.B. (1992a). Prolactin synthesized and secreted by human peripheral blood mononuclear cells: an autocrine growth factor for lymphoproliferation. Proc Natl Acad Sci U S A, 89, 7713-6. Sabharwal, P., Varma, S. & Malarkey, W.B. (1992b). Human thymocytes secrete luteinizing hormone: an autocrine regulator of T-cell proliferation. Biochem Biophys Res Commun, 187, 1187-92. Sabo, E., Miselevich, I., Bejar, J., Segenreich, M., Wald, M., Moskovitz, B. & Nativ, O. (1997). The role of vimentin expression in predicting the long-term outcome of patients with localized renal cell carcinoma. Br J Urol, 80, 864-8. Sanchez-Carbayo, M., Herrero, E., Megias, J., Mira, A. & Soria, F. (1999). Evaluation of nuclear matrix protein 22 as a tumour marker in the detection of transitional cell carcinoma of the bladder. BJU Int, 84, 706-13. Sarkis, A.S., Dalbagni, G., Cordon-Cardo, C., Zhang, Z.F., Sheinfeld, J., Fair, W.R., Herr, H.W. & Reuter, V.E. (1993). Nuclear overexpression of p53 protein in transitional cell bladder carcinoma: a marker for disease progression. J Natl Cancer Inst, 85, 53-9. Sauter, G., Gasser, T.C., Moch, H., Richter, J., Jiang, F., Albrecht, R., Novotny, H., Wagner, U., Bubendorf, L. & Mihatsch, M.J. (1997). DNA aberrations in urinary bladder cancer detected by flow cytometry and FISH. Urol Res, 25 Suppl 1, S37-43. Schapers, R.F., Smeets, A.W., Pauwels, R.P., van den Brandt, P.A. & Bosman, F.T. (1993). Cytogenetic analysis in transitional cell carcinoma of the bladder. Br J Urol, 72, 88792. Schmetter, B.S., Habicht, K.K., Lamm, D.L., Morales, A., Bander, N.H., Grossman, H.B., Hanna, M.G., Jr., Silberman, S.R. & Butman, B.T. (1997). A multicenter trial evaluation of the fibrin/fibrinogen degradation products test for detection and monitoring of bladder cancer. J Urol, 158, 801-5. Schröder, J., Suomalainen, H.A., Parkkinen, E., Lundqvist, C., Wada, G. & Koskimies, S. (1982). Specificity of monoclonal antibodies to an EBV transformed B-cell line. Med Biol, 60, 255-9. See, W.A. & Fuller, J.R. (1992). Staging of advanced bladder cancer. Current concepts and pitfalls. Urol Clin North Am, 19, 663-83. Serretta, V., Lo Presti, D., Vasile, P., Gange, E., Esposito, E. & Menozzi, I. (1998). Urinary NMP22 for the detection of recurrence after transurethral resection of transitional cell carcinoma of the bladder: experience on 137 patients. Urology, 52, 793-6. Shalev, M., Gdor, Y., Leventis, A., Radnay, J., Shapiro, H., Bernheim, J. & Nissenkorn, I. (2001). The prognostic 52 value of DNA ploidy in small renal cell carcinoma. Pathol Res Pract, 197, 7-12. Skinner, D.G., Colvin, R.B., Vermillion, C.D., Pfister, R.C. & Leadbetter, W.F. (1971). Diagnosis and management of renal cell carcinoma. A clinical and pathologic study of 309 cases. Cancer, 28, 1165-77. Smith, D.J., Evans, H.J., Newman, J. & Chapple, C.R. (1994). Ectopic human chorionic gonadotrophin (HCG) production: is the detection by serum analysis of HCG of clinical relevance in transitional cell carcinoma of the bladder? Br J Urol, 73, 409-12. Smith, E.M., Harbour-McMenamin, D. & Blalock, J.E. (1985). Lymphocyte production of endorphins and endorphin-mediated immunoregulatory activity. J Immunol, 135, 779s-782s. Smith, S.J., Bosniak, M.A., Megibow, A.J., Hulnick, D.H., Horii, S.C. & Raghavendra, B.N. (1989). Renal cell carcinoma: earlier discovery and increased detection. Radiology, 170, 699-703. Sobin, L.H. & Wittekind, C.H. (1997). TNM classification of Malignant tumors. International union against cancer. Wiley-Liss: New York. Soloway, M.S. (1983). Surgery and intravesical chemotherapy in the management of superficial bladder cancer. Semin Urol, 1, 23-33. Soloway, M.S. (1990). Invasive bladder cancer: selection of primary treatment. Semin Oncol, 17, 551-4. Soloway, M.S., Briggman, V., Carpinito, G.A., Chodak, G.W., Church, P.A., Lamm, D.L., Lange, P., Messing, E., Pasciak, R.M., Reservitz, G.B., Rukstalis, D.B., Sarosdy, M.F., Stadler, W.M., Thiel, R.P. & Hayden, C.L. (1996). Use of a new tumor marker, urinary NMP22, in the detection of occult or rapidly recurring transitional cell carcinoma of the urinary tract following surgical treatment. J Urol, 156, 363-7. Soloway, M.S. & Perito, P.E. (1992). Superficial bladder cancer: diagnosis, surveillance and treatment. J Cell Biochem Suppl, 16I, 120-7. Song, Z., Sun, X. & Wu, D. (1995). Significance of flow cytometry in the diagnosis and treatment of bladder tumors. Chin Med Sci J, 10, 38-41. Sorahan, T., Lancashire, R.J. & Sole, G. (1994). Urothelial cancer and cigarette smoking: findings from a regional case-controlled study. Br J Urol, 74, 753-6. Sozen, S., Biri, H., Sinik, Z., Kupeli, B., Alkibay, T. & Bozkirli, I. (1999). Comparison of the nuclear matrix protein 22 with voided urine cytology and BTA stat test in the diagnosis of transitional cell carcinoma of the bladder. Eur Urol, 36, 225-9. Steineck, G., Plato, N., Norell, S.E. & Hogstedt, C. (1990). Urothelial cancer and some industry-related chemicals: an evaluation of the epidemiologic literature. Am J Ind Med, 17, 371-91. Steiner, G., Schoenberg, M.P., Linn, J.F., Mao, L. & Sidransky, D. (1997). Detection of bladder cancer recurrence by microsatellite analysis of urine. Nat Med, 3, 6214. Stenman, U.H., Alfthan, H., Ranta, T., Vartiainen, E., Jalkanen, J. & Seppala, M. (1987). Serum levels of human chorionic gonadotropin in nonpregnant women and men are modulated by gonadotropin-releasing hormone and sex steroids. J Clin Endocrinol Metab, 64, 730-6. Stenman, U.H., Alfthan, H., Vartiainen, J. & Lehtovirta, P. (1995). Markers supplementing CA 125 in ovarian can- References cer. Ann Med, 27, 115-20. Sufrin, G., Mirand, E.A., Moore, R.H., Chu, T.M. & Murphy, G.P. (1977). Hormones in renal cancer. J Urol, 117, 433-8. Syrigos, K.N., Fyssas, I., Konstandoulakis, M.M., Harrington, K.J., Papadopoulos, S., Milingos, N., Peveretos, P. & Golematis, B.C. (1998). Beta human chorionic gonadotropin concentrations in serum of patients with pancreatic adenocarcinoma. Gut, 42, 88-91. Taback, B., Chan, A.D., Kuo, C.T., Bostick, P.J., Wang, H.J., Giuliano, A.E. & Hoon, D.S. (2001). Detection of occult metastatic breast cancer cells in blood by a multimolecular marker assay: correlation with clinical stage of disease. Cancer Res, 61, 8845-50. Takahashi, M., Rhodes, D.R., Furge, K.A., Kanayama, H., Kagawa, S., Haab, B.B. & Teh, B.T. (2001). Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification. Proc Natl Acad Sci U S A, 98, 9754-9. Talmadge, K., Boorstein, W.R. & Fiddes, J.C. (1983). The human genome contains seven genes for the beta-subunit of chorionic gonadotropin but only one gene for the beta-subunit of luteinizing hormone. Dna, 2, 281-9. Talmadge, K., Boorstein, W.R., Vamvakopoulos, N.C., Gething, M.J. & Fiddes, J.C. (1984a). Only three of the seven human chorionic gonadotropin beta subunit genes can be expressed in the placenta. Nucleic Acids Res, 12, 8415-36. Talmadge, K., Vamvakopoulos, N.C. & Fiddes, J.C. (1984b). Evolution of the genes for the beta subunits of human chorionic gonadotropin and luteinizing hormone. Nature, 307, 37-40. Tannapfel, A., Hahn, H.A., Katalinic, A., Fietkau, R.J., Kuhn, R. & Wittekind, C.W. (1996). Prognostic value of ploidy and proliferation markers in renal cell carcinoma. Cancer, 77, 164-71. Tetu, B., Allard, P., Fradet, Y., Roberge, N. & Bernard, P. (1996). Prognostic significance of nuclear DNA content and S-phase fraction by flow cytometry in primary papillary superficial bladder cancer. Hum Pathol, 27, 922-6. Thrash-Bingham, C.A., Salazar, H., Freed, J.J., Greenberg, R.E. & Tartof, K.D. (1995). Genomic alterations and instabilities in renal cell carcinomas and their relationship to tumor pathology. Cancer Res, 55, 6189-95. Torti, F.M. & Lum, B.L. (1984). The biology and treatment of superficial bladder cancer. J Clin Oncol, 2, 505-31. Trias, I., Campo, E., Benasco, C., Palacin, A. & Cardesa, A. (1991). Human chorionic gonadotropin in esophageal carcinomas. An immunohistochemical study. Pathol Res Pract, 187, 503-7. Tribukait, B. & Esposti, P.L. (1978). Quantitative flowmicrofluorometric analysis of the DNA in cells from neoplasms of the urinary bladder: correlation of aneuploidy with histological grading and the cytological findings. Urol Res, 6, 201-5. Tsui, K.H., Shvarts, O., Smith, R.B., Figlin, R., de Kernion, J.B. & Belldegrun, A. (2000). Renal cell carcinoma: prognostic significance of incidentally detected tumors. J Urol, 163, 426-30. Tsuji, M., Kojima, K., Murakami, Y., Kanayama, H. & Kagawa, S. (1997). Prognostic value of Ki-67 antigen and p53 protein in urinary bladder cancer: immunohistochemical analysis of radical cystectomy specimens. Br J Urol, 79, 367-72. UICC. (1978). TNM classification of malignant tumors, Harmer, M. (ed) pp. 113-117. WHO: Geneva. Utz, D.C., Farrow, G.M., Rife, C.C., Segura, J.W. & Zincke, H. (1980). Carcinoma in situ of the bladder. Cancer, 45, 1842-8. van Rhijn, B.W., Lurkin, I., Kirkels, W.J., van der Kwast, T.H. & Zwarthoff, E.C. (2001). Microsatellite analysis— DNA test in urine competes with cystoscopy in followup of superficial bladder carcinoma: a phase II trial. Cancer, 92, 768-75. Varkarakis, M.J., Gaeta, J., Moore, R.H. & Murphy, G.P. (1974). Superficial bladder tumor. Aspects of clinical progression. Urology, 4, 414-20. Vartiainen, J., Alfthan, H., Lehtovirta, P. & Stenman, U.H. (1998). Identification of choriocarcinoma by the hCG beta-to-hCG proportion in patients with delayed diagnosis caused by contraceptive use. Contraception, 57, 25760. Vartiainen, J., Lehtovirta, P., Finne, P., Stenman, U.H. & Alfthan, H. (2001). Preoperative serum concentration of hCGbeta as a prognostic factor in ovarian cancer. Int J Cancer, 95, 313-6. Vasavada, S.P., Novick, A.C. & Williams, B.R. (1998). P53, bcl-2, and Bax expression in renal cell carcinoma. Urology, 51, 1057-61. Velickovic, M., Delahunt, B., Storkel, S. & Grebem, S.K. (2001). VHL and FHIT locus loss of heterozygosity is common in all renal cancer morphotypes but differs in pattern and prognostic significance. Cancer Res, 61, 48159. Venencie, P.Y., Meduri, G., Pissard, S., Jolivet, A., Loosfelt, H., Milgrom, E. & Misrahi, M. (1999). Luteinizing hormone/human chorionic gonadotrophin receptors in various epidermal structures. Br J Dermatol, 141, 438-46. Vogelzang, N.J. (1987). Prognostic factors in metastatic testicular cancer. Int J Androl, 10, 225-37. Vogelzang, N.J., Lipton, A. & Figlin, R.A. (1993). Subcutaneous interleukin-2 plus interferon alfa-2a in metastatic renal cancer: an outpatient multicenter trial. J Clin Oncol, 11, 1809-16. von Eyben, F.E., Blaabjerg, O., Hyltoft-Petersen, P., Madsen, E.L., Amato, R., Liu, F. & Fritsche, H. (2001). Serum lactate dehydrogenase isoenzyme 1 and prediction of death in patients with metastatic testicular germ cell tumors. Clin Chem Lab Med, 39, 38-44. Wada, Y., Igawa, M., Shiina, H., Shigeno, K., Yokogi, H., Urakami, S., Yoneda, T. & Maruyama, R. (1998). Comparison of chromosomal aberrations detected by fluorescence in situ hybridization with clinical parameters, DNA ploidy and Ki 67 expression in renal cell carcinoma. Br J Cancer, 77, 2003-7. Wang, Y.X., Schwartz, P.E., Chambers, J.T. & Cole, L.A. (1988). Urinary gonadotropin fragments (UGF) in cancers of the female reproductive system. II. Initial serial studies. Gynecol Oncol, 31, 91-102. Webb, A., Scott-Mackie, P., Cunningham, D., Norman, A., Andreyev, J., O’Brien, M. & Bensted, J. (1995). The prognostic value of CEA, beta HCG, AFP, CA125, CA19-9 and C-erb B-2, beta HCG immunohistochemistry in advanced colorectal cancer. Ann Oncol, 6, 581-7. Wehmann, R.E., Blithe, D.L., Akar, A.H. & Nisula, B.C. (1990). Disparity between beta-core levels in pregnancy urine and serum: implications for the origin of urinary beta-core. J Clin Endocrinol Metab, 70, 371-8. 53 Kristina Hotakainen Weintraub, B.D. & Rosen, S.W. (1973). Ectopic production of the isolated beta subunit of human chorionic gonadotropin. J Clin Invest, 52, 335-42. Weissbach, L., Bussar-Maatz, R. & Mann, K. (1997). The value of tumor markers in testicular seminomas. Results of a prospective multicenter study. Eur Urol, 32, 16-22. Wilson, T.M., Yu-Lee, L.Y. & Kelley, M.R. (1995). Coordinate gene expression of luteinizing hormone-releasing hormone (LHRH) and the LHRH-receptor after prolactin stimulation in the rat Nb2 T-cell line: implications for a role in immunomodulation and cell cycle gene expression. Molecular Endocrinology, 9, 44-53. Wittke, F., Hoffmann, R., Buer, J., Dallmann, I., Oevermann, K., Sel, S., Wandert, T., Ganser, A. & Atzpodien, J. (1999). Interleukin 10 (IL-10): an immunosuppressive factor and independent predictor in patients with metastatic renal cell carcinoma. Br J Cancer, 79, 1182-4. Wojcik, E.M., Miller, M.C., O’Dowd, G.J. & Veltri, R.W. (1998). Value of computer-assisted quantitative nuclear grading in differentiation of normal urothelial cells from low and high grade transitional cell carcinoma. Anal Quant Cytol Histol, 20, 69-76. Wolf, H. & Hojgaard, K. (1983). Prognostic factors in local surgical treatment of invasive bladder cancer, with special reference to the presence of urothelial dysplasia. Cancer, 51, 1710-5. Wolk, A., Lindblad, P. & Adami, H.O. (1996). Nutrition and renal cell cancer. Cancer Causes Control, 7, 5-18. Wyczolkowski, M., Klima, W., Bieda, W. & Walas, K. (2001). Spontaneous regression of hepatic metastases after nephrectomy and metastasectomy of renal cell carcinoma. Urol Int, 66, 119-20. Yamaguchi, A., Ishida, T., Nishimura, G., Kumaki, T., Katoh, M., Kosaka, T., Yonemura, Y. & Miyazaki, I. (1989). Human chorionic gonadotropin in colorectal cancer and its relationship to prognosis. Br J Cancer, 60, 3824. Yamakawa, K., Morita, R., Takahashi, E., Hori, T., Ishikawa, 54 J. & Nakamura, Y. (1991). A detailed deletion mapping of the short arm of chromosome 3 in sporadic renal cell carcinoma. Cancer Res, 51, 4707-11. Yaman, O., Baltaci, S., Arikan, N., Ozdiler, E., Gogus, O. & Muftuoglu, Y.Z. (1996). Serum neuron specific enolase: can it be a tumour marker for renal cell carcinoma? Int Urol Nephrol, 28, 207-10. Yoshimi, T., Strott, C.A., Marshall, J.R. & Lipsett, M.B. (1969). Corpus luteum function in early pregnancy. J Clin Endocrinol Metab, 29, 225-30. Young, A.N., Amin, M.B., Moreno, C.S., Lim, S.D., Cohen, C., Petros, J.A., Marshall, F.F. & Neish, A.S. (2001). Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am J Pathol, 158, 1639-51. Zhang, Z.F., Sarkis, A.S., Cordon-Cardo, C., Dalbagni, G., Melamed, J., Aprikian, A., Pollack, D., Sheinfeld, J., Herr, H.W., Fair, W.R. & et al. (1994). Tobacco smoking, occupation, and p53 nuclear overexpression in early stage bladder cancer. Cancer Epidemiol Biomarkers Prev, 3, 19-24. Zippe, C., Pandrangi, L., Potts, J.M., Kursh, E., Novick, A. & Agarwal, A. (1999). NMP22: a sensitive, cost-effective test in patients at risk for bladder cancer. Anticancer Res, 19, 2621-3. Öberg, K. & Wide, L. (1981). hCG and hCG subunits as tumour markers in patients with endocrine pancreatic tumours and carcinoids. Acta Endocrinol (Copenh), 98, 25660. Öge, O., Atsu, N., Sahin, A. & Ozen, H. (2000). Comparison of BTA stat and NMP22 tests in the detection of bladder cancer. Scand J Urol Nephrol, 34, 349-51. Özen, H., Uygur, C., Sahin, A., Tekgul, S., Ergen, A. & Remzi, D. (1995). Clinical significance of serum ferritin in patients with renal cell carcinoma. Urology, 46, 494-8. Özkardes, H., Ergen, A., Ozen, H.A., Ayhan, A. & Remzi, D. (1991). Immunohistochemical detection of beta-human chorionic gonadotropin in urothelial carcinoma. Int Urol Nephrol, 23, 5-11.
© Copyright 2018