Vascular Endothelial Growth Factor in Renal Cell Carcinoma

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 1006 ISSN 0346-6612 ISBN 91-7264-029-4

Departments of Surgical and Perioperative Sciences, Urology and Andrology; Medical Biosciences, Clinical Chemistry; Radiation Sciences, Oncology, and Medical Biosciences, Pathology, Umeå University, Umeå, Sweden

Vascular endothelial growth factor in renal cell carcinoma

Jan Jacobsen

Umeå 2006

Printed by Hemströms Offset-Boktryck Härnösand, Sweden, 2006

This thesis is dedicated to Ida and Heini

– Den lige vej er den närmeste – men oftest når man den kun ad lange omveje. Robert Storm Petersen Danish artist (1882 - 1949)

ABSTRACT

VASCULAR ENDOTHELIAL GROWTH FACTOR IN RENAL CELL CARCINOMA Jan Jacobsen, Departments of Surgical and Perioperative Sciences, Urology and Andrology, Medical Biosciences, Clinical Chemistry; Radiation Sciences, Oncology, and Medical Biosciences, Pathology, Umeå University Medical Dissertations. New Series No. 1006 ISSN 0346-6612 ISBN 91- 7264-029-4. Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno- sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S- VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels comparable to those from kidney cortex, while VEGF165 was lower. In PRCC and ChRCC, VEGF189 levels correlated inversely with advancing tumour stage, and in PRCC, VEGF165 levels correlated inversely with increasing tumour size. VEGF189 was an independent prognostic factor for survival in patients with PRCC. Conclusions. S-VEGF has a stronger association to progression in RCC than platelet count. CRCC showed high levels of VEGF mRNA isoforms and VEGF-R1 mRNA compared to PRCC. VEGF mRNA isoforms expression decreased with advancing stage. IHC demonstrated VEGF expression in cell cytoplasm related to tumour growth, particular in CRCC. Different VEGF isoform patterns were found in different RCC types. Protein VEGF189 expression was associated with tumour stage and was an independent prognostic factor in PRCC. Protein VEGF165 expression was generally low and had no prognostic value. The trend for decreasing levels of VEGF isoforms in advanced tumour stages may indicate that angiogenic activity is an early event in tumour growth induced by VEGF, but that during later tumour progression the role of VEGF is less clear. Key words: VEGF, isoforms, quantitative RT-PCR, immunohistochemistry, tissue microarray, Western blot, stage, survival, renal cell carcinoma. 4

ORIGINAL PAPERS

This thesis is based on the following papers, which are referred to in the text by their roman numerals:

I Jacobsen J, Rasmuson T, Grankvist K and Ljungberg B. Vascular endothelial growth factor as prognostic factor in renal cell carcinoma. Journal of Urology 2000; 163(1): 343-7. Reprinted with permission from American Urological Association.

II Jacobsen J, Grankvist K, Rasmuson T, Ljungberg B. Prognostic importance of serum vascular endothelial growth factor in relation to platelet and leukocyte counts in human renal cell carcinoma. European Journal of Cancer Prevention 2002; 11(3): 245-52. Reprited with permission from Lippincott Williams & Wilkins.

III Ljungberg B, Jacobsen J, Häggström-Rudolfssson S, Rasmuson T, Lindh G, Grankvist K. Tumour vascular endothelial growth factor (VEGF) mRNA in relation to serum VEGF protein levels and tumour progression in human renal cell carcinoma. Urological Research 2003; 31(5): 335-40. Reprinted with kind permission of Springer Science and Business Media.

IV Jacobsen J, Grankvist K, Rasmuson T, Bergh A, Landberg G, Ljungberg B. Expression of vascular endothelial growth factor protein in human renal cell carcinoma. British Journal of Urology International 2004; 93(3): 297-302. Reprinted with kind permission of Blackwell Publishing.

V Jacobsen J, Grankvist K, Rasmuson T, Ljungberg B. Different isoform patterns for vascular endothelial growth factor between clear cell and papillary renal cell carcinoma. British Journal of Urology International, accepted for publication 2005. Printed with kind permission of Blackwell Publishing.

CONTENTS

ABSTRACT ...... 4

ORIGINAL PAPERS ...... 5

CONTENTS ...... 6

ABBREVIATIONS...... 8

INTRODUCTION ...... 10 Background ...... 10 Incidence ...... 10 Environmental risk factors...... 10 Inherited genetic vulnerability...... 11 Clinical and pathological prognostic factors...... 13 Histopathological grading and classification ...... 13 Angiogenesis in cancer ...... 16 Tumour secreted VEGF-A...... 17 Synergistic role of bFGF...... 18 Miscellaneous role of PDGF...... 18 AIMS OF THE INVESTIGATION ...... 20

MATERIAL AND METHODS ...... 21 Patients...... 21 Tumour staging ...... 21 Morphologic classification ...... 21 Methods...... 22 Blood analysis ...... 22 Serum analysis...... 23 Tissue collection and preparation ...... 24 Competitive quantitative RT-PCR ...... 24 Immunohistochemestry ...... 26 Protein extraction ...... 28 Western blotting ...... 28 Statistical methods ...... 29 RESULTS AND COMMENTS...... 31 Paper I...... 31 Comments on Paper I ...... 31 Paper II...... 31 Comments on Paper II ...... 32 Paper III...... 33 Comments on Paper III ...... 33 Paper IV...... 34 Comments on paper IV ...... 34

Study V...... 35 Comments on paper V ...... 35 FUTURE DIRECTIONS...... 37

CONCLUSIONS...... 39 Paper I...... 39 Paper II...... 39 Paper III...... 39 Paper IV...... 39 Paper V...... 39 ACKNOWLEDGEMENTS ...... 40

REFERENCES ...... 42

ABBREVIATIONS

AJCC American Joint Committee on Cancer Bcl-2 Gene encodes inhibitors of apoptosis Bcl-2 Protein encoded of Bcl-2 gene bFGF (basic) Fibroblast Growth Factor BHD Birt Hogg Dub’e syndrome cDNA (complementary) Deoxyribonucleic Acid ChRCC Chromophobe Renal Cell Carcinoma CRCC Conventional Renal Cell Carcinoma CT Computerised Tomography Cyclins D and E Promote cyclin-dependent kinases to activate cell cycle DNA Deoxyribonucleic Acid ECM Extra Cellular Matrix EGF Epidermal Growth Factor EGF-R Epidermal Growth Factor Receptor ELISA Enzyme Linked Immuno Sorbent Assay erb-B Gene encodes EGF-R erb-B Protein encoded by erb-B gene ESR Erythrocyte sedimentation rate FGF Fibroblast Growth Factor FH Gene encodes Fumarate Hydratase involved in citric acid cycle FH Protein encoded by FH gene HER-2 Synonymous to erb-B gene encodes EGF-R HER-2 Synonymous to erb-B HIF Hypoxia Inducible Factor HLRCC Hereditary Leiomyoma Renal Cell Carcinoma HPRC Hereditary Papillary Renal Cell Carcinoma IGF Insulin like Growth Factor IHC Immunohistochemistry IS Internal Standard bp Base Pairs kDA (kilo) Dalton met Gene encodes receptor with tyrosine kinase activity met Protein encoded by met gene MMP Matrix Metallo Proteinase MRI Magnetic Resonance Imaging mRNA (messenger) Ribonucleic Acid MVD Micro Vessel Density myc Gene encodes transcription activators and stimulating growth signals

8 myc Protein encoded by myc gene p27 Gene encodes transcription inhibitors of cell cycle p53 Gene encodes transcription inhibitors of cell cycle and activates apoptosis p53 Protein encoded by p53 gene PAD Pathological Anatomical Diagnosis PBS Phosphate Buffered Saline PDGF Platelet Derived Growth Factor pRb Gene encodes nuclear transcription factor once activated stimulates cell cycle PRCC Papillary Renal Cell Carcinoma pVHL (protein) VHL RCC Renal Cell Carcinoma RNA Ribonucleic Acid RT-PCR Reverse Transcription Polymerase Chain Reaction SDS Sodium Dodectyl Sulfate TGF Transforming Growth Factor TMA Tissue Micro Array TNF Tumour Necrosis Factor TNM Tumour Node Metastasis TS Tissue Section or Slide UICC Union Internationale Contre le Cancer VEGF Vascular Endothelial Growth Factor VEGF-R1 VEGF receptor 1 (flt-1) VEGF-R2 VEGF receptor 2 (KDR/flk-1) VHL von Hippel-Lindau VHL Gene encodes VHL promoting transcription of VEGF VHL Protein encoded by VHL gene WB Western Blot

INTRODUCTION

Background techniques such as ultrasonography, com- Renal cell carcinoma (RCC) is a puterised tomography (CT), and magnetic heterogeneous group of tumours, which resonance imaging (MRI) [8-10]. demonstrates wide variation in histopathological features, as well as clinical Environmental risk factors diversity, with unpredictable tumour Several epidemiologic studies have behaviour. The mortality-incidence ratio demonstrated an association between is higher in RCC than in other urological RCC and tobacco smoking and obesity malignancies [1]. In only 50-60% of RCC [11, 12], factors which have been asso- tumours which are localized to kidney is ciated in 40% of RCC cases in our high- surgical treatment curative [2, 3]. New risk society [13]. In rat experiments, the approaches in cancer treatment are needed, constituents from tobacco smoke, which and through genetic and molecular biolo- are excreted to the urine are thought to gical research novel molecular therapeutic cause RCC by VHL-mutations [14, 15]. targets have been identified [4, 5]. Moreover, gene-environment interactions Tumours seem to be influenced by serial have been recognized among smokers gene mutations and methylation with who have slow acetylator genotype for N- phenoltypical expression as dysfunctional acetyl-transferase, where there is an cell signalling, cell proliferation, inhi- increased risk for development of RCC bition of apoptosis, and promotion of [16]. A high body mass index or high angiogenesis during the growth [6]. body weight has been consistently linked Molecular targeting of dysfunction in cell to RCC [17, 18], predominantly in women signalling or angiogenesis is a potential [19]. Obesity itself or factors associated novel therapy, and the knowledge is with obesity may have a stressful effect essential in order to design new anti- on renal cell metabolism including angiogenic therapies for patients with through insulin resistance, elevated RCC. growth factors (EGF), free circulating Incidence estrogens, and insulin-like growth factor RCC constitutes 2-3% of adult (IGF-I), all of which may be involved in malignancies [7]. Almost 900 new cases development of RCC [20, 21]. Hyper- of RCC are observed annually in Sweden, tension seems to be associated with RCC, with 600 deaths each year attributed to but difficulties in separate possible side RCC. Men are affected more commonly effects from medication prevents classi- than women, with a ratio of 3:2. RCC is fying hypertension as an independent risk primarily a disease of the elderly, and is factor [22, 23]. Use of diuretics as well as usually detected in the sixth or seventh beta-blockers have been implicated as decade of life [1]. Worldwide, there is im- promoting the development of RCC, proved and earlier detection of localised though it remains unclear whether the tumours, largely due to expanded access medications or hypertension itself is the to better quality in radiological imaging active factor [22, 23]. The combination of

10 obesity and hypertension [24] might stress that code for proteins involved in re- the kidney and lead to renal damage with cognition and restitution of the damaged proliferation. Metabolic or functional DNA. They also initiate cell apoptosis. changes in renal tubules may result in Otherwise, continuous cell division would genetic alteration caused by exposure and transmit genetic defects, which could be increasing susceptibility to carcinogens. intensified by increasing susceptibility Other environmental risk factors are less due to carcinogenic exposure. The p53 well defined, including occupational gene codes for the protein p53, which exposure to trichloroethelene, nutritional when inactivated or impaired makes cells factors, chronic renal failure, urinary tract less able to restore damaged DNA [41, infection, use of analgesics or reproduce- 42]. Inactivation of the VHL gene induces tive hormones, and alcohol [13, 20, 22, mRNA transcription of growth factor, 25-29]. which generates anti-apoptotic signalling and promotes angiogenesis [43, 44] Inherited genetic vulnerability New cytogenetic and molecular gene- The genetic alterations in RCC may be tic data have established the association a consequence of activation of oncogenes between genetic characterisation and or inactivation of tumour suppressor gene. histopathological classification of RCC Since both copies of genes must be dis- [45]. Four histopathological types of RCC torted in order for a tumour to develop, a have been described and linked to genetic series of events are necessary [6]. diseases such as von Hippel-Lindau (VHL) Oncogenes are mutated proto-oncogenes disease, hereditary papil-lary renal carci- that normally code for proteins involved noma (HPRC), Birt-Hogg-Dubé (BHD) in control of cell division and differ- syndrome, and hereditary leiomyoma RCC rentiation. Intensified signalling in these (HLRCC) [46]. Hence, RCC can be divi- pathways can lead to uncontrolled cell ded into predominantly sporadic and non- division and make the cell become inherited form or an hereditary form, cancerous. An activated oncogene that which accounts only a fraction 2-5% of codes for erbB receptors, for example, can diagnosed RCC [47-50]. cause an over-receptive condition for Clear cell or conventional renal cell growth factors as an early stage event in carcinoma (CRCC) is the most pre- RCC [30]. The myc oncogene facilitates dominate renal tumour and accounts for signal transcription in the nucleus and about 70% of diagnosed RCC tumours have been found to be elevated as a late [45, 51]. The sporadic type of CRCC is stage event in RCC [6]. On the other genetically characterised by deletion of hand, lack of growth factors lead to chromosome 3p, which is believed to be apoptosis [31]. The oncogene that code the early-onset event in renal carcino- for met tyrosine kinase receptors generate genesis [45]. Inactivation of both VHL anti-apoptotic signalling by mitogen- alleles is mandatory for development of activated protein kinase pathways [32- CRCC [50]. Studies have shown that this 36]. Several oncogenes involved sig- mutation occurs in about 50% followed nalling pathways for cell growth control soon after by loss of heterozygoty 10-20% have been reported in RCC [37-40]. and then, as a late event, silencing of the Tumour suppressor genes are genes second VHL allele by hypermethylation

20% [51-53]. Other genetic alterations, 5q families have mutations in this proto- duplication as well as 6q, 8p, 9p, and 14q oncogene with late-onset. Histopatho- deletion, have been implicated in carcino- logically, the familiar and commonly genesis of sporadic CRCC [53-55]. Loss sporadic form of PRCC are both referred of the tumour suppressor gene (VHL) to as type I PRCC [53]. The hereditary increases expression of transforming growth leiomyomatosis syndrome (HLRCC) was factor (TGF-α and β) and vascular endo- recently characterised as an autosomal thelial growth factor (VEGF). Increased dominant tumour syndrome caused by expression of onco-gene myc with ab- germline mutations in the fumarate hydra- normal receptor signalling, despite the tase (FH) gene on 1q42 [66]. Benign lack of growth factors, has also been rela- leiomyomas of the skin and uterus are ted to CRCC [56]. The age-related inci- often seen with predisposition to early- dence of CRCC increases after 35 years onset type 2 PRCC in addition to uterine of age and peaks in the sixth decade. leiomyosarcoma [67]. In contrast to other Early-onset of CRCC indicates an here- PRCCs, the HLRCC tumours are usually ditary cause [57]. A familiar form of solitary and unilateral. The morphological CRCC has been described where there is characteristics of type 2 PRCC are inde- early-onset with balanced 3p translocation pendently associated with poorer survival but no traceable VHL changes, which ex- [68, 69]. cludes mutations in the VHL gene as the Chromophobe renal cell carcinoma mechanism for cancer development [56, (ChRCC) is a third carcinoma originated 58-60]. However, a majority of familiar from tubular epithelium, and accounts for CRCC is associated with von Hippel- 5% of diagnosed RCC [45, 51]. It has a Lindau syndrome. Inheritance is autosomal- better prognosis than other variants of dominant with high penetrance, with an RCC [70]. A low chromosome number is incidence of 1 in 36,000 live births [61, characteristics of ChRCC, due to frequent 62]. The mutation affects the tumour occurrence of loss of chromosomes 1, 2, suppressor gene (VHL) located on the 6, 10, 13, 17, and 21 [45, 55, 71]. The short arm of chromosome 3. Up to 45% of Birt-Hogg-Dubé (BHD) syndrome shows these patients develop CRCC, and often autosomal dominant inheritance for benign the early-onset form [52]. cutaneous tumours with predisposition for Papillary renal cell carcinoma (PRCC) ChRCC and oncocytomas [72]. Pulmo- is the second most frequent type of nary cysts are seen causing spontaneous carcinoma, and accounts for about 10- pneumothorax in 25% of patients [73]. 15% of diagnosed RCC [45, 51]. Papillary The germline mutation gene is located at tumours are characterised by chromo- chromosome 17p11 [74]. somal trisomies, most often 3q, 7, 8, 12, Renal cell carcinoma, unclassified, is 16,17, and 20, with lost of the Y chromo- a diagnostic category used when a RCC some [45, 51, 53, 56, 63]. Hereditary does not fit into one of the others cate- PRCC is a very rare, and is characterized gories. This group represent about 4-5% as an autosomal dominate inheritance of diagnosed RCC [45, 51]. No consistent with high penetrance [64, 65]. The muta- pattern of genetic abnormalities has been tion affects proto-oncogene (met) loca-ted established [53]. Due to variability in on chromosome 7q31. About 80% of morphological patterns this might also

12 include the collecting duct carcinoma, I, a significant number of late recurrences which accounts for less than 1% of renal was observed when 10- and 15-years sur- neoplasms [45, 51, 53]. vival rates are taken into account [84]. After such a long follow-up periods, no Clinical and pathological significant differences in survival has prognostic factors been observed between TNM stages I and Patient-related factors such as gender II or II and pT3b [81, 84]. The clinical and age have no predictive importance (TNM) and pathological (pTNM) classi- [6]. Inherited genetic diseases that pre- fication was modified in 2002 [85] so as dispose for RCC are rare [49]. Biological to better stratify patients with favourable parameters and serum factors such as prognosis [86] for the clinical setting of ESR, C-reactive protein, haptoglobin, and nephron-sparing surgery [87]. The T1 ferritin have been correlated to survival, category was then subdivided into T1a but only ESR has been shown to have (less than 4 cm) and T1b (4 to 7 cm) [87, independent prognostic value [75, 76]. In 88]. However, tumour size alone is not a some studies abnormal hemoglobin va- definite predictor of prognosis. Sarco- lues, thrombocytosis, and elevated liver matoid variants seen in all histological function tests have been correlated to a subtypes of RCC are associated with a worse prognosis [77]. Patient performance markedly worse prognosis, even if the status together with tumour presentation primary tumour is small [51, 89]. In at the time of diagnosis is more relevant, ChRCC, the probability of metastatic however, for prediction of prognosis [76, spread is very low, and in PRCC the 78]. lower stages tends to have a better Tumour related prognostic factors prognosis than CRCC [90]. include macroscopic and histologic features. The use of TNM (tumour-node- Histopathological grading and metastasis) classification as proposed by classification Union International Contre Le Cancer The nuclear grading system in multi- (UICC) and American Joint Committee variate analysis has proved to be the on Cancer (AJCC) [79] is the most second most important classification important tool to predict clinical be- (after TNM) for predicting the clinical haviour and outcome for RCC [77]. It outcome for RCC. Particular in low stages takes into account the size of the tumour, (T1-T2) the nuclear grading system was growth beyond the renal capsule, venous found to be an independent prognostic involvement, adrenal metastasis, lymph factor for survival [91-95]. Different node metastases, and distant metastases grading systems have been proposed for (Table 1). After partial or radical nephrec- RCC [91, 96]. Most evaluations empha- tomy [76, 80-83], the prognosis for size the nuclear features and not the survival at 5 years is 90-100% for TNM morphology of cytoplasm or histological stage I, 75-95% for TNM stage II, 60- formation. The worst area of the tumour, 70% for TNM stage III, and less than rather than over-all impression, defines 10% for distant metastatic RCC [2, 3]. the grade. Both approaches are taken into Although the 5-year-survival rate is very account in the Skinner and Fuhrman favourable for patients within TNM stage systems [91, 96] but the Fuhrman grading

13 system is currently the most used system in grade IV [3]. [91, 97] (Table 2). Both systems suffer New cytogenetic and molecular gene- from interobserver variability and pro- tic data have established the histologic blems in reproducibility [98-100]. The 5- classification of RCC [51, 79, 101] which year survival rates for Fuhrman grades 1– was presented at a consensus meeting in 4 are 65–76% in grade 1, 30–72% in Heidelberg in 1996 [45] and approved by grade 2, 20–50% in grade III and 10–35% the UICC and AJCC in 1997 [51].

Table 1. TNM classification and stage grouping of RCC [85] Primary Tumor (T) TX Primary tumor cannot be assessed. T0 No evidence of primary tumor. T1 Tumor 7 cm or less in greatest dimension, limited to the kidney. T1a Tumor 4 cm or less in greatest dimension, limited to the kidney. Tumor more than 4 cm but not more than 7 cm in greatest dimension; limited to the T1b kidney. T2 Tumor more than 7 cm in greatest dimension; limited to the kidney. Tumor extends into major veins or invades adrenal gland or perinephric tissues, but T3 not beyond Gerota’s fascia. Tumor directly invades adrenal gland or perirenal and/or renal sinus fat, but not T3a beyond Gerota´s fascia. Tumor grossly extends into the renal vein or its segmental (muscle-containing) T3b branches, or vena cava below the diaphragm. Tumor grossly extends into vena cava above diaphragm or invades the wall of the T3c vena cava. T4 Tumor invades beyond Gerota’s fascia. Regional Lymph Nodes (N)* NX Regional lymph nodes cannot be assessed. N0 No regional lymph node metastases. N1 Metastases in a single regional lymph node. N2 Metastasis in more than one regional lymph node. Distant Metastasis (M) MX Distant metastasis cannot be assessed. M0 No distant metastasis. M1 Distant metastasis. Stage grouping Stage I T1 N0 M0 Stage II T2 N0 M0 Stage III T1, T2 N1 M0 T3 N0, N1 M0 Stage IV T4 N0, N1 M0 Any T N2 M0 Any T Any N M1 * Not affected by laterality. Regional lymph nodes includes renal hilar, paracaval, aortic (para- aortic, peri-aortic).

Table 2. Nuclear grading systems Fuhrman grading system [91] G1 – Nuclei are small, round and uniform (10 μm), with inconspicuous or absent nucleoli. G2 – Nuclei are slightly irregular (15 μm), with small nucleoli. G3 – Nuclei are very irregular (20 μm), with large and prominent nucleoli. G4 – Nuclei exhibit large and pleomorphic often poly-lobed and bizarre (> 20 μm).

Skinner grading system [96] G1 – Nuclei are small, indistinguishable from those seen in normal tubular cells G2 – Nuclei are slightly irregular and frequently pyknotic without abnormal nucleoli G3 – Nuclei are irregular, enlarged and pleomorphic with prominent nucleoli G4 – Nuclei are extremely giant and bizarre

Clear cell or conventional renal cell growth pattern that can become solid in carcinoma (CRCC) that appears in light undifferentiated tumour areas. The papil- microscopy with a lucid cytoplasm appears lary structure consists of delicate fibro- more or less empty after staining with vascular cores with focal lipid-loaded hemotoxylin and eosin (H&E). This is an macrophages which are covered by single effect of intense intracytoplasmatic accu- layer of neoplastic cells [90, 107]. Type 1 mulation of glycogen and phospholipids PRCC exhibits a faint basophilic stained due to increased glucose-6-phosphate cytoplasm with small centrally located levels caused by activated glycolysis and nuclei [108]. The cytoplasm is pre- reduced gluconeogenesis [102, 103]. The dominantly surrounded by endoplasmic nuclei of well-differentiated tumour cells reticulum. Type 2 PRCC represents a are condensed, while in less differentiated more aggressive form [68] with enlarged tumour cells the nuclei demonstrate poly- and prominent nucleoli in addition to a morphism and prominent nucleoli [104]. eosinophilic and granular cytoplasm rela- Another variant of CRCC is the eosino- ted to accumulation of mitochondria philic or granular appearance of the cyto- [109]. This histological sub-typing of plasm, due to augmentation of mito- PRCC has prognostic value since the chondria. The architecture is characteris- morphological characteristics of type-2 is tically variable within the same tumour, independently associated with poor sur- with acinar or tubular growth patterns vival [69, 109]. associated with poor defined stroma de- Chromophobe renal cell carcinoma spite being surrounded by rich branching (ChRCC) is characterised by large poly- of delicate vasculature. [51, 89, 105]. gonal cells with transparent cytoplasm There is a positive correlation between the and prominent cell membranes [110, extent of lymphocytic infiltration and in- 111]. The cytoplasm is filled with glycol- creasing grade of malignancy [106]. gen deposits and numerous microvesicles. Papillary renal cell carcinoma (PRCC) Some tumour cells have an eosinophilic is characterized by a distinct papillary or granular cytoplasm due to an accu-

15 mulation of mitochondria [111]. Nuclear may respond differently to treatment size and shape varies, with lack of cyto- [121]. plasm colouring by routine dyes, which have been described as perinuclear ‘halos’. Angiogenesis in cancer The microvesicles can be stained blue by Angiogenesis is the formation of new the Hale colloidal iron technique [89]. capillaries by outgrowth of endothelial The architectural pattern is usually solid cells from pre-existing blood vessels. [51]. ChRCC has a better prognosis than Folkman and coworkers demonstrated other variants of RCC [89, 105, 107, 110]. that tumours could only grow up to 1.75 Collecting duct RCC has a tubular struc- mm when nutrition was obtained solely ture combined with a microcytic, pseudo- by diffusion. Growth beyond this size papillary, and solid growth patterns asso- required a supply by new blood vessels ciated with an indefinite stroma and gra- [122]. The onset of angiogenesis depends nulocytic infiltration. It has a clinically on a shift in the equilibrium in extra- aggressive course, often demonstrating cellular matrix (ECM) created by metastases at presentation and rapid pro- numerous of inhibitors and stimulators gression [112, 113]. No specific genetic towards an activation of angiogenesis alterations have been described in the rare [123]. Once activated, a sequence of collecting duct RCC [113]. Unclassified events is required in order for formation RCC is a category containing a hetero- of new blood vessels to occur [124]. First, geneous group of tumours that cannot be a local de-gradation of basement mem- classified as any of the previously brane is required to facilitate the mentioned subtypes [51]. Sarcomatoid migration of endothelial cells, and this is changes can occur in all subtypes of RCC followed by alignment of proliferated [51, 89]. endothelial cells which form new ca- The distinct histological subtypes have pillaries towards an angiogenic stimulus different biological and clinical behaviour [124]. A growing tumour needs to recruit [114-116]. The most common type is CRCC, additional blood supply in order to which has a 55-60% 5-year survival rate maintain sufficient oxygen and other [117, 118]. PRCC has a 80-90% 5-year nutrient availability for rapidly dividing survival rate [90, 107, 109]. ChRCC is cells. This onset of angiogenesis may be less common and therefore survival data brought about by a hypoxic or hypo- are limited, but ChRCC is overall glycaemic condition, or may be caused by associated with favourable prognosis [70]. increased angiogenic stimulators due to Collecting duct RCC show an extremely genetic faults [123, 125]. Several growth aggressive behaviour with no reported 5- factors with angiogenic signalling have year survivors [112, 113]. However by been identified [126]. These include basic using multivariate analysis, the histo- fibroblast growth factor (bFGF) and logical subtype was not found to be an platelet-derived growth factors (PDGF), independent prognostic factor for survival which have signalling activity for several [119, 120]. Recent studies indicated a different types of cells, whereas vascular clinical utility for classifying RCC in endothelial growth factor (VEGF) affects different histological subtypes since they primarily vascular endothelial cells [127].

Tumour secreted VEGF-A R2, respectively [138]. Exons 6 and 7 VEGF is a disulfide-bonded dimeric provide the binding affinity to heparin or glycoprotein with a molecular mass 34-46 heparan sulphate proteoglycans [143]. In kDa, and it is the most potent of all addition, exon 7 encodes the binding domain growth factors that stimulate vascular to receptor of Neurophilin-1 [144]. Exon permeability and endothelial cell 8 are required for the stimulation of mito- proliferation [128-131]. Structurally, VEGF sis [141,145]. Thus, alternative splicing of exhibits an sequence amino acid homo- VEGF mRNA generates forms that differ logy to platelet-derived growth factor in solubility, and longer forms are (PDGF) [132-134]. In cultured vascular retained by proteoglycans at cell surface smooth muscle cells, three natural occur- or extracellular matrix [143]. ring isoforms of VEGF-A have been iden- Alterations in the extracellular en- tified through analysis of isolated cDNA vironment can cause affected cells to clones to predict sequences of 189, 165 release VEGF, and extracellular stimuli and 121 amino acids [135]. Applying the include, for example, hypoxia [146], and reverse transcription polymerase chain hypoglycaemia [147], the presence of in- reaction (RT-PCR) technique to different flammatory cytokines [148], platelet- carcinoma cell lines has helped to identify derived growth factor (PDGF) [149], additional VEGF isoforms [136]. To date, basic fibroblast growth factor (bFGF)

7 different mRNA forms (VEGF121, 145, 165, [150], epidermal growth factor (EGF) 183, 189, and 206) have been described [136- [151], and insulin-like growth factor 139]. All VEGF isoforms are generated (IGF) [152, 153]. The mechanisms behind from the same gene, which is located on these different growth factors leading to the short arm of chromosome 6 [140]. VEGF promotion of angiogenesis are Recently, Bates et al. described a variant unknown. Uncontrolled cell proliferation of VEGF with 165 amino acid isoforms is a causal event to produce VEGF and termed VEGF165b [141]. The VEGF165b allow cell survival. Uncontrolled cell pro- demonstrated inhibitory properties on proliferation may be driven by an activated liferation, migration, and vasodilatation in oncogene [154] or though series of gene- endothelial cells. This indicates that tic events due to inactivated or impaired alternative spliced variants of VEGF have tumour suppressor gene [155], by ab- different functions or activities. All VEGF normal cell signalling in response to isoforms exhibit exons 1-5 and a terminal growth factors (myc) [6, 153], amplifying exon 8 although altered in VEGF165b growth recaptor signalling (erb-B/EGF-R, [141]. Exons 1-5 and terminal exon 8 met) [30, 32-36]., interfering in cell cycle encodes VEGF121. Inclusion of exon 7 or regulation (Cyclins D and E, p27, p53, exons 6 and 7 encodes VEGF165 and pRb) [37-40, 156, 157], or preventing VEGF189, respectively [139]. The biolo- apoptosis (p53, Bc12)[158, 159]. In gical differences between VEGF isoforms contrast, an inactivated or impaired tumour are dependent on incorporated exons. suppressor gene (VHL) leads tumours to Exon 1 encodes the signal sequence, exon consistent production of VEGF [160]. 2 the N terminus, and exon 3 the dimeri- These tumours have an impaired von sation domain [142]. Exons 3 and 4 encodes Hippel-Lindau protein (pVHL), which binding domain to VEGF-R1 and VEGF- cause an accumulation of hypoxia indu-

17 cible factor (HIF-1α), and, as a con- EMC and basement membrane, creating a sequence, release of several growth factors window through which endothelial cells such as VEGF, PDGF and TGF-α into the can migrate [174-176]. extracellular matrix [150]. Co-expression between growth factors might have an Synergistic role of bFGF autocrine role as well as a paracrine bFGF (18 kDa) was earlier described function [161]. as an important angiogenic factor in- All isoforms of VEGF-A are able to volved in the angiogenic switch during stimulate endothelial cells through two early tumourigenesis [177], though re- receptor tyrosine kinases VEGF-R1 [162, cently VEGF has been advocated as 163] and VEGF-R2 [164, 165]. Structural responsible [178]. bFGF is produced by a dimerisation of VEGF is essential for variety of cells including tumour cells, biological activity, and particularly for endothelial cells, fibroblasts, and macro-

VEGF165, where the ligand pairing has a phages [179, 180]. Most bFGF that is high affinity to VEGF-R2 [142, 144]. secreted from these cells is sequestered by Several members of the VEGF family heparan sulphate in the extracellular (VEGF-A, -C, -D, and -E) can activate matrix, with the matrix serving as a VEGF-R2 and promote endothelial cells reservoir for growth factors [174, 179- to proliferate, migrate, and differentiate 181]. bFGF activates additional proteases [166-169]. On the other hand, VEGF-R1, from endothelial cells to dissolve the activated by VEGF-A, -B, and PDGF basement membrane (ECM) and thus stimulates endothelial cells to migrate facilitate and induce proliferation and [170-172]. VEGF and PDGF demonstrate migration of endothelial cells [127, 179, an amino acid sequence homology which 182]. bFGF is not as potent as VEGF in may in heterodimerisation provide a stimulating endothelial cells [127]. There potential diversity in signal transduction is evidence that VEGF and bFGF act through ligand-mediated paring with synergistically to promote angiogenesis receptors VEGF-R1 or PDGF-R [132, 171]. [182]. In serum, bFGF has been found at The functional consequences of hetero- significantly higher concentration levels dimerisation between the growth factors in patients with RCC than in patients are not quite understood. Thus, VEGF-R1 without tumours [183-185]. RCC some- regulates structural organisation and sta- how leads to secretion of bFGF [186], but bility of new-formed vessels, whereas no differences in expression could be VEGF-R2 regulates proliferation of endo- observed between tumour cells and thelial cells and appears to have a more normal renal tissue [187, 188]. bFGF was prominent role of promoting angio- found localised to ECM, whereas a small genesis [144, 169, 170]. The other family number of tumour cells expressed bFGF members in VEGF (VEGF-C, and -D) are [189]. The expression of bFGF in general involved in regulation of endothelial cells has been associated with poor survival, during formation of new lymphatic vessels but was not an independent factor related [166, 172, 173]. VEGF, PDGF, and bFGF to survival in RCC [189]. stimulates endothelial cells to release proteases such as matrix metallo- Miscellaneous role of PDGF proteinases (MMP), which degrade the PDGF (30 kDa) is stored in the plate-

18 let granules, and is released with platelet cells, and monocytes [192]. Although activation [190]. It can also be produced PDGF does not appear to be important for by a variety of cells, including activated early formation of blood vessels, it may macrophages, endothelial cells, smooth play a role at later stages through its muscle cells as well as tumour cells [191]. ability to recruit pericytes and stimulate PDGF is responsible for migration and development of vascular smooth muscle proliferation of fibroblasts, smooth muscle cells [4, 191].

AIMS OF THE INVESTIGATION

I. To analyse the prognostic value of VEGF in preoperatively collected sera from patients with RCC.

II. To investigate whether intercurrently diseases and medications affects VEGF in serum as a prognostic factor in patients with RCC.

III. To investigate the prognostic impact of VEGF mRNA isoforms and VEGF receptor mRNA in RCC

IV. To evaluate the clinical impact of immunohistochemical detected VEGF expression in RCC.

V. To investigate the prognostic impact of VEGF protein isoforms in different RCC types.

MATERIAL AND METHODS

Patients PRCC had a 47% 15-year survival rate The study included a total of 265 and the less common ChRCC had an 80% patients with histopathologically verified 15-year survival rate. Histological un- RCC after surgery between August 1982 classified RCC had an extremely aggres- and December 1999. Each patient parti- sive behaviour as no survival could be cipated after informed consent and the reported at 10-year follow-up. The 15- ethics committee at the Umeå University year survival rates for Skinner grades 1– 4 approved the studies. The patients were was 88% in grade 1, 75% in grade 2, 32% randomly assigned to the individual stu- in grade III, and 18% in grade IV. dies as indicated in figure 1. There were 156 men and 109 women, with a median Tumour staging age of 66.0 years (mean 65.4; range 25 – Tumour staging was performed 87 years). Among the 265 patients, 256 according to the TNM stage classification were operated with nephrectomy, seven system 1997 (Paper I-III) and 2002 (Paper with partial, and two with combined IV-V) [85]. TNM classification included partial and radical nephrectomy due to microscopic evaluation for tumour pre- bilateral RCC. None of the patients had sence in regional lymph node and peri- been treated with radiation, chemotherapy, nephric tissues as well as tumour invasion or immunotherapy before surgery. Most through the renal capsule into peri-renal of patients were followed-up according to fat or into major renal veins at the renal clinical routine [83] with clinical and hilum. There were 78 stage I tumours radiological examinations at regular inter- (29.4%), 37 stage II (14%), 57 stage III vals. Survival was determined from time (21.5%), and 93 stage IV tumours of nephrectomy to the latest follow-up, (35.1%). Among the 57 stage III tumours, where information was obtained from 12 invaded peri-nephric tissue or adrenal hospital records or from the Cancer gland (pT3a, pN0, M0), 23 major veins Registry of Sweden [1]. only (pT3b-c, pN0, M0) and 22 invaded At the latest follow-up, 65 patients peri-nephric tissues and major veins were alive with a median follow-up time (pT3a-b, pN0-1, M0). One patient with 133 months, (range 26 – 236 months), stage III tumour had metastasis in a single 138 had died of RCC with median regional lymph node. There was no appa- survival time of 17 month (range 1 – 168 rent shift in tumour stage distribution months), and 62 had died of unrelated during the 20 years of sample collection. causes with 55 month median survival Tumour size was measured as the maxi- time (range 1 – 203 months). After radical mal diameter on the surgical specimen or or partial nephrectomy the survival rate at by computerized tomography. The me- 5-years was 95% for TNM stage I, 80% dian tumour diameter was 75 mm (mean for TNM stage II, 45% for TNM stage III, 80; range 10 – 220 mm). and 5% for TNM stage IV. The distinct histological subtypes showed different Morphologic classification biological and clinical behaviour [116]. Histopathologic nuclear grading was CRCC had a 35% 15-year survival rate, performed according to Skinner and co-

Individual cases

No. patients 233 164 161 96 61 Paper IV I II V III

Figure 1. Depiction of the different studies in which 265 patients were included. The patients were arranged according number of assigned studies and not by time of nephrectomy. workers [96], characterized on the worse fication system by Professor G Kovacs [45]. histologic features, by pathologists at Umeå Methods University Hospital (Table 3). Eleven (4%) tumours were classified as grade 1, The methods used in this thesis are 55 (21%) were grade 2, 132 (50%) were described in material and methods sec- grade 3, and 67 (25%) were grade 4 tions of the individual papers. A more (Table 4). RCC tumour type was histo- general description is presented below. pathologic examined according to the Heidelberg classification system [45]. Blood analysis More than 75% tubulopapillary architec- Preoperative blood samples were ture was used for designation of tumour obtained as a routine before 10 a.m. The as a papillary RCC [193, 194]. There blood cell composition was analysed in were 203 (77%) conventional (clear cell), the Clinical Chemistry Laboratory with 36 (13%) papillary and 15 (6%) chromo- standard Coulter counter technique (Coulter phobe RCCs while 6 (2%) tumours were counters S-Plus STKR, Coulter Elec- not amenable to morphological classi- tronics, Luton, England, or Sysmex SE- fication. Five (2%) tumours were missing 9000, Toa Medical Electronics Co., Kobe, and not available for second microscopic Japan). Patients receiving preoperatively review according to Heidelberg classi- intravenous treatment were excluded.

Table 3. Pathologists who participated in the clinical examination of microscopic sections in this thesis. Nuclear grading according to Skinner [96] Grade 1 Grade 2 Grade 3 Grade 4

R Stenling 9 44 113 58 84.5 % L Boquist 1 3 7 2 4.9 % U Gerdes 3 3 2.3 % O Hassler 2 3 1.9 % J Vasko 2 2 1,5 % S Cajander 1 1 1 1.1 % F Bergman 2 0.8 % B Lundskog 2 0.8 % L Bjersing 1 1 0.8 % B Bozoky 1 1 0.8 % G Hallmans 1 0.4 % A Bergh 1 0.4 %

Table 4. Clinicopatholgoical characteristics of different RCC types involved in this thesis.

Conventional Papillary Chromophope Not classified Not accessible

Skinner Grade 1 5 5 1 Grade 2 35 11 6 1 2 Grade 3 101 18 9 3 1 Grade 4 62 2 1 2 TNM Stage I 57 14 5 1 1 Stage II 23 5 6 1 2 Stage III 47 4 3 2 1 Stage IV 76 13 2 2

Serum analysis VEGF was analysed by quantitative sand- wich enzyme immunoassay tech-nique The samples were routinely obtained (Quantikine, Human VEGF immuno-assay, from peripheral veins in patients. The R & D Systems, Minneapolis, MN). A recommendation was 5 min horizontal microplate precoated with mouse mono- bed resting and food restriction before clonal antibody against human VEGF, sample taking [195]. Samples were immobilized free VEGF in samples. After collected in tubes without additive and a new wash, an enzyme-linked secondary allowed to coagulate for 30 min and goat polyclonal antibody against VEGF centrifuged (1500 x g) for 15 min at room was added to the wells. After additional temperature. Obtained serum samples wash, a substrate solution was added to were stored at -80°C until analysis. Serum the wells and colour developed in

23 proportion to the amount of VEGF bound RNA of VEGF, flt-1, and cyclophilin. in initial step. Optical density was Each RNA sample was titrated with three determined on a microtiter plate reader different concentrations of IS for the (Multiskan MCC/340, Lab Systems, respective gene (in duplicates). During 30 Stockholm, Sweden). The results were cycles of PCR (94°C, 30 s; 61°C, 30 s; measured in duplicate, and a deviation 72°C, 45 s) templates were competitively more than 10% between measurements led amplified with cDNA for corresponding to re-measurement. IS. VEGF primers used in the PCR reaction were designed for the simul- Tissue collection and preparation taneous amplification of all VEGF iso- Tumour and kidney cortex tissue forms (Figure 2). The PCR products were samples were obtained from the surgical analysed by a laser fluorescence system specimen as described by Ljungberg et al (ABI PRISM 377 DNA sequencer, Perkin [196]. Each sample was divided into Elmer), and processed by the ABI PRISM smaller pieces. One part was snap frozen GenScan software (Perkin Elmer) (Figure in liquid nitrogen and stored in –80°C and 3). Messenger RNA levels were calcu- the other part was formalin fixed and lated from the linear regression by extra- paraffin embedded for immunohisto- polation at equivalent templates to IS chemical staining and morphologic exa- signals as previously described. The mination. VEGF and flt-1 values were corrected for the corresponding cyclophilin values in Competitive quantitative RT-PCR each RNA sample and expressed as Frozen tumour samples were homo- relative levels (fmol/amol cyclophilin). genised and total RNA was isolated using The measurements were performed twice, the TRIzol method (Life Technologies, and a deviation more than 10% between Stockholm, Sweden). Total RNA concen- measurements led to re-measurement. trations were measured spectrophoto- VEGF-A is a 34-46 kDa peptide for- metrically at 260 nm (Lambda 2 UV/VIS, med by two subunits (monomers) organi- Perkin Elmer, Stockholm, Sweden). The zed in anti parallel (homodimeric) manner mRNA levels of all isoforms of VEGF-A, by disulfide linked bridges. VEGF exists VEGF-R1 and cyclophilin were quanti- in different isoforms due to alternative fied by competitive RT-PCR, as described splicing after signal sequence cleavages previously [197]. Primer sequences were of transcript encode, mRNA [198]. The designed to quantify all isoforms simul- human VEGF gene is structured as 8 taneously from the human genes of exons, separated by 7 introns. The basic VEGF-A (5´-ATC TTC AAG CCG TCC sequence of mRNA consists of 8 exons TGT GTG C-3´ and 5´- TCA CCG CCT and encodes the longest variant of

CGG CTT GTC ACA T-3´) and flt-1 (5´- VEGF189 with 189 amino acid. The other AGG AGA GGA CCT GAA ACT GTC isoforms arise from alternative splicing of TT-3´and 5´-ATT CCT GGC TCT GCA mRNA regarding sequence of exons 6 and

GGC ATA G-3´). Briefly, 50 ng of total 7. VEGF165 lacks exon 6 and VEGF121 RNA was reverse-transcribed together lacks exons 6 and 7. Sense primer 5´ - with appropriate amounts of internal ATC TTC AAG CCG TCC TGT GTG C RNA-standards (IS), meaning truncated - 3´ corresponds with exon 3, and anti-

Reverse Transcription mRNA

5’ 3’

n n o

o Reverse transcription reaction

i i t

Sample t

p p

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homogenised r c

c 5’ 3’ s

in TRIzol s st n

n 3’ 5’ 1 strand cDNA

a a

r r T

Centrifuge - T

Reagents added e

s s r

Centrifuge - r

e e v

RNA v Rnase H cleavage of RNA

e e R

preparation R

RNA extraction

5’ 3’

3’ 5’

Polymerase Chain Reaction

Single stranded DNA

The primers formed (sense/antisense primer) n

a n o

o 5’ 3’

i i t

double strands with the complementary t c

a c 3’ 5’

a a e sequences of desired site of DNA amplification e

R R

Denatuering – double stranded DNA

n n i (annealing). Substrate and DNA polymerase were i

a denatured by heat to single strand

a a h

added and from primers site a DNA fragment was h

C C

a e

e Annealing – incoporation of s

synthesised (extension). A fluorescence was s a

a a r

r of of primer… e marked at sense primer (-5’) and PCR products e

a m

m Extension – synthetis

y y

l l o

were analysed by a laser fluorescence system. o Cycles

P P

Figure 2. Schematically illustration on reverse transcription polymerase chain reaction (RT-PCR).

Size (bp)

) units y arbitar ( ht g Peak hi

Figure 3. Chromatogram on VEGF analysis. Three isoforms of human VEGF were detected after RT-PCR and following analysis by ABI PRISM 377 DNA sequencer. Adapted from Häggström S [197].

25 sense primer 5´ - TCA CCG CCT CGG treated with standard procedures for de- CTT GTC ACA T -3´ with exon 8 of parafinating, rehydrating, microwave hea- human cDNA. ting and immunohistochemical (IHC) staining. Different antibodies were tested and a Immunohistochemestry mouse monoclonal anti-human VEGF antibody (NeoMarkers, Lab Vision Representative paraffin tumour blocks Corporation, Fremont, CA; VEGF Ab-3) were selected by primary evaluation of was selected. The remains of antibody haematoxylin/eosin- stained slides before bound to antigen through washing were tissue microarray (TMA) preparation. Two visualized through a colour reaction with tissue cores were taken with a sample biotinylated multilink secondary antibody needle (0.6 mm in diameter) from each (Vectastain Elite ABC kit, Vector Labora- tumour block and placed in a new tories, Inc., Burlingame, CA). Most recipient paraffin-block containing 98 favourable antibody was chosen on three tissue cores with 0,8 mm space between tissue slides selected by presence of (Beecher Instruments, USA). For micro- kidney tissue, adjacent to tumour tissue, scopic evaluation 4-μm thick paraffin which contained poor, intermediary or sections was slized. The slides were strong IHC VEGF expression. (Table 5).

Table 5. An overwiew of primary antibodies that have been used in this thesis.

Applications Antibody Manufacture Reactivity/Epitope

Serum Mouse, monoclonal Quantikine, Human Human VEGF165 and VEGF121 antibody, anti- VEGF immunoassay, homodimer (VEGF165/hPDGF VEGF R & D Systems, heterodimer exhibit 20 % cross- Minneapolis, MN reactivity) Goat polyclonal antibody, anti- VEGF WB Rabbit, polyclonal Santa Cruz, Human (Mouse and Rat) VEGF / antibody, anti- Biotechnology Inc, N-terminus of VEGF VEGF (A-20) Santa Cruz, CA WB* Rabbit, polyclonal NeoMarkers, Lab Human (Mouse, and Rat) VEGF / antibody, anti- Vision Corporation, Not determined VEGF (Ab-1) Fremont, CA IHC Mouse, monoclonal NeoMarkers, Lab Human (Rabbit) VEGF / antibody, anti- Vision Corporation, Not determined VEGF (Ab-3) Fremont, CA IHC* Rabbit, polyclonal Santa Cruz, Human (Mouse, and Rat) VEGF / antibody, anti- Biotechnology Inc, Antibody raised against a peptide VEGF (147) Santa Cruz, CA corresponding to amino acids 1- 140 of VEGF

* Not applicable to this study.

A B

Nucleus

A B

Figure 4. These images illustrate the evaluation of different profiles using a special grid to calculate percentage of fields with positive IHC staining in cells emphasized by presence of nucleus and the overlaying chosen area. Profiles intersected by inclusions- edges were counted in addition to enclosed ones. A. Cytoplasmic VEGF staining. B. Cell surface VEGF staining.

The positive and uniform IHC VEGF series. An additional control to confirm expressions in normal kidney tubular cells IHC VEGF expression was performed were used as control for adequate immuno- with the absence of the primary antibody histochemical application for evaluation and by a neutralizing primary VEGF positive IHC VEGF expression in tumour antibody with recombine VEGF peptide

(Recombinant human VEGF, 293 VE; dismembrator for 2 x 10 sec., and after R&D Systems Inc., Minneapolis, MN) slight thawing, the samples were sus- before incubation of the slides. For pended in chilled Tris lysis buffer con- quantifying IHC VEGF staining, a square- taining protease inhibitors. The homogenates latitude mounted in the eyepiece of a light were then centrifuged (13000 x g) for 30 microscope was used to evaluate the min at 4 Co. Protein in the supernatant density of VEGF stained tumour cells. was quantified by bicinchoinic acid assay Within framework of 121-point eyepiece (Pierce, Illinois). A standard curve created graticule at 200x magnification, the den- on absorbance of the control series was sity was defined as number of manually used to set the concentration of protein counted fields with cellular stained VEGF extractions. These measurements were and overlaying chosen area (Figure 4). In performed in duplicate, a deviation be- TMA the sampling area was demarcated tween measurements of more than 10% within framework of a 100-square grati- led to re-measurement. cule at 200x magnification, and area density of VEGF expression was defined Western blotting as maximal score of two samples from A Western blot (WB) procedure was each tumour. For tissue section (TS), the performed on 30 µg protein extracts to slides were examined at low magnify- which was added an equal volume of cation (100x), and three areas containing electrophoresis sample buffered with the highest VEGF expression were chosen reducing agents to accomplish a mono- as hot spot areas and then evaluated at meric form of VEGF. The samples were 200x magnification. The VEGF expression electrophoresed on 12% SDS poly- in the tissue sections was defined as mean acrylamide gel and then transferred to a score of the volume density from the three nitrocellulose membrane. Staining with hot spot areas. In TS, the staining Ponceau-red was performed to ensure a intensity of IHC VEGF was assessed total transfer of protein extract to according to a three-graded scale: weak membrane. The membrane was either staining- when visible at 200x magni- blocked in 10% horse serum or in 5% fication, moderate intensity- when visible milk powder and 2.5% bovine calf serum at 100x, and strong staining- when visible with PBS, 0.1% Tween-20 prior to at 20x. Staining intensity was evaluated incubation with antibodies. The mem- separately and independently three times, brane was primarily incubated with a and the quantification of volume density polyclonal rabbit antibody to human was also evaluated three times by one VEGF, and secondarily incubated with observer without knowledge to preceding horseradish peroxidase (HRP) labeled results. Any discordance was resolved by anti-rabbit antibody. VEGF was visua- re-examined by a pathologist. lised by enhanced chemiluminescence (ECL Plus, Amersham Int., England) and Protein extraction recorded on Hyperfilm-ECL (Amersham Imprints were made from frozen tissue Int., England) (Figure 5). Protein ex- and stained with Giemsa to verify pre- pression was quantified using Fluor-S sence of tumour cells. Frozen tissue Multi Imager scanning and Quantity One samples were homogenized with a micro- software analysis (Bio-Rad Laboratories,

Secondary Oxidized HRP-labelled antibody product

Peroxide O2 +

2 H2O Luminol Light VEGF Primary + antibody Enhancer nitrolulose membrane Hybond ECL Western Detection by HyperfilmECL

Figure 5. The membrane was soaked with ECL Plus detection reagent. Where the HRP- labeled secondary antibody was bound, a peroxidase-catalyzed oxidation of luminol was induced leading to chemiluminescence. This resulting light was detected on hyperfilm ECL Western.

Hercules, California). Relative protein ex- variables for two independent groups, and pression was evaluated against a linear the Kruskal-Wallis test was used for standard curve obtained by different comparison of more than two groups. dilutions on extracts of a standard tumour Spearman rank correlation test was used used in each gel (Figure 6). Detected to compare relationship between sets of protein bands were controlled by not non-parametric variables that did not adding primary antibody or by primary demonstrate a linear relation. Chi-Square antibody was blocked with human VEGF test was used to evaluate differences in peptide before incubation. At start of the proportions of observations between in- study, different primary VEGF antibodies dependent groups. Fisher’s exact test was were evaluated (Table 5). The molecular used when the sample size was too small weights of detected proteins were verified to use the chi-square test. The median by comparison with pre-stained SDS- value was chosen as the cut-off value. PAGE standards (161-0372, Bio-Rad Survival data was analysed using the Laboratories, Hercules, California). These Kaplan-Meier method, and comparison of measurements were performed in dupli- survival times for groups was performed cate, and a deviation of more than 10% with the log-rank test. The variables were led to re-measurement. Equal loading of dichotomously tested, and analysed as protein extracts was confirmed by the continuous in a univariate Cox regression actin expression. The band at 28-kDa was analysis. Multivariate analysis was per- defined as VEGF189, the band at 22-23- formed according to Cox proportional kDa as VEGF165 and the band at 18-kDa hazard model. Survival time was mea- was defined as VEGF121 [21, 22]. sured from the date of nephrectomy to date of death, or latest follow-up. In all Statistical methods tests, a two-tailed significance level was The Mann-Whitney U test was used to set to < 0.05. identify differences in non-parametic

Figure 6. Using Fluor-S Multi Imager scanning and Quantity One software analysis facilitate the determination of protein density from Hyperfilm ECL. The relative protein concentration was evaluated against a linear standard curve obtained by different dilutions and extracts of tumour loaded in each gel.

RESULTS AND COMMENTS

Paper I in patients with RCC. When Paper I was Serum VEGF concentration (S-VEGF) accepted for publication, there was dis- was significantly increased in patients cussion regarding the value of measuring with RCC compared to a control group. VEGF in sera [199-201]. Peripheral ve- No alteration in S-VEGF was observed nous blood samples showed higher con- due to storage time of the samples. There centration of VEGF in serum compared were no differences in distribution of with matched plasma samples [200, 202- tumour stage, grade, and size related to 207] due to VEGF release related to gender or age. Increasing S-VEGF corre- platelet activation and some contribution lated with higher tumour stage and grade. from white cells [200, 202-204, 207, 208]. Localized tumours (stages I-II) had low S- RCCs are thought to affect the bone VEGF compared to tumours with vein marrow leading to increased platelet invasion or extracapsular dissemination counts [208, 209]. Platelet activation in (stage III) as well as tumours with distant tumours [210-213] may lead to elabora- metastasis (stage IV). There was no tion of several angiogenic growth factors, relation between S-VEGF and extent of including VEGF [200]. The theoretical tumour-thrombus in vena renalis (pT3b) advantage of analysing VEGF in plasma or vena cava (pT3c). A weak correlation has limitations since circulating free was observed between S-VEGF and size VEGF in vivo is subject to degradation as of localized tumours (stages I-II). There well as binding to VEGF-binding protein was no difference in S-VEGF between levels, and may not directly reflect PRCC and CRCC, although ChRCC tumour secretion of VEGF [201]. S- results showed lower S-VEGF compared VEGF levels differ from plasma VEGF to CRCC but not PRCC. Increasing S- also due to the fact that platelets deliver VEGF correlated with shorter survival in VEGF to tumours [214-216]. It has been male patients, but not in female patients. suggested that PDGF reflects tumour In multivariate analysis, S-VEGF was not biological activity [217], and that serum an independent prognostic factor. samples are more useful for measurement circulating growth factors [205]. VEGF165 Comments on Paper I is the predominant isoform in serum [218] Considerable efforts have been made and is also the isoform predominantly to find biomarkers for progression of secreted by a variety of cancer types disease in order to monitor effect of [219]. In Papers I-II, analysed levels of S- cancer treatment. Measuring circulating VEGF165 reflect also the biological effects biomarkers in peripheral blood samples of VEGF121 and VEGF165/PDGF hetero- has practically appeal since access is easy dimer occurring in serum after platelet and results reliable when uniform agglutination. handling of blood samples is performed [199]. We demonstrated a significant Paper II correlation between S-VEGF levels and Approximately two-thirds of patients tumour stage, and that high S-VEGF with RCC were concurrently treated with levels were associated with poor outcome long-term medication because of chronic

31 diseases. No differences in distribution of levels and blood platelet counts in sur- tumour stage, grade, or size were ob- vival curves may be due to peripheral served related to gender, age group, or distribution of larger immature platelets presence of long-term medication. The with higher quantities of VEGF [207] or proportion of patients with long-term caused by other factors as acquired dys- medication increased with age for both function of platelets commonly associated genders. No difference in S-VEGF was with medication in elderly people [221]. found in relation to gender, age group or Moreover, decreased blood platelet counts presence of long-term me medication. have been associated with medication and Increasing S-VEGF was correlated with advancing age [222-224]. Epidemiologic higher tumour stage and grade, as well as studies have shown that established risk higher platelet and leukocyte counts. No factors such as obesity and cigarette difference in blood platelet counts was smoking can be confounding factors when found between male and female patients. examining effects of medication [225]. In However, platelet counts showed an in- addition, anti-hypertensive drugs and verse correlation to age, particularly in analgesics have been proposed as in- women. In male patients, platelet counts dependent risk factors for RCC develop- were lower in those on long-term ment [225, 226]. This paper showed that medication compared to those without. patients with RCC had decreased platelet Univariate analysis showed that increased counts that were associated with inter- S-VEGF and platelet counts correlated current chronic disease, presence of long- with shorter survival especially in male term medications, and advancing age. In patients. In contrast to S-VEGF, higher women, blood platelet counts were not blood platelet counts were only associated affected by chronic diseases or medica- to shorter survival in patients with RCC tions but decreased with advancing age. with no intercurrent chronic disease or In addition blood platelet counts had no long-term medication. Leukocyte counts predictive value for survival for women, were not associated with prognosis. Age, regardless of presence or absence of gender, or long-term medication had no chronic decease or long-term medications. association with survival time in patients In men with chronic diseases and long- with RCC. Using Cox proportional hazard term medications, blood platelet counts step-wise elimination, VEGF levels were were decreased, and no predictive value the last variable to be excluded, and only for survival was found in those subgroups stage and grade remained as independent of patients. It is noteworthy that RCC is prognostic factors. most common in patients at their sixth to seventh decades of life [1], but this ratio Comments on Paper II between gender tends to decrease with This paper demonstrated a correlation increasing age [227]. Whether or not between VEGF levels and blood platelet other health risk factors may initiate and leukocyte counts, as reported in other conversion of tumour dormancy to angio- malignancies [202]. Platelets have been genic phenotype is not clear [228]. S- revealed as the main source of VEGF in VEGF levels in peripheral blood samples serum [200, 207, 210, 211, 220]. However, suggest that S-VEGF has much of its the lack of relationship between S-VEGF origin from platelets and to some extent

32 from white blood cells. However, the tu- levels correlated with adverse survival. mour angiogenesis process may be For VEGF165 and VEGF-R1 mRNA levels dependent on platelets that deliver VEGF no predictive information was found. to tumours. Still, S-VEGF levels have been reported to be significantly higher in Comments on Paper III samples from tumour-bearing renal veins compared with samples from peripheral RT-PCR method allows detection and veins [229]. We found that S-VEGF quantification of small amounts of speci- levels were unaffected by intercurrent fic mRNA in RCC samples. Inappropriate chronic diseases or medications. In addi- preparation of tissue samples can lead to tion, there were no differences in S-VEGF invalid results due to tiny amounts DNA levels in relation to age or gender. In introduced through external contamina- contrast to men, S-VEGF levels had no tion. Optimal preservation, storing and predictive value for survival in women handling of samples are important to with RCC, and the reason for this is avoid mRNA degradation. Despite ade- unclear. Our results indicate that S-VEGF quate execution of the RT-PCR method, is more useful as a prognostic indicator or the interpretation of analysed mRNA biomarker, than platelet or leukocyte involves a mixture of cells from different counts. Measurement of VEGF in serum origin that includes tumour cells. The may have value for monitoring of surgical procedure, with ligation of the therapeutic effect or choice of therapeutic blood supply during nephrectomy and modality [230]. time to excision of tumour samples, inevitable effects VEGF expression. Paper III However, previous studies have shown The RCC samples demonstrated a that when handling tissue samples, HIF- wide range of mRNA levels for VEGF121, 1α levels remain constant up to 60 min VEGF165, and Flt-1 (VEGF-R1). Other VEGF after clamping of the renal artery [43]. isoforms were not be detected. A signi- Moreover, no significant up-regulation of ficant correlation was observed between mRNA VEGF or changes in protein mRNA levels for VEGF121 and VEGF165 VEGF content has been demonstrated and between mRNA levels for VEGF165 during 20 minutes of ischemic conditions and VEGF-R1. RCC confined to the in kidney tissue [231]. Although the kidney (pT1-2 N0 M0) had higher mRNA patient sample sizes was small in Paper levels for VEGF121 compared with locally III we demonstrated that VEGF mRNA advanced tumours (pT3 N0-1 M0). On the and VEGF-R1 mRNA levels were higher other hand, S-VEGF was lower in loca- in CRCC compared with PRCC, and the lised tumours (pT1-2 N0 M0) compared appearances of diversity between VEGF with locally advanced tumours (pT3 N0-1 mRNA and S-VEGF levels should be M0). There was no association between further examined. VEGF-R1 mRNA le- different isoform VEGF mRNA levels vels had no predictive information, which and S-VEGF. CRCC had significantly could be explained by endothelial cell higher VEGF121 and VEGF-R1 mRNA activation of VEGF-R1 brought about by levels compared with PRCC. In patients several growth factors, including VEGF- with locally advanced tumours (pT3a-c A, VEGF-B as well as PDGF released at

N0-1 M0) increased VEGF121 mRNA different stages in RCC.

Paper IV fying also the localization of protein Positive immunohistochemical (IHC) expressed within tissue sections. However, VEGF staining was observed in both RCC the outcome of immunohistochemical and kidney cortex samples. Kidney cortex examination is dependent on the quality usually showed a strong IHC VEGF ex- of the biopsy sample, presence of tissue pression in cytoplasm of tubular cells. necrosis, antigen loss due to delay in While samples from RCC showed fixation of samples, type of fixative, dura- considerable variation in IHC VEGF tion of fixation, processing of samples, expression on cell membranes as well as choice of antibody, and sensitivity of in cytoplasm of tumour cells. IHC VEGF detections method for antigen-antibody expression at cell membrane was affected reaction [232]. Fixation prevents the by the storage time of paraffin embedded degradation of tissues, preserves the mor- tumour specimens. Hence, membranous phology, and stabilizes cells and tissues. VEGF expression was not further eva- On the other hand, fixation causes a cross- luated. There were no differences in linking within protein molecules, which cytoplasm VEGF expressions between alters the structure of the proteins, possi- gender or different age groups. A signi- bly altering the epitope and masking the ficant correlation was found between antigen against potential antibody bin- VEGF expression in cytoplasm and the ding. Heat-induced antigen retrieval may size of tumours. In tumours smaller than 7 disrupt these cross-links but recovery of cm, increasing VEGF expression was native molecule structure may depend on observed between stage I (pT1 N0 M0), achieved temperature and length of stage III (pT3a-c N0-1 M0), and stage IV incubation. Complete recovery of native (N2 M1), but no such difference was molecule structure is particularly im- found in VEGF expression at tumours larger portant for monoclonal antibodies, which than 7 cm. Tumours invading through the recognise a single epitope, whereas poly- renal capsule (pT3a, N0-1, M0) had clonal antibodies recognise a number of higher VEGF expression compared with different epitopes. Polyclonal antibodies tumours confined to kidney (pT1-2, N0, may express increased sensitivity to target M0) or tumours invading veins only but may also contain increased risk for (pT3b-c, N0, M0). No difference in VEGF cross-reactivity. Monoclonal antibodies expression was recognized between on the other hand are targeted at a single differrent RCC types. In univariate ana- epitope on the antigen and are therefore lysis, increased VEGF expression corre- highly specific. However, a monoclonal lated with adverse survival in patients antibody might fail to bind the epitope if with CRCC. No correlation was found the antigen is vulnerable and unstable due between VEGF expression and survival to the fixation process. It has been demon- time in PRCC or in ChRCC. In multi- strated that the antigenicity is inversely variate analysis the cytoplasmatic IHC correlated to duration of exposure to VEGF expression was not an independent formaldehyde [233]. Quantification of prognostic factor. immunoreaction intensity is an error filled process, since no linear relationship exists Comments on paper IV between amount of antigen and the Immunohistochemistry allows identi- expressed antigen-antibody reaction. A

34 more valid form of quantification is VEGF189 and VEGF165 isoforms compared determining the percentage of estimated with kidney cortex. There were no corre- antigen-antibody specific cellular reaction. lations between isoforms and tumour Identifying hotspot areas and estimating stage or grade in CRCC, although relative area with immunoreactions is a VEGF189 was verified as low in tumours highly operator-dependent process, and with renal capsule invasion (pT3a, N0-1, other investigators have emphasized the M0) compared with those without advantage of using the methods described invasion (pT1-2, N0, M0). VEGF121 was by Chalkey [234]. In Paper IV, variation more frequently detected in smaller (pT1) in results obtained from different methods and localized tumours (stage I-II) was shown, but nevertheless a correlation compared with lager (pT2) and advanced between observations could be noted. tumour stages (stage III-IV). PRCC

Thus, the established VEGF expression in showed also lower levels of VEGF189 and cytoplasm as being predictor for survival VEGF165 compared with kidney cortex. In led us to further examine the role of VEGF PRCC, protein VEGF165 level correlated location in RCC. Cytoplasmic VEGF was inversely with tumour size, and VEGF189 demonstrated to be of prognostic signi- levels correlated inversely with tumour ficance only in smaller tumours. Further- stage and grade. In ChRCC, protein level more, in solid tumours, several other of VEGF189 was comparable to that in growth factors have been proposed as kidney cortex, while VEGF165 level was candidate to persuade the growth when significantly decreased. Also in ChRCC, tumours reached a certain volume [235]. the VEGF189 levels were lower in advanced tumour stages (stage III-IV) Study V compared with tumours confined to Both RCC and kidney cortex samples kidney (stage I-II). VEGF121 was not expressed immunoreactive VEGF bands. detected in chromophobe RCC. In Samples from kidney cortex expressed univariate analysis, VEGF165 showed no predictive information. However, low VEGF189 and VEGF165 isoforms, but not VEGF levels were associated with VEGF121. Both VEGF189 and VEGF165 189 isoforms were strongly expressed in kid- shorter survival in PRCC. In Cox multi- ney cortex, compared to RCC samples. In variate analysis, the VEGF189 expression remained an independent prognostic RCC samples, VEGF189 had highest factor in patients with PRCC, while TNM immunoreactive expression and VEGF165 stage was the last variable to be omitted intermediate, while VEGF121 was rarely after stepwise elimination analysis. detected. VEGF121 expression was considered positive when a band was visually detected on the Western blot. There was Comments on paper V considerable variation in VEGF189 ex- The WB-technique is based on protein pression between the different RCC types, extractions separated in SDS-PAGE gel. in contrary to VEGF165 expression, which An equal loading of samples is crucial was usually low. No differences in VEGF when WB is used for quantification. isoforms were found according to gender, Assessment of protein extract concen- age, or due to storage time of tumour tration was reproduced at least twice, and samples. CRCC had lower levels of both the loaded protein was controlled by

Ponceau-Red staining, and sample protein paper support the idea that VEGF189 may concentration re-assured through actin be the primary isoform reflecting tumour detection by WB. Each sample was angiogenesis. Although mRNA VEGF189 analysed in duplicate, and in this paper was barely detectable in Paper III, the with different blocking solutions, which high VEGF189 protein expression in were used due to the EU import restrict- tumour tissue can be interpreted that tion of hoarse serum from New Zealand VEGF189 protein is produced and most in 1995. Any discordance in duplicate likely stored locally in tumour cells. results led to re-analysis with available Expression of mRNA VEGF189 has been blocking solutions. Advantages with WB- demonstrated in tumours stage T3-T4, and technique are the option to evaluate furthermore is associated with higher different VEGF protein isoform by their microvessel density in contrast to other molecular weight separated in SDS- isoforms [236]. Angiogenesis is induced PAGE gel, as well as the possibility to by released VEGF and enhanced by quantify expression of each VEGF iso- increased VEGF receptor density on form. A weakness with the WB method is endothelial cells. In addition, angio- that protein extractions are based on genesis is thought to be promoted by expression from a mixture of cells of release of bioactive different growth different origin including some other than factors such as VEGF165, VEGF189, and tumour cells. Therefore imprints were bFGF during proteolytic degradation of made to verify all samples in fact con- ECM [143, 237]. tained tumour tissue. The findings in this

FUTURE DIRECTIONS

Molecular biomarkers are expected to currences of RCC exist or in combination have an important impact on future with drugs having different mechanisms diagnosis, prognostication and selection of action. Currently no prognostic bio- of therapeutic targets for RCC. Several markers are available to independently potential drugs appear promising to in- validate the therapeutic effect in indivi- hibit tumour angiogenesis in RCC. These duals with RCC. These evaluations still include targeting VEGF with monoclonal depend on traditional clinical surveillance. antibody (Bevacizumb) [238, 239] and The complexities of multiple different inhibition of VEGF-R2 and PDGF-R growth factors are not completely under- (BAY 43-9006 / Sorafenib) [240, 241], or stood, and their role and relationship re- multitargeted tyrosine kinase inhibitor of mains important as a subject of future VEGF-R1, VEGF-R2, Flt-3 and PDGF-R investigation in RCC. (SU11248) [240, 241], whereas Thalidomide There are several issues, which need [242] and TPN-470 [243] have provided to be further investigated, as whether less promising clinical results. Novel cancer cells have receptors for VEGF and targeted therapies such as matrix metalo- what significance this existence of auto- proteinase inhibitors (AG3340 / Primo- crine effect might have. Do different mastat), (BB2516 / Marimstat), AE-941 / VEGF isoforms have an auxiliary effect Neovastat) have undergone clinical tes- on potential target cells i.e. causing ting with positive results [244]. Unique vascular permeability to macromolecules approaches with the aim of selectively and oedema, hemodynamic alteration by inhibiting angiogenesis is provoking vasodilatation, and incitation of angio- apoptosis in tumour stimulated endo- genesis? Despite evidence of vascular thelial cells in order to undermine growth dilatation and increased permeability the and support of existing tumour vessels effects and regulation are not clear in and cause tumour cell death from ische- pathological angiogenesis. Does VEGF mia (ZD6126 / Combretastatin) [245]. independently correlate to increased Considering the number of different microvessel density, or are there other genetic defects in RCC, it is unlikely that factors that counteract or counterpart a single drug will be effective in treatment VEGF? Hypoxy inducible factor (HIF) of advanced RCC. More likely, these new affects transcription level of VEGF, angiogenesis inhibitors can be expected to however do there exist other factors that be most effective when used as adjuvant could be considered as regulatory of therapy to surgery where risk for re- VEGF in RCC?

Figure 7. Antiangiogenic drugs currently under evaluation in cancer therapy.

CONCLUSIONS

Paper I In RCC, S-VEGF level was significantly correlated to tumour stage and grade. Increased levels of S-VEGF were associated with shorter survival. Although, S-VEGF did not remain as an independent prognostic factor in multivariate analysis, the levels of S- VEGF were found useful for the identification of patients with potentially progressive disease especially those with vein invasion.

Paper II S-VEGF showed a stronger association to prognosis than blood platelets and leukocyte counts. Blood platelet count seems to be affected in patients with intercurrent chronic disease and long-term medication, and in this large group of patients blood platelets counts are not associated with survival. Blood leukocyte count had no prognostic value.

Paper III

No correlation was found between VEGF165 mRNA and S-VEGF levels. Both VEGF121 and VEGF165 mRNA isoforms and Flt-1 mRNA (VEGF-R1) levels were higher in CRCC compared with PRCC. The trend of decreased levels of isoforms VEGF121 and VEGF165 mRNA in locally advanced RCC indicates that initiation of angiogenic activity by VEGF might be an early event and during tumour progression other factors are more relevant.

Paper IV IHC VEGF staining was observed at cell membrane and cytoplasm in RCC cells. There was no difference in cytoplasm VEGF expression between the different RCC types. Associations between expression of cytoplasm VEGF and tumour size as well as prognosis indicate the significance of VEGF in growth and progression. The expression of IHC VEGF in cytoplasm might be useful for identification of potentially progressive RCC but was not an independent prognostic factor in multivariate analysis.

Paper V Different VEGF isoform patterns were found between the specific RCC types.

VEGF165 protein expression had no predictive value for survival and was observed in low levels in RCC samples. The protein VEGF189 expression was associated with tumour stage and grade and was an independent prognostic factor in PRCC.

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to all those who have supported me in this work. Especially I thank:

Börje Ljungberg, (my supervisor, for never failing support and who introduced me to the field of RCC), Professor at the Department of Surgical and Perioperative Sciences, Urology and Andrology

Kjell Grankvist, (my cosupervisor, for contribution to discussions and new ideas), Professor at the Department of Medical Biosciences, Clinical Chemistry

Torgny Rasmuson, (my cosupervisor and for valuable help with manuscripts), Assistant Professor at the Department of Radiation Sciences, Oncology

Anders Bergh, (my cosupervisor, for guiding me through immunohistochemical evaluation), Professor at the Department of Medical Biosciences, Pathology

Göran Landberg, (for introducing TMA to this thesis), Professor at Division of Pathology, Department of Laboratory Medicine, Lund University, Malmö University Hospital

Radisa Tomic, (for providing me with good working conditions and skilful guidance in surgery), Chief surgeon and former Head of the Department of Urology

Stina Häggström-Rudolfssson, (for introducing the RT-PCR to this thesis) Ph.D., Assistant at the Department of Surgical and Perioperative Sciences, Urology and Andrology

Gudrun Lindh, (for carrying out the RT-PCR technique), Assistant at the Department of Surgical and Perioperative Sciences, Urology and Andrology

Ylva Hedberg, (for advice and positive attitude in my first contact with lab facilities), Ph.D., Assistant at the Department of Medical Biosciences, Pathology

Britta Lindgren, (for carrying out the numerous slides and TMA to this thesis), Assistant at the Department of Medical Biosciences, Pathology

Michael Haney, (for effective help in revising the English text), Ph.D., M.D. at Department of Surgical and Perioperative Science, Anaesthesiology and Intensive Care Medicine

Solveig Isberg, (for taking care of economy and secretarial services), Secretary at the Department of Surgical and Perioperative Sciences, Urology and Andrology

Veronica Berglund, Kerstin Almroth and Britt-Inger Dahlin (for helping me with categorize blood and tissue samples), Reseach Assistants at the Department of Urology

My colleagues, all staff and friends at the Department of Urology, Umeå University Hospital, Umeå, Sweden

This work was supported by the Swedish Cancer Society (4431-B01-02XBB), the Maud and Birger Gustavsson Foundation, and the Lions Research Foundation, Umeå University, Umeå, Sweden.

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