Radiation sterilisation of cultured human brain tumour cells for clinical immune tumour therapy

The aim is to investigate the radiosensitivity of noninfected cultured human glioma cells to ascertain that intracutaneously administered cells are viable enough to produce interferon-gamma but not able to proliferate. Cell cultures were established from five patients undergoing brain tumour surgery. By karyotyping, we found four malignant (three glioblastoma multiforme (GBM), one giant cell glioma) and one normal. The cells were irradiated with (137)Cs-gamma rays at absorbed dose levels of 0, 20, 40, 60, 80, 100 and 120 Gy. The fraction of viable cells was examined by MTT incorporation assay. The average of the data obtained from three GBM cell cultures was fitted to an exponential model. The parameters were: extrapolation number n=0.85+/-0.10, mean lethal dose D(0)=12.4+/-3.2 Gy and an additional uncertainty parameter deltaS=0.14+/-0.03. By setting deltaS=0, the corresponding values of the parameters were n=0.86+/-0.16 and D(0)=30.0+/-8.1 Gy. The rate of proliferation was examined by (3)H-thymidine incorporation. The average of the proliferation data obtained from three GBM cell cultures was fitted to an exponential model yielding n=0.943+/-0.005 and D(0)=5.8+/-0.5 Gy for deltaS=0.057+/-0.005, and by setting deltaS=0, n=1.00+/-0.02 and D(0)=8.4+/-1.6 Gy. No outgrowth of plated cells was observed after 4 weeks at an absorbed dose of 100 Gy. This absorbed dose is recommended for irradiation of 2 x 10(6) glioma cells used for clinical immunisation.     

Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry

This study aimed to evaluate tumour hypoxia by comparing [(18)F]Fluoromisonidazole uptake measured using positron emission tomography ([(18)F]FMISO-PET) with immunohistochemical (IHC) staining techniques. Syngeneic rhabdomyosarcoma (R1) tumour pieces were transplanted subcutaneously in the flanks of WAG/Rij rats. Tumours were analysed at volumes between 0.9 and 7.3 cm(3). Hypoxic volumes were defined using a 3D region of interest on 2 h postinjection [(18)F]FMISO-PET images, applying different thresholds (1.2-3.0). Monoclonal antibodies to pimonidazole (PIMO) and carbonic anhydrase IX (CA IX), exogenous and endogenous markers of hypoxia, respectively, were used for IHC staining. Marker-positive fractions were microscopically measured for each tumour, and hypoxic volumes were calculated. A heterogeneous distribution of hypoxia was observed both with histology and [(18)F]FMISO autoradiography. A statistically significant correlation (P<0.05) was obtained between the hypoxic volumes defined with [(18)F]FMISO-PET and the volumes derived from the PIMO-stained tumour sections (r=0.9066; P=0.0001), regardless of the selected threshold between 1.4 and 2.2. A similar observation was made with the CA IX staining (r=0.8636; P=0.0006). The relationship found between [(18)F]FMISO-PET and PIMO- and additionally CA IX-derived hypoxic volumes in rat rhabdomyosarcomas indicates the value of the noninvasive imaging method to measure hypoxia in whole tumours.     

In vivo evaluation of [18F]fluoroetanidazole as a new marker for imaging tumour hypoxia with positron emission tomography

Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [(18)F]fluoroetanidazole ([(18)F]FETA) as a tumour hypoxia marker by positron emission tomography (PET). Cellular transport and retention of [(18)F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [(18)F]FETA, with an octanol-water partition coefficient of 0.16+/-0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60-120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO(2) values <5 mmHg (r=0.805, P=0.027) and carbogen breathing decreased [(18)F]FETA-derived radioactivity levels (P=0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30-60 min. Higher fractional retention of [(18)F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P=0.05) reflected higher percentage of pO(2) values <1 mmHg (P=0.023), lower vessel density (P=0.026), and higher radiobiological hypoxic fraction (P=0.008) of the HT1080/1-3C tumours. In conclusion, [(18)F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.     

A cyclin-dependent kinase inhibitor (p21(WAF1/CIP1)) affects thymidine incorporation in human liver cancer cells

p21(WAF1/CIP1) is a universal cyclin-dependent kinase inhibitor. To investigate the role of p21(WAF1/CIP1) in proliferation of human liver cancer cells, we examined the expression of p53, p21(WAF1/CIP1), cdk2 and cdk4 expression in two human liver cancer cell lines (HepG2 and PLC/PRF/5 cells). The effects of p21(WAF1/CIP1) on [(3)H]thymidine incorporation and cdks were also examined in these cells. HepG2 cells expressed all these proteins with lower level of cdk4. PLC/PRF/5 cells expressed the other proteins except p21(WAF1/CIP1). Transfection of p21(WAF1/CIP1) gene inhibited [(3)H]thymidine incorporation of both cells with different extent. Although the transfection of p21(WAF1/CIP1) did not affect cdk2 and cdk4 expression, it did reduce cdk2 kinase activity by 20%. These results suggest that: (a) p21(WAF1/CIP1) involved in DNA synthesis of human liver cancer cells; (b) p21(WAF1/CIP1) could be a target gene for the treatment of human hepatocellular carcinoma.     

Monitoring HSVtk suicide gene therapy: the role of [(18)F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [(18)F]FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [(18)F]FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [(18)F]FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour-bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2-chloroadenosine did not significantly affect the [(18)F]FHPG uptake in vitro. Thymidine and uridine significantly decreased [(18)F]FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [(18)F]FHPG uptake in C6tk and C6 tumours decreased from 3.0+/-0.5 to 1.0+/-0.2 after infusion of adenine. Thus, in our tumour model, [(18)F]FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier.     

Irradiation of rat brain reduces P-glycoprotein expression and function

The blood-brain barrier (BBB) hampers delivery of several drugs including chemotherapeutics to the brain. The drug efflux pump P-glycoprotein (P-gp), expressed on brain capillary endothelial cells, is part of the BBB. P-gp expression on capillary endothelium decreases 5 days after brain irradiation, which may reduce P-gp function and increase brain levels of P-gp substrates. To elucidate whether radiation therapy reduces P-gp expression and function in the brain, right hemispheres of rats were irradiated with single doses of 2-25 Gy followed by 10 mg kg(-1) of the P-gp substrate cyclosporine A (CsA) intravenously (i.v.), with once 15 Gy followed by CsA (10, 15 or 20 mg kg(-1)), or with fractionated irradiation (4 x 5 Gy) followed by CsA (10 mg kg(-1)) 5 days later. Additionally, four groups of three rats received 25 Gy once and were killed 10, 15, 20 or 25 days later. The brains were removed and P-gp detected immunohistochemically. P-gp function was assessed by [(11)C]carvedilol uptake using quantitative autoradiography. Irradiation increased [(11)C]carvedilol uptake dose-dependently, to a maximum of 20% above non irradiated hemisphere. CsA increased [(11)C]carvedilol uptake dose-dependently in both hemispheres, but more (P<0.001) in the irradiated hemisphere. Fractionated irradiation resulted in a lost P-gp expression 10 days after start irradiation, which coincided with increased [(11)C]carvedilol uptake. P-gp expression decreased between day 15 and 20 after single dose irradiation, and increased again thereafter. Rat brain irradiation results in a temporary decreased P-gp function.     

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.     

Analysis of resveratrol as a lung cancer chemopreventive agent in A/J mice exposed to benzo[a]pyrene

Resveratrol inhibits PAH bioactivation through reduced expression of the CYP1A1 and CYP1B1 genes in human bronchial epithelial cells. Ad libitum access to a diet containing resveratrol showed no effect on benzo[a]pyrene-induced lung tumorigenesis in A/J mice. Also, resveratrol did not change CYP1A1 and CYP1B1 gene expression or benzo[a]pyrene protein adduct levels in the lung tissue. The lack of chemopreventive activity may have been caused by insufficient concentrations or nonreactive forms of resveratrol in the lungs.     

Alpha-fetoprotein and human chorionic gonadotrophin-beta as prognostic markers in neuroendocrine tumour patients

Serum chromogranin A is the most useful general and prognostic tumour marker available for neuroendocrine tumour (NET) patients. The role of other tumour markers is less clear. In order to determine the diagnostic and prognostic value of serum alpha-fetoprotein (AFP) and human chorionic gonadotrophin-beta (hCGbeta) in NETs, a database containing biochemical, histological, and survival data on 360 NET patients was constructed. This data was statistically assessed, using Statistical Package for the Social Sciences, to determine the utility of commonly measured tumour markers with particular emphasis on AFP and hCGbeta. Alpha-fetoprotein and hCGbeta were raised in 9.5 and 12.3% of patients respectively and jointly raised in 9.1% of patients in whom it was measured. Alpha-fetoprotein levels associated strongly and positively with tumour grade, serum CgA and hCGbeta levels, and worse survival. Human chorionic gonadotrophin-beta levels also associated strongly and positively with serum CgA and AFP levels, and worsening survival. Alpha-fetoprotein and hCGbeta are elevated in high-grade NETs, with a rapidly progressive course and poorer survival. They also correlate with chromogranin-A, which is known to be a marker of tumour burden and to have prognostic value. Thus AFP and hCGbeta are clinically important in NETs and when elevated are poor prognostic markers.     

Identification of novel helper epitopes of MAGE-A4 tumour antigen: useful tool for the propagation of Th1 cells

MAGE-A4 has been considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy. It has been well accepted that T-helper type 1 (Th1) cell-dominant immunity is critical for the successful induction of antitumour immunity in a tumour-bearing host. The adoptive Th1 cell therapy has been shown to be an attractive strategy for inducing tumour eradication in mouse systems. However, Th1-cell therapy using human tumour-specific Th1 cells, which were expanded from peripheral blood mononuclear cells (PBMCs) in a clinically useful protocol, has never been performed. Here, we first identified MAGE-A4-derived promiscuous helper epitope, peptide (MAGE-A4 280–299), bound to both HLA-DPB1^*0501 and DRB1^*1403. Using the peptide, we established a suitable protocol for the propagation of MAGE-A4-specific Th1 cells in vitro. Culture of CD4⁺ T cells with IFN-γ-treated PBMC-derived adherent cells in the presence of helper epitope peptide resulted in a great expansion of MAGE-A4-reactive Th cells producing IFN-γ , but not IL-4. Moreover, it was shown that ligation of MAGE-A4-reactive Th1 cells with the cognate peptide caused the production of IFN-γ and IL-2. Thus, our identified MAGE-A4 helper epitope peptide will become a good tool for the propagation of tumour-specific Th1 cells applicable to adoptive immunotherapy of human cancer.     

Preclinical studies of targeted alpha therapy for breast cancer using 213Bi-labelled-plasminogen activator inhibitor type 2

The control of micrometastatic breast cancer remains problematic. To this end, we are developing a new adjuvant therapy based on (213)Bi-PAI2, in which an alpha-emitting nuclide ((213)Bi) is chelated to the plasminogen activator inhibitor-2 (PAI2). PAI2 targets the cell-surface receptor bound urokinase plasminogen activator (uPA), which is involved with the metastatic spread of cancer cells. We have successfully labelled and tested recombinant human PAI2 with the alpha radioisotope (213)Bi to produce (213)Bi-PAI2, which is highly cytotoxic towards breast cancer cell lines. In this study, the 2-day postinoculation model, using MDA-MB-231 breast cancer cells, was shown to be representative of micrometastatic disease. Our in vivo efficacy experiments show that a single local injection of (213)Bi-PAI2 can completely inhibit the growth of tumour at 2 days postcell inoculation, and a single systemic (i.p.) administration at 2 days causes tumour growth inhibition in a dose-dependent manner. The specific role of uPA as the target for (213)Bi-PAI2 therapy was determined by PAI2 pretreatment blocking studies. In vivo toxicity studies in nude mice indicate that up to 100 microCi of (213)Bi-PAI2 is well tolerated. Thus, (213)Bi-PAI2 is successful in targeting isolated breast cancer cells and preangiogenic cell clusters. These results indicate the promising potential of (213)Bi-PAI2 as a novel therapeutic agent for micrometastatic breast cancer.     

Expression level of integrin alpha 5 on tumour cells affects the rate of metastasis to the kidney

Tumour metastasis is known clinically to have organ specificity. We hypothesised that integrins might be involved in determining the organ specificity of tumour metastasis. Here, we report the results of spontaneous metastasis tested in nude mice that were inoculated with Chinese hamster ovary (CHO) cells expressing integrin alpha 5 beta 1 at various levels. The growth of the primary tumour inversely correlated with the alpha 5 expression level on CHO cells, which is consistent with a previous report (Schreiner et al, 1991). The rates of pulmonary, lymph node, and adrenal metastases that developed in nude mice were not related to changes of the alpha 5 expression level on CHO cells. Kidney metastasis developed in 40% of nude mice inoculated with alpha 5B2 cells (CHO cells overexpressing alpha 5) and in 20% of mice with CHO-K1 cells (CHO cells expressing native alpha 5), whereas inoculation with CHO-B2 cells (alpha 5-defective mutants) and alpha 5CHO cells with the highest expression of alpha 5 did not lead to development of kidney metastasis. Furthermore, alpha 5CHO, which shows the slowest growth of these cell types, did not lead to primary tumours in nude mice. These findings suggest that there is an appropriate level of alpha 5 expression on tumour cells that leads to metastasis. Microscopic observations revealed that micrometastasis in the kidney was formed in glomeruli. An adhesion assay using frozen sections of the kidney demonstrated that alpha 5B2 cells, but not CHO-B2 cells, effectively adhered to glomeruli. Kidney metastasis in vivo and the adhesion of alpha 5B2 to glomeruli shown ex vivo were significantly suppressed by the administration of GRGDS peptide. Finally, we conclude that the interaction of alpha 5 beta 1 on tumour cells with fibronectin in kidney glomeruli is involved in kidney metastasis and that the tumour has appropriate levels of integrins crucial for metastasis.     

Cytostatic potential of novel agents that inhibit the regulation of intracellular pH

Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH (pHi) through the activity of membrane-based ion exchange mechanisms including the Na(+)/H(+) antiport and the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger. Inhibition of these regulatory mechanisms has been proposed as an approach to tumour therapy. Previously available inhibitors of these exchangers were toxic (e.g. 4,4-diisothiocyanstilbene-2,2-disulphonic acid), and/or non-specific (e.g. 5-N-ethyl-N-isopropyl amiloride). Using two human (MCF7, MDA-MB231) and one murine (EMT6) breast cancer cell lines, we evaluated the influence of two new agents, cariporide (an inhibitor of the Na(+)/H(+) antiport) and S3705 (an inhibitor of the Na(+)-dependent Cl(-)/HCO(3)(-) exchanger) on the regulation of intracellular pH (pHi). The cytotoxicity of the two agents was assessed by using clonogenic assays. Our results suggest that cariporide has similar efficacy and potency to 5-N-ethyl-N-isopropyl amiloride for inhibition of Na(+)/H(+) exchange while S3705 is more potent and efficient than 4,4-diisothiocyanstilbene-2,2-disulphonic acid in inhibiting Na+-dependent Cl(-)/HCO3(-) exchange. The agents inhibited the growth of tumour cells when they were incubated at low pHe (7.0-6.8), but were non-toxic to cells grown at doses that inhibited the regulation of pHi. Our results indicate that cariporide and S3705 are selective cytostatic agents under in vitro conditions that reflect the slightly acidic microenvironment found in solid tumours.     

The transcription factor DEC1 (stra13, SHARP2) is associated with the hypoxic response and high tumour grade in human breast cancers

DEC1, also known as SHARP-2 or Stra13, plays important roles in embryonic development, proliferation, apoptosis and cell differentiation in the mouse. DEC1 was recently identified as hypoxically induced in cDNA microarray studies of the human renal carcinoma cell line RCC4, to be regulated through hypoxia-inducible factor (HIF)-1alpha and via HIF-1alpha, able to block adipocyte differentiation. Nevertheless, its distribution and role in hypoxia and differentiation in human breast cancer are unknown. We therefore examined the pattern and level of expression of DEC1 using immunohistochemistry in whole tissue sections in normal, in situ and invasive breast carcinomas, and correlated the level of expression of DEC1 and clinicopathological factors and hypoxic tumour markers in 253 invasive carcinomas on tissue microarrays. We observed an increase in DEC1 expression during progression from normal to in situ and invasive carcinoma. Expression was not restricted to the tumour cell element but was also observed in endothelial, fibroblasts and inflammatory cells. There was a significant positive correlation between DEC1 and tumour grade (P=0.01), HIF-1alpha (P=0.04) and the hypoxically regulated gene angiogenin (P<0.0001), but no significant associations were observed with patient age (P=0.15), lymph node status (P=0.8), tumour size (P=0.3), oestrogen receptor (P=0.45), epidermal growth factor receptor (P=0.27) or Chalkley vessel count (P=0.45). There was no difference in relapse-free (P=0.84) or overall (P=0.78) survival. These findings suggest that DEC1 plays an important role in the progression to invasive breast cancer and that it may provide a mechanism by which hypoxia blocks tumour differentiation, and may contribute to a more aggressive phenotype. Reversing this phenotype may alter the biological behaviour of individual tumours.     

Cell cycle checkpoint status in human malignant mesothelioma cell lines: response to gamma radiation

Knowledge of the function of the cell cycle checkpoints in tumour cells may be important to develop treatment strategies for human cancers. The protein p53 is an important factor that regulates cell cycle progression and apoptosis in response to drugs. In human malignant mesothelioma, p53 is generally not mutated, but may be inactivated by SV40 early region T antigen (SV40 Tag). However, the function of p53 has not been investigated in mesothelioma cells. Here, we investigated the function of the cell cycle checkpoints in six human mesothelioma cell lines (HMCLs) by studying the cell distribution in the different phases of the cell cycle by flow cytometry, and expression of cell cycle proteins, p53, p21(WAF1/CIP1) and p27(KIP1). In addition, we studied p53 gene mutations and expression of SV40 Tag. After exposure to gamma-radiation, HMCLs were arrested either in one or both phases of the cell cycle, demonstrating a heterogeneity in cell cycle control. G1 arrest was p21(WAF1/CIP1)- and p53-dependent. Lack of arrest in G1 was not related to p53 mutation or binding to SV40 Tag, except in one HMCL presenting a missense mutation at codon 248. These results may help us to understand mesothelioma and develop new treatments.     

Potentiation of the anti-tumour effects of Photofrin-based photodynamic therapy by localized treatment with G-CSF

Photofrin-based photodynamic therapy (PDT) has recently been approved for palliative and curative purposes in cancer patients. It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing.     

Specific recognition and inhibition of Ewing tumour growth by antigen-specific allo-restricted cytotoxic T cells

Background:

The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient''s immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8⁺ T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis.

Methods:

Following repetitive CHM1³¹⁹ (VIMPCSWWV) and EZH2⁶⁶⁶ (YMCSFLFNL) peptide-driven stimulations with HLA-A^*0201⁺ dendritic cells (DC), allo-restricted HLA-A^*0201⁻ CD8⁺ T cells were stained with HLA-A^*0201/peptide multimers, sorted and expanded by limiting dilution.

Results:

Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A^*0201 and killed HLA-A^*0201⁺ ET lines expressing the antigen while HLA-A^*0201^– ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2^−/−γ_C^−/− mice. Within this context, we identified the CHM1³¹⁹ peptide as a new candidate target antigen for ET immunotherapy.

Conclusion:

These results clearly identify the ET-derived antigens, EZH2⁶⁶⁶ and CHM1³¹⁹, as suitable targets for protective allo-restricted human CD8⁺ T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation.     

Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer

Overexpression of fibroblast growth factors (FGFs) has been implicated in prostate carcinogenesis. FGFs function via their high-affinity interactions with receptor tyrosine kinases, FGFR1-4. Expression of FGFR1 and FGFR2 in prostate cancer (CaP) was not found to be associated with clinical parameters. In this report, we further investigated for abnormal FGFR expression in prostate cancer and explore their significance as a potential target for therapy. The expression levels of FGFR3 and FGFR4 in CaP were examined and corroborated to clinical parameters. FGFR3 immunoreactivity in benign prostatic hyperplasia (BPH) and CaP (n=26 and 57, respectively) had similar intensity and pattern. Overall, FGFR4 expression was significantly upregulated in CaP when compared to BPH. A significant positive correlation between FGFR4 expression and Gleason score was noted: Gleason score 7-10 tumours compared to BPH (P<0.0001, Fisher's exact test), Gleason score 4-6 tumours compared to BPH (P<0.0004), and Gleason 7-10 compared to Gleason 4-6 tumours (P<0.005). FGFR4 overexpression was associated with an unfavourable outcome with decreased disease-specific survival (P<0.04, log rank test). FGF-induced signalling is targeted using soluble FGF receptor (sFGFR), potent inhibitor of FGFR function. We have previously shown that sFGFR expression via a replication-deficient adenoviral vector (AdlllcRl) suppresses in vitro FGF-induced signalling and function in human CaP DU145 cells. We tested the significance of inhibiting FGF function along with conventional therapeutic modalities in CaP, and confirmed synergistic effects on in vitro cell growth (proliferation and colony formation) by combining sFGFR expression and treatment with either Paclitaxel (Taxol) or gamma-irradiation. In summary, our data support the model of FGF system as valid target for therapy in CaP.     

Degree of tumour vascularity correlates with drug accumulation and tumour response upon TNF-alpha-based isolated hepatic perfusion

Isolated hepatic perfusion (IHP) with melphalan with or without tumour necrosis factor alpha (TNF-alpha) is currently performed in clinical trials in patients with hepatic metastases. Previous studies led to the hypothesis that the use of TNF-alpha in isolated limb perfusion causes specific destruction of tumour endothelial cells and thereby induces an increased permeability of tumour vasculature. However, whether TNF-alpha contributes to the therapeutic efficacy in IHP still remains unclear. In an in vivo rat liver metastases model we studied three different tumours: colon carcinoma CC531, ROS-1 osteosarcoma and BN-175 soft-tissue sarcoma which exhibit different degrees of vascularisation. IHP was performed with melphalan with or without the addition of TNF-alpha. IHP with melphalan alone resulted, in all tumour types, in a decreased growth rate. However in the BN-175 tumour addition of TNF-alpha resulted in a strong synergistic effect. In the majority of the BN-175 tumour-bearing rats, a complete response was achieved. In vitro cytoxicity studies showed no sensitivity (CC531 and BN-175) or only minor sensitivity (ROS-1) to TNF-alpha, ruling out a direct interaction of TNF-alpha with tumour cells. The response rate in BN-175 tumour-bearing rats when TNF-alpha was coadministrated with melphalan was strongly correlated with drug accumulation in tumour tissue, as only in these rats a five-fold increased melphalan concentration was observed. Secondly, immunohistochemical analysis of microvascular density (MVD) of the tumour showed a significantly higher MVD for BN-175 tumour compared to CC531 and ROS-1. These results indicate a direct relation between vascularity of the tumour and TNF-alpha mediated effects. Assessment of the tumour vasculature of liver metastases would be a way of establishing an indication for the utility of TNF-alpha in this setting.     

Mutation of the PIK3CA oncogene in human cancers

It is now well established that cancer is a genetic disease and that somatic mutations of oncogenes and tumour suppressor genes are the initiators of the carcinogenic process. The phosphatidylinositol 3-kinase signalling pathway has previously been implicated in tumorigenesis, and evidence over the past year suggests a pivotal role for the phosphatidylinositol 3-kinase catalytic subunit, PIK3CA, in human cancers. In this review, we analyse recent reports describing PIK3CA mutations in a variety of human malignancies, and discuss their possible implications for diagnosis and therapy.