Adenovirus-mediated herpes simplex virus thymidine kinase ... - Nature

Cancer Gene Therapy (2002) 9, 917 – 924 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com / cgt

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy in BT4C rat glioma model Kristiina Tyynela¨,1,2 Anu-Maaria Sandmair,1,3 Marita Turunen,1,2 Ritva Vanninen,4 Pauli Vainio,4 Risto Kauppinen,1,5 Risto Johansson,2,6 Matti Vapalahti,3,7 and Seppo Yla¨ -Herttuala1,6,7 1

AI Virtanen Institute, University of Kuopio, Kuopio, Finland; Departments of 2Oncology, 3Neurosurgery and Clinical Radiology, Kuopio University Hospital, Kuopio, Finland; 5NMR Research Group, University of Kuopio, Kuopio, Finland; 6Department of Medicine; and 7Gene Therapy Unit, Kuopio University Hospital, Kuopio, Finland.

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Adenovirus ( Adv ) - mediated herpes simplex virus thymidine kinase ( adv / tk ) gene therapy combined with ganciclovir ( GCV ) medication is a promising approach for the treatment of malignant glioma. However, optimal administration and the effect of possible adjuvant treatments have not been fully examined. In the present study, we examined the efficacy of adv / tk / GCV gene therapy in a syngeneic BT4C rat malignant glioma model, either as a single administration or given as three injections during three consecutive days. The effect of combined adv - mediated macrophage colony - stimulating factor ( MCSF ) and adv / tk gene transfer was also studied. BT4C malignant glioma cells were injected into the right corpus callosum of BDIX rats ( n = 112 ). Before gene therapy, the presence of tumors was verified by MRI. The rats were divided into eight groups as follows: group I ( n = 20 ) received a single adv / tk gene transfer ( total dose 4108 pfu ) and GCV treatment for 14 days; group II ( n = 5 ) received the same gene transfer without GCV; group III ( n = 28 ) received three adv / tk injections ( total dose 4108 pfu ) on three consecutive days and GCV for 14 days; group IV ( n = 5 ) received three similar adv / tk injections without GCV medication; group V ( n = 13 ) received three adv / MCSF injections ( total dose 2108 pfu ) on three consecutive days and GCV medication; group VI ( n = 12 ) received three adv / tk and adv / MCSF ( total dose 6108 pfu ) injections on three consecutive days followed by GCV medication; and group VII ( n = 12 ) the same treatment without GCV. Group VIII ( n = 17 ) consisted of wild - type BT4C malignant glioma tumors without any treatment. Treatment effect and tissue responses were characterized by general histology, immunohistochemistry, MRI, and survival of the study groups. The best treatment effect and survival was found in rats treated with adv / tk gene transfer once a day for three consecutive days ( P < .05 ). No improvement of the treatment effect was seen after the combined adv / tk and adv / MCSF gene transfer compared with the repeated adv / tk gene transfer. The results show that 20% of the rats can be cured ( survival >6 months ) after optimized adv / tk gene therapy. It is concluded that repeated intratumoral administration of adv / tk is a promising approach for the treatment of malignant glioma tumors in vivo. Cancer Gene Therapy ( 2002 ) 9, 917 – 924 doi:10.1038/sj.cgt.7700515 Keywords: gene transfer; thymidine kinase; macrophage colony - stimulating factor; glioma; MRI imaging

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espite improvements in current treatment strategies malignant glioma has a very poor prognosis.1,2 Gene therapy may offer a new strategy for the treatment of malignant brain tumors. Herpes simplex virus thymidine kinase ( tk ) gene therapy with ganciclovir (GCV ) medication has been successfully used for the treatment of malignant glioma in animal experiments,3 – 6 and recently encouraging results have been obtained from a phase I /II human study7 where adenovirus (adv ) was used for the delivery of

Received August 8, 2002. Address correspondence and reprint requests to: Dr Seppo Yla¨ - Herttuala, A.I. Virtanen Institute, University of Kuopio, P.O.B. 1627, FIN - 70211 Kuopio, Finland.

thymidine kinase (adv /tk ) into the tumor cavity. However, optimal administration of the treatment has not yet been fully examined. Several rat malignant glioma models have been used for experimental studies. However, many widely used syngeneic models, such as 9L, can be immunogenic.8 Another common model, C6 glioma, is not syngeneic and induces immunoresponses in the host.8 In this study, we used a syngeneic BT4C rat malignant glioma model.6,9 The BT4C glioma model was chosen because it resembles human malignant glioma in many aspects, including its ability to grow without inducing any significant tissue reactions in the recipient animals. The model has also predicted the outcome of human tk gene therapy trials.7,9,10 In this study, adv/tk gene therapy was given either as a single intratumoral injection or as three intratumoral

Malignant glioma gene therapy K Tyynela¨ et al

918 injections during three consecutive days. As an adjuvant treatment, macrophage colony - stimulating factor ( MCSF ) adenovirus was used to see whether it can augment the treatment effect. Before gene therapy, the presence of brain tumors was verified by MRI. The best treatment effect on survival was seen after repeated administrations of adv /tk during three consecutive days. Materials and methods Adenoviral vectors

Production of tk adenoviruses containing herpes simplex virus tk cDNA ( GeneBank V00470) under CMV promoter has been described earlier.7 Briefly, recombinant E1 partially E3 - deleted first - generation adenoviruses were generated through homologous recombination.11 Adenovirus containing human MCSF cDNA (GeneBank NM 000757 ) under CMV promoter was generated in a similar way. Human clinical grade adenoviruses were produced in 293 cells and the virus lysates were purified through two CsCl gradients, dialyzed, and stored at 808C.7 The ability of adenoviruses to produce functional tk was verified in cultured BT4C glioma cells as their ability to kill glioma cells in the presence of GCV. Functionality of the MCSF adenoviruses was verified with an ELISA assay (R&D Systems, Minneapolis, MN ) and in in vitro colony formation assay.12 The absence of wild - type replication- competent viruses was analyzed by a cytopathic effect assay on HeLa ( ATCC CCL 2 ) and A549 (ATCC CCL185 ) cells.13 Virus preparations were also tested for the presence of mycoplasma, other microbiological contaminants, and lipopolysaccharide ( Limulus assay; Sigma, St. Louis, MO ).13 Titers of the purified virus lots were adv/tk 2.01010 pfu /mL and Adv /MCSF 4.01010 pfu /mL. Experimental model

BT4C rat malignant glioma cells were grown in Dulbecco’s modified Eagle’s medium ( DMEM; GIBCO BRL, Paisley, Scotland ) with 10% fetal calf serum (GIBCO ), 2 mM glutamine, 2 mM sodium pyruvate, and 50 g/ mL genta-

micin at 378C in the presence of 5% CO2. Stereotactic injections of the BT4C glioma cells were done into the right corpus callosum of BDIX rats (160 – 350 g ) (n =112) as described.9 BT4C cells were resuspended in Optimem ( GIBCO - BRL, Gaithersburg, MD ) to a final concentration of 10 000 cells / 5L. Rats were anesthetized intraperitoneally ( i.p. ) with 0.4 mL/ 100 g of the solution containing 4.25% chloral hydrate and 0.97% pentobarbital. Rats were placed into a stereotactic apparatus ( Kopf, Berlin, Germany ) and injected with 104 BT4C cells in 5L of Optimem with a 25L Hamilton syringe ( Hamilton, Bonaduz, Switzerland ). Injections were made to the depth of 2.5– 3 mm into the right corpus callosum.14 To avoid back flow of the tumor cells the injections were given during one minute and the needle was left in place for 10 minutes before removal. Skin incision was closed with stitches (4 -0 Dexon, Davis and Geck, Hampshire, UK ). Rats were divided into experimental groups as shown in Table 1. Before the treatments, the presence of tumors was verified by MRI (see below ). One day after the first MRI gene transfers were made into the tumors with a volume of 10 L into the depth of 2.5 mm, followed by another 10 -L injection after 10 minutes. These injection volumes were previously found to give maximal penetration of viruses in the tumor area. To prevent back flow, the needle was left in place for another 10 minutes before removal. As indicated in Table 1, some animals received i.p. GCV treatment 50 mg /kg per day divided into two doses per day for 2 weeks. GCV treatment was started 5 days after the first adenovirus injection to allow sufficient time for the production of tk enzyme in the transfected tissue before GCV treatment. Study groups were as follows (Table 1): Group I (n =20 ) received a single injection of adv /tk ( total dose 4108 pfu ) and GCV medication. Group II (n = 5) received the same adv/ tk gene transfer without GCV. Group III (n =28 ) received adv /tk injections ( total dose 4108 pfu ) once a day for three consecutive days and GCV medication. Group IV ( n= 5 ) received similar adv /tk injections as group III but no GCV medication. Group V ( n= 13 ) received adv / MCSF injections ( total dose 2108 pfu ) once a day for three consecutive days and GCV medication. Group VI (n =12)

Table 1 Study groups Number of animals Group

Injection I

Injection II

Injection III

Medication

Total

Sacrificed

Survival

I II III IV V VI VII VIII Total

Adv / tk Adv / tk Adv / tk Adv / tk Adv / MCSF Adv / tk / Adv / MCSF Adv / tkAdv / MCSF –

– – Adv / tk Adv / tk Adv / MCSF Adv / tkAdv / MCSF Adv / tkAdv / MCSF –

– – Adv / tk Adv / tk Adv / MCSF Adv / tkAdv / MCSF Adv / tkAdv / MCSF –

GCV – GCV – GCV GCV – –

20 5 28 5 13 12 12 17 112

12 5 7 5 7 5 5 – 46

8 – 21 – 6 7 7 17 66

BT4C malignant glioma cells were injected stereotactically into BDIX rat right corpus callosum. Adv / tk, adv / MCSF, or their combination were stereotactically injected into the tumors either once ( injection I ) or once a day for three consecutive days ( injections I – III ). Rats in groups I, III, V, and VI received GCV medication i.p. ( 50 mg / kg per day ) for 14 days. Randomly selected rats ( n = 46 ) were sacrificed after the GCV treatment, and the rest of the animals ( n = 66 ) were left for a survival study ( total n = 112 ).

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919 Magnetic resonance imaging

Figure 1 MRI of BT4C malignant glioma tumors. T2 - weighted coronal views of the brain were used to visualize the tumor growth before and after gene therapy. A: BT4C glioma tumor ( arrow ) in the right corpus callosum 12 days after cell injection at the beginning of gene therapy. B: The same rat as in ( A ) 1 month after the BT4C cell injection after a single administration of adv / tk gene therapy and GCV medication. Some necrosis is seen in the central area of the tumor. The growing tumor causes hydrocephalus. C: BT4C glioma tumor ( arrow ) in the right corpus callosum 12 days after cell injection. Injection canal is seen above the corpus callosum. D: The same rat as in ( C ) 1 month after the BT4C cell injection after adv / tk gene therapy given once a day during three consecutive days, followed by GCV medication. A scar area, large ventricles, and hydrocephalus are visible after the gene therapy. E: The same rat as in ( C ) and ( D ) 3 months after the BT4C cell injection. The scar area ( arrow ) and very large ventricles are seen but no glioma tumor is present. The rat survived 6 months without any severe neurological symptoms. No glioma cells were detected in histological analysis of the brain ( data not shown ). F: A control rat with a large wild - type glioma tumor without any treatment 1 month after implantation of the BT4C cells.

received combined adv /tk and adv/ MCSF ( total dose 6108 pfu ) as a single injection during three consecutive days and GCV medication. Group VII ( n =12 ) received the same adv/ tk and adv/MCSF treatment as group VI but no GCV. Group VIII with wild - type BT4C cells (n =17 ) received no treatment and showed the natural course of the disease. Some rats were sacrificed for histological analyses after the GCV treatment and the rest of the rats were left for the survival study (Table 1 ). If the rats had no severe neurological symptoms, the follow -up time in the survival study was 6 months. Malignant glioma was considered cured if the rats survived 6 months.

MRI was used for imaging of the tumors. Rats weighing less than 250 g were imaged in a 9.4 T vertical magnet ( Oxford Instruments, Oxford, UK ) equipped with actively shielded field gradients (Magnex Scientific, Abdincton, UK ) interfaced to a microimaging SMIS console (Surrey Medical Imaging Systems, Guildford, UK ). Imaging was made with a transmit – receive birdcage coil ( internal diameter 38 mm ). The rats were fixed to the holder and were continuously kept under halothane anesthesia (induction 3% and maintenance 1 – 2% ). T2 -weighted images were taken in frontal orientation (repetition time 4000 and echo time 60 ms ). Field of view was 40 mm with 256128 imaging matrix and the slice thickness was 1 mm. Rats weighing over 250 g were imaged with a superconducting whole- body system ( Siemens, Vision, Erlangen, Germany ) operating at 1.5 T using a small field view surface coil. T1 and T2 -weighted spin - echo sequences were used. The imaging parameters for the T1 -weighted images were as follows: 500 /20 (repetition time, TR /echo time, TE; ms ) with four acquisitions, imaging time 8:36 minutes. Proton and T2 -weighted images were acquired with TR /TE of 2000/20 and 76 ms, imaging time 8:36 with one acquisition. Slice thickness was 2 mm with both sequences and a 60- to 100- mm field of view was used. A 256256 imaging matrix yielded in - plane resolution of 0.23– 0.39 mm. For imaging all rats received contrast enhancer i.p. (Magnevist, Schering Pharma, Berlin, Germany; 0.3 mL in 0.7 mL in physiological salt solution ).6 Analysis of blood samples

Blood samples were taken before the adv /tk gene transfer, and 7, 14, and 35 days after the treatment. Hemoglobin, white blood cells, platelets, C - reactive protein (CRP ), creatinine, aspartylaminotransferase (AST ), alanine aminotransferase (ALT ), alkaline phosphatase (AP ), and bilirubin were monitored using routine clinical chemistry assays ( Specific, Kone Instruments, Finland and Celtac Auto MEK -8118K, Nihon Kohden, Japan ) at Kuopio University Hospital laboratory. Anti-adenovirus antibodies (adv -ab) were measured using a standard assay (BioWhittaker, Walkersville, MD ).

Table 2 Tumor volumes in different treatment groups as measured using image analysis system from histological sections

Treatment groups I III V VI VIII

Adv / tk1 + GCV Adv / tk3 + GCV Adv / MCSF3 + GCV Adv / tk / MCSF3 + GCV Controls

Tumor volume 35 days after the treatment ( mean ± SE ) 0.50 ± 0.03 ( ns ) 0.21 ± 0.27 ( P = .01 ) 0.42 ± 0.08 ( P < .01 ) 0.34 ± 0.32 ( P < .05 ) 0.66 ± 0.1

Statistical significance compared with the control group ( Mann Whitney test ). ns = not significant.

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920 Histology and immunohistochemistry

Rats were sacrificed with an overdose of anesthetic and perfused with 150 mL phosphate - buffered saline (PBS ) and

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4% paraformaldehyde solution for 10 minutes. The brains were dissected and divided at the injection site into two coronal pieces. Anterior samples were rinsed with PBS overnight and embedded in OCT compound ( Miles


921

Figure 3 Clinical chemistry of adv / tk3 treated rats ( mean ± SD ) was analyzed from serum samples before the gene therapy and 7, 14, and 35 days after the treatment as described in Materials and methods. Aspartylaminotransferase, alkaline phosphatase, bilirubin, and anti - adenovirus antibodies increased after the gene therapy.

Scientific, Elkhart, IL). Posterior samples were further fixed in 4% paraformaldehyde solution for 12– 18 hours and

rinsed in 15% sucrose overnight. The posterior samples were embedded in paraffin and sections (6 - to 8- m thick ) were

Figure 2 Characterization of the adv / tk and GCV treatment effect in BT4C malignant glioma model. Hematoxylin – eosin staining in ( A ), ( D ), ( G ), ( J ), immunostaining for GFAP in ( B ), ( E ), ( H ), ( K ), and OX - 42 in ( C ), ( F ), ( I ), ( L ). A – C: Group III rat with BT4C glioma tumor after adv / tk gene therapy and GCV medication 1 month after the BT4C cell injection. A: A big tumor is present with a necrotic area inside the tumor. B: A weak GFAP reactivity is present near the injection site. C: Some macrophages are seen in the tumor area. D – F: Group III rat, which survived 6 months. D: No glioma cells are seen in the treated area. E: A strong GFAP reactivity is present in the scar area. F: No macrophages were detected in the scar area. G – I: Group VI rat glioma tumor with adv / tk and adv / MCSF gene therapy and GCV medication 1.5 months after the BT4C cell injection. G: Necrotic areas were present inside the tumor. H: GFAP reactivity was present in the tumor area near the injection site. I: Many macrophages were detected in the tumor area. J – L: Group VIII control rat without any treatment 1 month after the BT4C cell injection. J: A large glioma tumor is present. K: Some GFAP reactivity is seen around the tumor, ( L ) a few macrophages are present in the tumor area. Control immunostainings with class - and species - matched immunoglobulins and incubations where primary antibodies were omitted did not show any staining ( data not shown ). Original magnification: 40, except 4 in ( A ), ( G ), and ( J ).

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922 either stained with hematoxylin –eosin (BDH, Poole Dorset, England ) or immunostained with monoclonal antibodies ( mAbs ) against glial fibrillary acid protein (GFAP ) ( dilution 1:300, Boehringer- Mannheim, Mannheim, Germany ) or OX - 42 (dilution 1:200, Serotec, Kidlington, Oxon, UK ). Microwave oven incubation in citric acid buffer was used to improve the immunostainings. Avidin –biotin – horseradish peroxidase system (Zymed, Histostain- Plus, San Francisco, CA, USA ) was used for signal detection. Negative controls included incubations with glass - and species- matched unrelated immunoglobulins and incubations where primary antibodies were omitted. Tumor volumes were determined with computerized image analysis system ( MCID Softwear, Imaging Research, St Catharines, Ontario, Canada ) from histological sections. Tumor areas were measured as pixels at the level of the injection site and standardized against the total brain area measured from the same section.6 Statistical analysis

Statistical analysis of the tumor volumes was done with Mann -Whitney test for SPSS. Statistics of the clinical chemistry results were evaluated with Kruskal -Wallis test for SPSS. Survival data were analyzed using log rank test for Kaplan -Mayer plots for SPSS with Bonferroni correction.

inside the tumors near the adenovirus injection site (Fig 2B). Very strong astrogliosis was seen in group III rats in the scar area where the glioma tumor had been present before gene therapy (Fig 2E ). Weak microglia responses were seen in OX - 42 immunostainings after adv/tk gene therapy (Fig 2C ). However, a clearly enhanced OX - 42 reactivity was seen after adv -mediated MCSF gene therapy ( Fig 2I). Safety of gene therapy

Clinical chemistry showed elevations in aspartylaminotransferase, alkaline phosphatase, and bilirubin, but not in C reactive protein, creatinine, or alanine aminotransferase. Anti-adenovirus antibodies increased after adenovirus injections (P < .05) (Fig 3). No changes were seen in hemoglobin, white cell count, and platelets ( data not shown ). Survival

Survival of the treatment groups was analyzed by Kaplan Mayer log rank test (Fig 4). The mean survival time in group I was 51.6± 2.8 days, in group III 77.7 ± 11.0 days, in group V 42.7 ± 1.1 days, in group VI 71.3 ± 13.5 days, in group VII 49.7± 2.2 days, and in group VIII (controls ) 49.5 ± 1.7 days ( ± SE ). Statistically significant differences were present in the survival times between groups III and VIII (P < .05 ) (Fig 4 ). Twenty percent of the animals were cured ( survival >6 months) in group III ( Fig 4 ).

Results MRI imaging and tumor volumes

Treatment effect in MRI ( Fig 1) was seen at 2 weeks after adv /tk gene transfer and GCV treatment. High signal intensity areas were found inside the tumors although the tumors were still present ( Fig 1B ). In group III, 20% of the animals survived 6 months. Large ventricles and hydrocephalus were present although they did not cause any major neurological symptoms (Fig 1D and E ). Upon histological examination, no tumors were detected in the surviving animals but scar tissue was seen in the place of the tumor (Fig 2 ). Follow -up of the tumor growth with MRI showed that after adv/ tk, adv/MCSF, and adv/tk /MCSF gene transfer once a day for three consecutive days the tumors tended to grow more slowly than control tumors. A single adv /tk gene transfer did not improve the outcome of the treatment ( Table 2 ). Histological findings

Hematoxylin – eosin stainings showed large malignant glioma tumors in group VIII wild -type animals (Fig 2 ). Necrotic areas were present inside the tumors after adv /tk and combined adv /tk and adv/MCSF gene therapy. These areas were probably due to the treatment effect, but the tumors still progressed. No glioma cells were found in the brains of rats that survived 6 months after the repeated adv / tk gene therapy. Instead, scar tissue was seen in the place of the tumor (Fig 2D ). Astrogliosis with positive GFAP immunostaining was seen around large wild -type glioma tumors (Fig 2H ). Weak GFAP reactivity was also present

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Figure 4 Kaplan - Mayer survival curves of rats after implantation of BT4C cells, followed by injection of adv / tk gene once ( group I, n = 8 ), once a day during three consecutive days ( group III, n = 21 ), adv / MCSF injection once a day for three consecutive days ( group V, n = 6 ), and adv / tk together with adv / MCSF once a day for three consecutive days ( group VI, n = 7 ). Control rats with wild - type tumors in group VIII ( n = 17 ). A significant difference was found between group III versus group VIII ( P < .05, log rank test ).


923 Discussion

With current treatment strategies the prognosis of malignant glioma is still very poor.1,2 Gene therapy may offer a new treatment strategy for malignant glioma. We used the syngeneic BT4C rat malignant glioma model, which, unlike 9L, C6, or some other glioma models, does not involve any significant host immune response against the tumor.6,8 MRI was used to ensure the presence of tumors before gene therapy and to follow the tumor growth and the treatment response. We have previously evaluated the efficacy of retrovirusmediated thymidine kinase gene therapy in the BT4C rat glioma model using PA317 retrovirus producer cell injections, followed by GCV treatment.9 Unfortunately, retroviral gene therapy was not effective for the treatment of BT4C glioma. Similar results have recently been reported from human trials.7,10,15,16 In this study, adenovirus mediated gene therapy was used for the treatment of wild -type tumors using direct stereotactic injections into the tumors. The presence of the tumors was verified by MRI. Single intratumoral injections were compared with intratumoral injections given during three consecutive days. Different modes of administration were tested because very little information is available about the optimal treatment protocol for malignant glioma.7,17 It was found that repeated treatments with adv /tk, followed by GCV medication, significantly prolonged survival of rats with malignant glioma, and that 20% of the animals were cured using this treatment protocol. Rats tolerated relatively high virus doses without any major problems and it is unlikely that high viral loads per se had any major effects on the results. Because BT4C glioma grows in the brain without inducing much microglia reactivity or activation of host immune system, we tested the efficacy of the combined treatment with adv /tk and adv /MCSF. The rationale for the use of adv /MCSF treatment was to induce monocyte macrophage accumulation and activation in the tumor area, which could lead to improved detection and presentation of tumor antigens to the host immune system. Even though increases in macrophage numbers were detected in the adv /MCSF- treated tumors, survival of the animals with combined adv /tk and adv /MCSF therapy was not improved compared with the repeated administration of adv/tk. Thus, it is concluded that temporary stimulation of monocyte / macrophages in the BT4C rat malignant glioma model does not lead to any significant improvement in the treatment effect. The repeated adv /tk therapy is the most efficient treatment for the BT4C rat malignant glioma. However, repeated injections may not be given more than 7 – 10 days apart because induction of neutralizing anti -adenovirus antibodies may theoretically reduce the efficacy of the treatment. Encouraging results of the adv /tk gene therapy have been recently reported from a human controlled phase I/ II study.7 Even though experimental animal studies may never accurately predict outcome of human clinical trials, the present results suggest that maximal cytotoxic effect induced

by the adv /tk gene therapy and GCV treatment may be achieved by giving repeated injections of adv /tk into the treated tumors.

Acknowledgments

This study was supported by grants from Finnish Academy, Kuopio University Hospital ( EVO Grants 5022 and 5118 ), Sigrid Juselius Foundation, Northern Savo Cancer Foundation, Finnish Society of Oncology, and Ark Therapeutics. The authors thank Aila Erkinheimo, Mervi Nieminen, Tommi Heikura, and Sirpa Laitinen for skillful technical assistance, and Marja Poikolainen for preparing the manuscript.

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