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Isolation and Characterization of Stem Cells in Head and Neck Cancer
Proceedings of the 52nd Annual ASTRO Meeting
279
Gene Profiling of Cancer Stem Cells-like Cells Reveals IGF-1R and IGFBP3 Pathway as a Promising Therapeutic Target for Lung Cancer
Y. Sun, S. Zheng, C. Speirs, P. Kopsombut, Z. Zhao, B. Lu Vanderbilt University, Nashville, TN Purpose/Objective(s): Combination of platinum-based chemotherapy and radiation is currently the standard treatment for locally advanced non-small cell lung cancer (NSCLC) patients. However, this approach often results in therapeutic resistance and disease relapse, partly because of the presence of cancer stem cells (CSCs) within the tumor. To investigate the CSCs hypothesis of chemoradiation resistance in NSCLC, we used high-throughput microarray assay to profile CSCs-like cells versus parental cancer cells to identify the candidate genes for cancer therapy. Materials/Methods: Cisplatin-resistant H460 cells (CDDP-cells) were established by exposing normal H460 cells to 3mM cisplatin for 7 days, followed by 0.8% methycellulose supplement with basal growth factors every 2 days for 14 consecutive days. Cells growing as spheres were then collected and expanded. RNAs were extracted and affymetrix gene array were performed. Immunoblots were used to determine expression levels of proteins. Cell radiosensitivity and cytotoxicity were determined by clonogenic and MTS assays. CDDP-cells and H460 mouse xenograft models were used for in vivo studies. Results: We found that CDDP-cells had CSCs characteristics such as self-renewing and expressed higher level of stem cell markers, including CD133 and ALDH. CDDP-cells were resistant to cisplatin- and etoposide-induced apoptosis and to high radiation dose (20 Gy). In clonogenic assays, CDDP-cells were more resistant to radiation than parental H460 cells (1.21, p \ 0.01). In vivo, CDDP-cells were significantly more tumorigenic than parental H460 cells (p \ 0.001). By comparing 30,000 human genes with microarray techniques, a total of 180 genes were identified as differentially expressed genes (DEGs), of which 73 were upregulated and 107 downregulated. Comprehensive protein interaction networks analysis revealed IGFBP3 as a highly ranked hub protein which plays an important role in the mechanism of cisplatin resistance, suggesting IGF-1R pathway was a promising target of overcome drug resistant. The specific targeting of IGF-1R using siRNA resulted in significant sensitization of CDDP-cells (DER = 1.17, p \ 0.05) to radiation compared to parental cells. Conclusions: Our findings suggest that CDDP-cells have all the characteristics of CSCs, and constitute a ‘‘suitable’’ model to study lung CSCs. Profiling of CSCs-like H460 cells and H460 parental cells by microarray resulted in the identification of IGFBP3 as an important pathway for chemo- and radiotherapy resistance in NSCLC. The targeting of this pathway has the potential to overcome the resistance observed in lung CSCs-like cells. Author Disclosure: Y. Sun, None; S. Zheng, None; C. Speirs, None; P. Kopsombut, None; Z. Zhao, None; B. Lu, None.
280
Tumor Specific Cell-free DNA in the Plasma is a Marker of the Tumor Response to Radiation in an Experimental Mouse Model
C. Cheng1, M. Omura-MInamisawa1, M. Natsuhori2, Y. Kang1, T. Hara3, I. Koike1, M. Hata1, T. Inoue1 1 Department of Radiology, Yokohama City University School of Medicine, Yokohama, Japan, 2Department of Radiology, Japan Animal Referral Medical Center, Kawasaki, Japan, 3Clinical Oncology Center, Fukushima Medical University, Fukushima, Japan
Purpose/Objective(s): The cell-free plasma DNA levels are increased in cancer patients and decrease in response to effective treatments. These nucleic acids thus represent a potential new tumor marker. In our previous study, we measured the total cell-free plasma DNA levels in cancer patients undergoing radiation therapy and found dynamic changes in these levels with treatment progression and an overall decrease at the end of the therapy. In our current study, we investigated whether radiation for transplanted tumors in mice altered the levels of cell-free tumor specific DNA in the plasma and whether this correlated with the tumor response to radiation. Materials/Methods: Solitary tumors were produced in the leg of nude mice by inoculation of the human tumor cells SQ5 (squamous cell carcinoma) or A549 (adenocarcinoma). Tumors that had reached a size of 200-250mm3 were locally irradiated with a single dose of 15 Gy or 22.5 Gy using a 4MV linear accelerator. Serial blood samples were collected before and at 18, 48, and 96 hours after radiation, and then twice per week up to 21 days after radiation. The cell-free tumor specific DNA levels in the plasma were then quantified by PCR amplification of the human specific beta-actin gene. Results: Serial analyses of the mouse blood samples revealed increased levels of cell-free tumor specific DNA in plasma upon tumor growth which were suppressed by radiation. These suppressive effects of radiation were greater for the 22.5 Gy than the 15 Gy. We further found a significant correlation between the cell-free tumor specific DNA levels and the tumor volume at 21 days after radiation. A good inverse correlation was also found between these DNA levels at 21 days after radiation and the period required for the tumors to grow to a 500 mm3 volume after radiation. Some mice with SQ5 tumor showed a transient increase in cellfree tumor specific DNA levels after 18-96 hours of radiation (up to 16-fold), followed by a rapid decrease. This transient increase was observed more frequently in mice that received 22.5 Gy of radiation (6 of 8 mice) compared with animals who received 15 Gy (2 of 8 mice). This suggests that the rapid cell death caused by radiation induces the release of tumor cell DNA into the circulation. Conclusions: The cell-free tumor specific DNA levels in the plasma of tumor bearing mice are significantly associated with the tumor volume after radiation. The dynamics of tumor specific DNA in the plasma may thus reflect tumor burden and serial analyses of this DNA could thus be a potent tool for monitoring the tumor response to radiation. Additional prospective studies will be required to further elucidate the potential clinical utility and biological implications of the cell-free tumor specific DNA levels in the plasma of cancer patients during radiation therapy. Author Disclosure: C. Cheng, None; M. Omura-Minamisawa, None; M. Natsuhori, None; Y. Kang, None; T. Hara, None; I. Koike, None; M. Hata, None; T. Inoue, None.
281
Isolation and Characterization of Stem Cells in Head and Neck Cancer
G. D. Wilson, S. Galoforo, B. Marples, T. Geddes, B. Thibodeau, S. Park, J. Akervall
S131
I. J. Radiation Oncology d Biology d Physics
S132
Volume 78, Number 3, Supplement, 2010
William Beaumont Hospital, Royal Oak, MI Purpose/Objective(s): There is growing evidence that stem cells exist in tumors and contribute to treatment resistance. In this study, we have utilized low passage head and neck tumor (H&N) cell lines, isolated cancer stem cells (CSC) using different methods and subjected them to gene expression analysis. The goal is to find new biomarkers for stem cells for prognostic use and to discover new targets for treatment. Materials/Methods: A panel of five (UT14 UT16A, UT24A, UT30 and UT33) low passage H&N cancer lines was studied. CSC (and non CSC) were isolated from these by high-speed cell sorting using two different flow cytometry techniques 1) Hoechst 33342 ‘‘side’’ population and 2) the Aldefluor assay. RNA was extracted from the sorted populations for gene expression analysis and further experimental studies of radiosensitivity. Differentially expressed genes were determined by utilizing Affymetrix Human Exon 1.0 ST microarrays and subsequent analysis in Partek’s Genomics Suite. Results: The two methods of isolation identified different proportions of ‘‘stem’’ cells. The ‘‘side’’ population was less prevalent and ranged from 0.3 to 3.7% in the 5 cell lines whereas Aldefluor positive cells ranged from 6 to 31.4%. Only UT33 had a similar proportion of cells (3.7 vs. 6%) by the two methods. In the analysis so far using ANOVA with a significance of p#0.05 and a foldchange cutoff of 1.5, 83 differentially expressed genes were identified by comparing ‘‘side’’ population to non-‘‘side’’ population samples. of the 45 genes that were up-regulated, 22 were associated with histones. Conclusions: The high incidence of histone gene overexpression in ‘‘side’’ population cells is consistent with the known involvement of chromatin packaging in the maintenance of the stem cell phenotype and the involvement of histones in DNA repair may relate to the increased radioresistance of stem cells. Further studies on the epigenetic regulation of histones in stem cells may provide future avenues for therapeutic targets to be used in conjunction with chemoradiation. Author Disclosure: G.D. Wilson, None; S. Galoforo, None; B. Marples, None; T. Geddes, None; B. Thibodeau, None; S. Park, None; J. Akervall, None.
282
Fast and Accurate Verification of Proton Range, Position, and Intensity in IMPT with a 3D Detector System
L. Archambault1, S. Beddar2, N. Sahoo2, A. Lee2, M. T. Gillin2, R. Mohan2 1 Centre Hospitalier Universitaire de Quebec, Quebec, QC, Canada, 2University of Texas, M. D. Anderson Cancer Center, Houston, TX
Purpose/Objective(s): Intensity-modulated proton therapy (IMPT) using scanned proton spots relies on the delivery of a large numbers of spots to shape the dose distribution in a highly conformal manner. We have developed a 3D system based on liquid scintillator (LS) to evaluate the spot position, intensity (dose) and range (energy) of proton spots in real time. Material/Methods: The LS is a tissue equivalent material emitting scintillation light when irradiated by ionizing radiation. The LS detector system is comprised of a 202020 cc volume of LS in a light tight enclosure to prevent against ambient light contamination. The LS detector is rigidly coupled to a CCD camera. This camera continuously acquires images of the light emitted by the LS therefore allowing a quantitative characterization of the proton beam. Image acquisitions are triggered by a signal from the proton accelerator. Irradiations were conducted on a proton spot scanning beam line for which one monitor unit (MU) is defined as 1 c Gy in the middle of a 10 cm spread-out Bragg peak for a field of 10 cm by 10 cm with a range of 30.6 cm in water. Scintillation emission is expected to be dependent on the proton energy due to the quenching effect. Therefore, irradiations were done to evaluate the magnitude of this effect and test correction strategies. Results: Range: Irradiations with protons of known ranges were used for calibration. We found that one centimeter of LS was equivalent to 0.87 cm of water. Measurement of proton range with the LS detector system was found to be within 0.1 mm (0.3 mm maximum error) for protons with nominal range between 6.6 cm and 10.5 cm. Moreover, proton range measurements did not require a quenching correction. Position: Measurement of lateral position of proton spots was found to be accurate to within 0.4 mm (0.6 mm maximum error). Imaging artifacts such as vignetting did not impact our ability to measure spot position. However, it was necessary to correct for image distortion caused by the objective lens. Intensity: The system was sensitive enough for accurate measurements of spot intensity delivered at the lowest (0.005 MU) and highest (0.04 MU) dose per proton spot. The precision of the detector was 3.3% and 0.5% for 0.005 MU and 0.04 MU, respectively. Quenching had a strong effect on relative spot intensity measurement, however, a Monte Carlo based correction could be used to completely account for this effect. Conclusions: Our LS detector system is well suited for fast and accurate verification of proton treatments using scanned proton beams such as IMPT. Proton spots range and position could be measured with sub-millimeter accuracy. The system was sufficiently sensitive to measure individual proton spots in real-time. This new device has potential to both improve and simplify the quality assurance process of IMPT treatments. Author Disclosure: L. Archambault, None; S. Beddar, None; N. Sahoo, None; A. Lee, None; M.T. Gillin, None; R. Mohan, None.
283
OPTIS2: A Second Generation Horizontal Beam Line for Ocular Proton Therapy using a 250 MeV Cyclotron - First Patients Treated 25 Years after Start of the OPTIS Program at Paul Scherrer Institute
J. Verwey1, F. Assenmacher1, J. Heufelder2, M. van Goethem3, M. Grossmann1, G. Goitein1, T. Lomax1, A. Tourovsky1, L. Zografos4, E. Hug1 1 Paul Scherrer Institut, Villigen, Switzerland, 2Charite - U¨niversita¨tsmedizin Berlin, Berlin, Germany, 3KVI, Groningen, Netherlands, 4Hopital Ophtalmique Jules Gonin, Lausanne, Switzerland Purpose/Objective(s): In 1984 proton therapy was introduced to Europe with start of the OPTIS program at Paul Scherrer Institute. A prototype ‘‘low energy’’ 72 MeV cyclotron provided protons of sufficient energy to treat ocular lesions. To date more than 5000 uveal melanoma patients have been treated with local tumor control of 97% at 5 years. To continue the program using the horizontal beam line connected to ‘‘high energy’’ 250 MeV cyclotron COMET a new facility (OPTIS2) was designed and constructed. As the current generation of high energy cyclotrons provide a beam current at 70 MeV that is dramatically lower than
279
Gene Profiling of Cancer Stem Cells-like Cells Reveals IGF-1R and IGFBP3 Pathway as a Promising Therapeutic Target for Lung Cancer
Y. Sun, S. Zheng, C. Speirs, P. Kopsombut, Z. Zhao, B. Lu Vanderbilt University, Nashville, TN Purpose/Objective(s): Combination of platinum-based chemotherapy and radiation is currently the standard treatment for locally advanced non-small cell lung cancer (NSCLC) patients. However, this approach often results in therapeutic resistance and disease relapse, partly because of the presence of cancer stem cells (CSCs) within the tumor. To investigate the CSCs hypothesis of chemoradiation resistance in NSCLC, we used high-throughput microarray assay to profile CSCs-like cells versus parental cancer cells to identify the candidate genes for cancer therapy. Materials/Methods: Cisplatin-resistant H460 cells (CDDP-cells) were established by exposing normal H460 cells to 3mM cisplatin for 7 days, followed by 0.8% methycellulose supplement with basal growth factors every 2 days for 14 consecutive days. Cells growing as spheres were then collected and expanded. RNAs were extracted and affymetrix gene array were performed. Immunoblots were used to determine expression levels of proteins. Cell radiosensitivity and cytotoxicity were determined by clonogenic and MTS assays. CDDP-cells and H460 mouse xenograft models were used for in vivo studies. Results: We found that CDDP-cells had CSCs characteristics such as self-renewing and expressed higher level of stem cell markers, including CD133 and ALDH. CDDP-cells were resistant to cisplatin- and etoposide-induced apoptosis and to high radiation dose (20 Gy). In clonogenic assays, CDDP-cells were more resistant to radiation than parental H460 cells (1.21, p \ 0.01). In vivo, CDDP-cells were significantly more tumorigenic than parental H460 cells (p \ 0.001). By comparing 30,000 human genes with microarray techniques, a total of 180 genes were identified as differentially expressed genes (DEGs), of which 73 were upregulated and 107 downregulated. Comprehensive protein interaction networks analysis revealed IGFBP3 as a highly ranked hub protein which plays an important role in the mechanism of cisplatin resistance, suggesting IGF-1R pathway was a promising target of overcome drug resistant. The specific targeting of IGF-1R using siRNA resulted in significant sensitization of CDDP-cells (DER = 1.17, p \ 0.05) to radiation compared to parental cells. Conclusions: Our findings suggest that CDDP-cells have all the characteristics of CSCs, and constitute a ‘‘suitable’’ model to study lung CSCs. Profiling of CSCs-like H460 cells and H460 parental cells by microarray resulted in the identification of IGFBP3 as an important pathway for chemo- and radiotherapy resistance in NSCLC. The targeting of this pathway has the potential to overcome the resistance observed in lung CSCs-like cells. Author Disclosure: Y. Sun, None; S. Zheng, None; C. Speirs, None; P. Kopsombut, None; Z. Zhao, None; B. Lu, None.
280
Tumor Specific Cell-free DNA in the Plasma is a Marker of the Tumor Response to Radiation in an Experimental Mouse Model
C. Cheng1, M. Omura-MInamisawa1, M. Natsuhori2, Y. Kang1, T. Hara3, I. Koike1, M. Hata1, T. Inoue1 1 Department of Radiology, Yokohama City University School of Medicine, Yokohama, Japan, 2Department of Radiology, Japan Animal Referral Medical Center, Kawasaki, Japan, 3Clinical Oncology Center, Fukushima Medical University, Fukushima, Japan
Purpose/Objective(s): The cell-free plasma DNA levels are increased in cancer patients and decrease in response to effective treatments. These nucleic acids thus represent a potential new tumor marker. In our previous study, we measured the total cell-free plasma DNA levels in cancer patients undergoing radiation therapy and found dynamic changes in these levels with treatment progression and an overall decrease at the end of the therapy. In our current study, we investigated whether radiation for transplanted tumors in mice altered the levels of cell-free tumor specific DNA in the plasma and whether this correlated with the tumor response to radiation. Materials/Methods: Solitary tumors were produced in the leg of nude mice by inoculation of the human tumor cells SQ5 (squamous cell carcinoma) or A549 (adenocarcinoma). Tumors that had reached a size of 200-250mm3 were locally irradiated with a single dose of 15 Gy or 22.5 Gy using a 4MV linear accelerator. Serial blood samples were collected before and at 18, 48, and 96 hours after radiation, and then twice per week up to 21 days after radiation. The cell-free tumor specific DNA levels in the plasma were then quantified by PCR amplification of the human specific beta-actin gene. Results: Serial analyses of the mouse blood samples revealed increased levels of cell-free tumor specific DNA in plasma upon tumor growth which were suppressed by radiation. These suppressive effects of radiation were greater for the 22.5 Gy than the 15 Gy. We further found a significant correlation between the cell-free tumor specific DNA levels and the tumor volume at 21 days after radiation. A good inverse correlation was also found between these DNA levels at 21 days after radiation and the period required for the tumors to grow to a 500 mm3 volume after radiation. Some mice with SQ5 tumor showed a transient increase in cellfree tumor specific DNA levels after 18-96 hours of radiation (up to 16-fold), followed by a rapid decrease. This transient increase was observed more frequently in mice that received 22.5 Gy of radiation (6 of 8 mice) compared with animals who received 15 Gy (2 of 8 mice). This suggests that the rapid cell death caused by radiation induces the release of tumor cell DNA into the circulation. Conclusions: The cell-free tumor specific DNA levels in the plasma of tumor bearing mice are significantly associated with the tumor volume after radiation. The dynamics of tumor specific DNA in the plasma may thus reflect tumor burden and serial analyses of this DNA could thus be a potent tool for monitoring the tumor response to radiation. Additional prospective studies will be required to further elucidate the potential clinical utility and biological implications of the cell-free tumor specific DNA levels in the plasma of cancer patients during radiation therapy. Author Disclosure: C. Cheng, None; M. Omura-Minamisawa, None; M. Natsuhori, None; Y. Kang, None; T. Hara, None; I. Koike, None; M. Hata, None; T. Inoue, None.
281
Isolation and Characterization of Stem Cells in Head and Neck Cancer
G. D. Wilson, S. Galoforo, B. Marples, T. Geddes, B. Thibodeau, S. Park, J. Akervall
S131
I. J. Radiation Oncology d Biology d Physics
S132
Volume 78, Number 3, Supplement, 2010
William Beaumont Hospital, Royal Oak, MI Purpose/Objective(s): There is growing evidence that stem cells exist in tumors and contribute to treatment resistance. In this study, we have utilized low passage head and neck tumor (H&N) cell lines, isolated cancer stem cells (CSC) using different methods and subjected them to gene expression analysis. The goal is to find new biomarkers for stem cells for prognostic use and to discover new targets for treatment. Materials/Methods: A panel of five (UT14 UT16A, UT24A, UT30 and UT33) low passage H&N cancer lines was studied. CSC (and non CSC) were isolated from these by high-speed cell sorting using two different flow cytometry techniques 1) Hoechst 33342 ‘‘side’’ population and 2) the Aldefluor assay. RNA was extracted from the sorted populations for gene expression analysis and further experimental studies of radiosensitivity. Differentially expressed genes were determined by utilizing Affymetrix Human Exon 1.0 ST microarrays and subsequent analysis in Partek’s Genomics Suite. Results: The two methods of isolation identified different proportions of ‘‘stem’’ cells. The ‘‘side’’ population was less prevalent and ranged from 0.3 to 3.7% in the 5 cell lines whereas Aldefluor positive cells ranged from 6 to 31.4%. Only UT33 had a similar proportion of cells (3.7 vs. 6%) by the two methods. In the analysis so far using ANOVA with a significance of p#0.05 and a foldchange cutoff of 1.5, 83 differentially expressed genes were identified by comparing ‘‘side’’ population to non-‘‘side’’ population samples. of the 45 genes that were up-regulated, 22 were associated with histones. Conclusions: The high incidence of histone gene overexpression in ‘‘side’’ population cells is consistent with the known involvement of chromatin packaging in the maintenance of the stem cell phenotype and the involvement of histones in DNA repair may relate to the increased radioresistance of stem cells. Further studies on the epigenetic regulation of histones in stem cells may provide future avenues for therapeutic targets to be used in conjunction with chemoradiation. Author Disclosure: G.D. Wilson, None; S. Galoforo, None; B. Marples, None; T. Geddes, None; B. Thibodeau, None; S. Park, None; J. Akervall, None.
282
Fast and Accurate Verification of Proton Range, Position, and Intensity in IMPT with a 3D Detector System
L. Archambault1, S. Beddar2, N. Sahoo2, A. Lee2, M. T. Gillin2, R. Mohan2 1 Centre Hospitalier Universitaire de Quebec, Quebec, QC, Canada, 2University of Texas, M. D. Anderson Cancer Center, Houston, TX
Purpose/Objective(s): Intensity-modulated proton therapy (IMPT) using scanned proton spots relies on the delivery of a large numbers of spots to shape the dose distribution in a highly conformal manner. We have developed a 3D system based on liquid scintillator (LS) to evaluate the spot position, intensity (dose) and range (energy) of proton spots in real time. Material/Methods: The LS is a tissue equivalent material emitting scintillation light when irradiated by ionizing radiation. The LS detector system is comprised of a 202020 cc volume of LS in a light tight enclosure to prevent against ambient light contamination. The LS detector is rigidly coupled to a CCD camera. This camera continuously acquires images of the light emitted by the LS therefore allowing a quantitative characterization of the proton beam. Image acquisitions are triggered by a signal from the proton accelerator. Irradiations were conducted on a proton spot scanning beam line for which one monitor unit (MU) is defined as 1 c Gy in the middle of a 10 cm spread-out Bragg peak for a field of 10 cm by 10 cm with a range of 30.6 cm in water. Scintillation emission is expected to be dependent on the proton energy due to the quenching effect. Therefore, irradiations were done to evaluate the magnitude of this effect and test correction strategies. Results: Range: Irradiations with protons of known ranges were used for calibration. We found that one centimeter of LS was equivalent to 0.87 cm of water. Measurement of proton range with the LS detector system was found to be within 0.1 mm (0.3 mm maximum error) for protons with nominal range between 6.6 cm and 10.5 cm. Moreover, proton range measurements did not require a quenching correction. Position: Measurement of lateral position of proton spots was found to be accurate to within 0.4 mm (0.6 mm maximum error). Imaging artifacts such as vignetting did not impact our ability to measure spot position. However, it was necessary to correct for image distortion caused by the objective lens. Intensity: The system was sensitive enough for accurate measurements of spot intensity delivered at the lowest (0.005 MU) and highest (0.04 MU) dose per proton spot. The precision of the detector was 3.3% and 0.5% for 0.005 MU and 0.04 MU, respectively. Quenching had a strong effect on relative spot intensity measurement, however, a Monte Carlo based correction could be used to completely account for this effect. Conclusions: Our LS detector system is well suited for fast and accurate verification of proton treatments using scanned proton beams such as IMPT. Proton spots range and position could be measured with sub-millimeter accuracy. The system was sufficiently sensitive to measure individual proton spots in real-time. This new device has potential to both improve and simplify the quality assurance process of IMPT treatments. Author Disclosure: L. Archambault, None; S. Beddar, None; N. Sahoo, None; A. Lee, None; M.T. Gillin, None; R. Mohan, None.
283
OPTIS2: A Second Generation Horizontal Beam Line for Ocular Proton Therapy using a 250 MeV Cyclotron - First Patients Treated 25 Years after Start of the OPTIS Program at Paul Scherrer Institute
J. Verwey1, F. Assenmacher1, J. Heufelder2, M. van Goethem3, M. Grossmann1, G. Goitein1, T. Lomax1, A. Tourovsky1, L. Zografos4, E. Hug1 1 Paul Scherrer Institut, Villigen, Switzerland, 2Charite - U¨niversita¨tsmedizin Berlin, Berlin, Germany, 3KVI, Groningen, Netherlands, 4Hopital Ophtalmique Jules Gonin, Lausanne, Switzerland Purpose/Objective(s): In 1984 proton therapy was introduced to Europe with start of the OPTIS program at Paul Scherrer Institute. A prototype ‘‘low energy’’ 72 MeV cyclotron provided protons of sufficient energy to treat ocular lesions. To date more than 5000 uveal melanoma patients have been treated with local tumor control of 97% at 5 years. To continue the program using the horizontal beam line connected to ‘‘high energy’’ 250 MeV cyclotron COMET a new facility (OPTIS2) was designed and constructed. As the current generation of high energy cyclotrons provide a beam current at 70 MeV that is dramatically lower than