- Email: [email protected]
33 In vivo radiosensitization of human tumors expressing mutant ras by farnesyltransferase inhibitors
Proceedings
of the 4 1st Annual
ASTRO
Meeting
163
biology to cancer therapy have provided new opportunities to target specific molecular pathways which modulate tumor growth characteristics during treatment. One such approach in patients with XC of the H&N involves modulation of radiation response by EGF receptor blockade using the anti-EGFR monoclonal antibody C225 which inhibits activation of the EGF receptor tyrosine kinase. Methods and Materials: In vitro and in vivo pre-clinical studies regarding the capacity of EGFR blockade to modulate epithelial tumor cell growth characteristics are described. The specific capacity of C225 to influence radiation response in human SCCs derived from H&N epithelium is reviewed. Experimentation in cell culture and in xenograft model systems has been performed across a series of human SCCs to demonstrate specific molecular and growth modulating effects. The design and rationale is provided for recently completed Phase I/II clinical trials, and for a new international Phase III clinical trial in H&N cancer patients which examines the combination of EGFR blockade plus radiation therapy. Results: Pre-clinical studies identify C225 as a potent antiproliferative agent in human SCCs capable of inhibiting tumor cell growth kinetics. EGFR blockade with C225 induces Gl cell cycle arrest with an associated decrease in the S-phase fraction. The mechanism of cell cycle arrest and growth inhibition induced by C225 involves accumulation of the Gl cyclin dependent kinase (CDK) inhibitor p27kipl and inhibition of CDK-2 activity with subsequent accumulation of hypophosphorylated retinoblastoma protein. Exposure of SCCs to C22,5 enhances the in vitro radiosensitivity of cells using both single dose and fractionated radiation schemes. Amplification of radiation-induced apoptosis (approximately three-fold) is also observed in the presence of C225. The capacity of C225 to augment the in vivo radiation response of SCC xenografts in athymic mice is demonstrated with experiments showing complete regression of fully-established tumor xenografts in mice receiving the combination of C225 and radiation therapy. Phase I/II clinical trial work examining C225 in combination with high dose radiation for advanced H&N cancer patients has clarified optimal drug dosing, confirmed a modest toxicity profile and produced a high complete tumor response rate (greater than 90%). The international Phase III randomized multicenter trial now underway in advanced H&N cancer patients is previewed. Conclusions: The concept of molecular therapy has been developed over the last radiation therapy in EGFR-rich epithelial in selected breast cancer and lymphoma Rituxan), EGFR blockade plus radiation now commencing Phase III clinical trial
32 Gupta
C-RAF-l EMBRYO AK’,
University
Bernhard
PROTEIN KINASE FIBROBLASTS EJ’, Bakanauskas
of Pennsylvania,
Philadelphia,
targeting of the EGF receptor via monoclonal antibody blockade as an adjuvant cancer decade. The rationale and preliminary data supporting the use of EGFR blockade with tumors such as SCC of the H&N are very strong. Similar to recent therapy successes patients with molecules that target specific growth receptor blockade (e.g., Herceptin, in SCC of the H&N represents a promising new molecular cancer therapy approach evaluation.
IS NOT VJ’,
ESSENTIAL
Zimmer
PA, USA’;
A’,
FOR
Muschel
National
RJ’,
Institute
RAS
TRANSFORMATION
McKenna
of Health,
IN MOUSE
WG’ Bethesda,
MD,
lJSA2
Purpose: Mutations of ras are found in nearly one-third of human cancers and result in the acquisition of the transformed cell phenotype. Ras is known to have multiple effector proteins and the pathway by which transformation occurs is not entirely clear. An important contribution of the raf-MEKK-MAP kinase pathway in ras transformation of rodent hbroblasts has been determined. In this study, we further examined the role of ras-raf interaction in transformation. Materials and Methods: Fibroblasts were derived from homoaygous knock out c-raf-1 mouse embryos (20KO) and wild-type c-raf-1 mouse embryos (16WT). The raf kinase assay was performed in these cell lines to confirm the raf status before irradiation and 5 minutes after 6 Gy irradiation using the c-raf-1 immunoprecipitation kinase cascade assay kit (Upstate Biotechnology). These cells were transfected by the lipofectamine method with H-ras plus the cooperating oncogene V-myc using the plasmids pEJ and pMC29. Stable clones expressing myc and ras in both the 20K0 (20MR) and 16WT (16MR) were selected. Cell growth curves for the parental and oncogene transfected cells were carried out. 16MR and 20MR cells were also assessed for tumorigenicity. Results: The raf kinase assay was performed in 20K0 and 16WT cells without irradiation and 5 minutes after irradiation, Neither the 20K0 or 16WT cells had basal raf kinase activity. Raf kinase activity was induced with irradiation in 16WT cells but not in 20K0 cells confirming the raf knock out status of 20K0 cells. Oncogene transfection resulted in morphologic changes consistent with transformation in both cell lines. Cell growth curves showed that the doubling times of 16WT, 16MR, 20K0, and 20MR cells were 20.6, 14.5, 40.4, and 20.8 hours respectively. Both 16MR and 20MR cells were able to form tumors in nude mice, however, tumor latency was prolonged in 20MR (mean 51 days) relative to 16MR (mean 13 days) cells. The doubling time of 16MR and 20MR tumors was 1.9 and 4.1 days respectively, Conclusions: differentiation, morphologically
33
Although the ras-raf pathway has been shown to play a role in the regulation of cellular proliferation and raf is not required for ras transformation in this system. Raf knock out cells transfected with ras were transformed and tumorigenic.
IN VIVO RADIOSENSITIZATION FARNESYLTRANSFERASE
OF HUMAN INHIBITORS
TUMORS
EXPRESSING
MUTANT
RAS
BY
Cohen Jonathan-Moyal EL’, Muschel RJ’, McKenna WGr: Hamilton AD2, Sebti SM3, Gibbs J4, Oliff A4, Cerniglia G’, Mick RI, Bernhard EJ’ University qf Pennsylvania, Philadelphia, PA, USA’; Yale University, New Haven, CT, USA’; University of South Florida, Tampa, FL, lJSA3; Merck Research Laboratories, West Point, PA, USA4 Purpose: Ras activity depends upon membrane binding mediated by post-translational farnesylation of ras which can be blocked by farnesyltransferase inhibitors (FTI). These inhibitors have been shown to radiosensitize H-ras transformed rodent cells and human tumor cells expressing activated H- or K-ras in tissue culture. Activated ras appears to be the target of
164
I. J. Radiation
Oncology
0 Biology
l
Physics
Volume
45. Number
3 Supplement
1999
FTI-inhibition that results in radiosensitization. To determine whether treatment with FTIs may be clinically useful, we have studied the effect of farnesyltransferase inhibitor treatment on the radiosensitivity of two human tumor xenografts in nude mice: T24 bladder carcinoma cells expressing a mutated H-ras and HT-29 colon carcinoma cells expressing wild-type ras. Materials and Methods: Ras prenylation was inhibited by intraperitoneal injection or osmotic pump administration of two different FTI (FTI-276 or L744;832). The effect of FTI on radiation survival was determined by two methods, clonogenic survival and comparison of the plating efficiency between tumors treated with drug or with carrier alone, and by regrowth delay experiments. Results: FTI treatment resulted in significant radiosensitization in T24 tumors (p
34 Beavis
A COMBINATION DELIVERY BASED ON SPECIFIC PATIENT AW’,‘:
Princess Royal CA, USA3
Whitton
VJ’,‘,
Hospital,
Hull,
Xing
of radiosensitizing human tumors in viva. Our studies also show that damage to cells expressing wild-type ras. These results suggest that of human tumors expressing mutated ras.
MODE FOR CASES
INTENSITY
MODULATED
RADIATION
THERAPY
L’
United
Kingdom’;
University
of Hull,
Hull,
United
Kingdom
‘. , Stanford
University,
Stanford,
Purpose/Objective: Currently there are two methods available for the delivery of Intensity Modulated Radiation Therapy (IMRT) beams by Multileaf Collimators (MLC): 1) Step and Shoot method and 2) Dynamic method. The aim of this study was to critically examine the role of these methods and identify clinically relevant scenarios within which each has its limitations, A method has been developed for delivery which utilises a combination of these two approaches while attempting to maintain a higher level of accuracy and efficiency. Materials and Methods: In IMRT highly optimised beams are computed by the Inverse Planning system based on the required dose coverage of the planning target volume and the tolerance of the sensitive structures. This criterion is designed by the radiation oncologist. The delivery of these beams requires the superposition of a large number of shaped sub-beams and is always an estimation of the ideal beam. The simplest method of achieving this superposition is known as the ‘Step and Shoot’ method where each sub-field is defined and delivered in sequence. The main disadvantage of this method is a sacrifice of accuracy in the delivery of desired dose distributions that have steep gradients. This may be significant in close proximity to critical organs, such as spinal cord, which the treatment is designed to avoid. The ‘Dynamic’ method delivers the required distribution by sweeping the leaf pair across the beam. In principle this is a more efficient method of delivery. However, since the leaves are continuously moving whilst the beam is on, accuracy in delivery will be compromised in that ‘flat’ profiles are rendered undeliverable. The IMRT plans for ten patients have been studied. It is seen from IMRT plans generated for clinical cases that this situation may often arise. We implemented an algorithm combing these two methods utilising the advantages of each. The scheme measures the slope of each segment of the intensity profile and chooses the suitable delivery component. Finally, the corresponding leaf sequence is generated. Results: We have shown how the required intensity profile and resulting leaf sequence may be divided, by our algorithm. into a combination of ‘Step and Shoot’ and ‘Dynamic’ components. We have calculated the magnitude of the discrepancies between the idealised intensity profile and that delivered by the two existing methods and the combination method for the ten patients. It is seen that the combination method can always improve the delivery in comparison to the Step and Shoot method. It was found that the improvement can be of the order of 6%. It was noticed that several intensity distributions could not be executed using dynamic technique alone, whereas the combination method provided a solution. Conclusions: In general the dynamic method is seen to be more accurate however, clinical situations exist where the dynamic leaf sequence fails to produce the required distribution because of the limitation of maximum leaf speed; Unless a sliding window is used. Our combination method is a viable approach for these cases. It is quite general, selecting the optimal appropriate delivery method. It reduces discrepancy between the idealised intensity profile and that actually delivered, which is significant where sensitive organs lie in close proximity to the tumour volume.
35
INVESTIGATION OF THE PATIENT HETEROGENEITIES
Pawlicki
T. Guerrero
Stanford
University
T, Jiang School
DOSE
PERTURBATIONS
SB, Deng J, Li J, Boyer
of Medicine,
Stanford,
AL;
IN IMRT
TREATMENT
PLANS
CAUSED
BY
Ma CM
CA, USA
Purpose: The use of IMRT is becoming a prevalent treatment option for cases where 3DCRT can not adequately cover the target volume or spare critical structures. However, the dose calculation algorithms in commercial inverse planning systems contain approximations that give uncertainty to the final calculated dose distribution. The purpose of this work was to investigate the perturbation of dose in heterogeneous regions of patient treatment plans that were calculated by a commercial inverse planning system. The resultant dose perturbations were investigated using Monte Carlo simulation. We considered several clinical cases where IMRT was chosen primarily as a method to spare critical structures.
of the 4 1st Annual
ASTRO
Meeting
163
biology to cancer therapy have provided new opportunities to target specific molecular pathways which modulate tumor growth characteristics during treatment. One such approach in patients with XC of the H&N involves modulation of radiation response by EGF receptor blockade using the anti-EGFR monoclonal antibody C225 which inhibits activation of the EGF receptor tyrosine kinase. Methods and Materials: In vitro and in vivo pre-clinical studies regarding the capacity of EGFR blockade to modulate epithelial tumor cell growth characteristics are described. The specific capacity of C225 to influence radiation response in human SCCs derived from H&N epithelium is reviewed. Experimentation in cell culture and in xenograft model systems has been performed across a series of human SCCs to demonstrate specific molecular and growth modulating effects. The design and rationale is provided for recently completed Phase I/II clinical trials, and for a new international Phase III clinical trial in H&N cancer patients which examines the combination of EGFR blockade plus radiation therapy. Results: Pre-clinical studies identify C225 as a potent antiproliferative agent in human SCCs capable of inhibiting tumor cell growth kinetics. EGFR blockade with C225 induces Gl cell cycle arrest with an associated decrease in the S-phase fraction. The mechanism of cell cycle arrest and growth inhibition induced by C225 involves accumulation of the Gl cyclin dependent kinase (CDK) inhibitor p27kipl and inhibition of CDK-2 activity with subsequent accumulation of hypophosphorylated retinoblastoma protein. Exposure of SCCs to C22,5 enhances the in vitro radiosensitivity of cells using both single dose and fractionated radiation schemes. Amplification of radiation-induced apoptosis (approximately three-fold) is also observed in the presence of C225. The capacity of C225 to augment the in vivo radiation response of SCC xenografts in athymic mice is demonstrated with experiments showing complete regression of fully-established tumor xenografts in mice receiving the combination of C225 and radiation therapy. Phase I/II clinical trial work examining C225 in combination with high dose radiation for advanced H&N cancer patients has clarified optimal drug dosing, confirmed a modest toxicity profile and produced a high complete tumor response rate (greater than 90%). The international Phase III randomized multicenter trial now underway in advanced H&N cancer patients is previewed. Conclusions: The concept of molecular therapy has been developed over the last radiation therapy in EGFR-rich epithelial in selected breast cancer and lymphoma Rituxan), EGFR blockade plus radiation now commencing Phase III clinical trial
32 Gupta
C-RAF-l EMBRYO AK’,
University
Bernhard
PROTEIN KINASE FIBROBLASTS EJ’, Bakanauskas
of Pennsylvania,
Philadelphia,
targeting of the EGF receptor via monoclonal antibody blockade as an adjuvant cancer decade. The rationale and preliminary data supporting the use of EGFR blockade with tumors such as SCC of the H&N are very strong. Similar to recent therapy successes patients with molecules that target specific growth receptor blockade (e.g., Herceptin, in SCC of the H&N represents a promising new molecular cancer therapy approach evaluation.
IS NOT VJ’,
ESSENTIAL
Zimmer
PA, USA’;
A’,
FOR
Muschel
National
RJ’,
Institute
RAS
TRANSFORMATION
McKenna
of Health,
IN MOUSE
WG’ Bethesda,
MD,
lJSA2
Purpose: Mutations of ras are found in nearly one-third of human cancers and result in the acquisition of the transformed cell phenotype. Ras is known to have multiple effector proteins and the pathway by which transformation occurs is not entirely clear. An important contribution of the raf-MEKK-MAP kinase pathway in ras transformation of rodent hbroblasts has been determined. In this study, we further examined the role of ras-raf interaction in transformation. Materials and Methods: Fibroblasts were derived from homoaygous knock out c-raf-1 mouse embryos (20KO) and wild-type c-raf-1 mouse embryos (16WT). The raf kinase assay was performed in these cell lines to confirm the raf status before irradiation and 5 minutes after 6 Gy irradiation using the c-raf-1 immunoprecipitation kinase cascade assay kit (Upstate Biotechnology). These cells were transfected by the lipofectamine method with H-ras plus the cooperating oncogene V-myc using the plasmids pEJ and pMC29. Stable clones expressing myc and ras in both the 20K0 (20MR) and 16WT (16MR) were selected. Cell growth curves for the parental and oncogene transfected cells were carried out. 16MR and 20MR cells were also assessed for tumorigenicity. Results: The raf kinase assay was performed in 20K0 and 16WT cells without irradiation and 5 minutes after irradiation, Neither the 20K0 or 16WT cells had basal raf kinase activity. Raf kinase activity was induced with irradiation in 16WT cells but not in 20K0 cells confirming the raf knock out status of 20K0 cells. Oncogene transfection resulted in morphologic changes consistent with transformation in both cell lines. Cell growth curves showed that the doubling times of 16WT, 16MR, 20K0, and 20MR cells were 20.6, 14.5, 40.4, and 20.8 hours respectively. Both 16MR and 20MR cells were able to form tumors in nude mice, however, tumor latency was prolonged in 20MR (mean 51 days) relative to 16MR (mean 13 days) cells. The doubling time of 16MR and 20MR tumors was 1.9 and 4.1 days respectively, Conclusions: differentiation, morphologically
33
Although the ras-raf pathway has been shown to play a role in the regulation of cellular proliferation and raf is not required for ras transformation in this system. Raf knock out cells transfected with ras were transformed and tumorigenic.
IN VIVO RADIOSENSITIZATION FARNESYLTRANSFERASE
OF HUMAN INHIBITORS
TUMORS
EXPRESSING
MUTANT
RAS
BY
Cohen Jonathan-Moyal EL’, Muschel RJ’, McKenna WGr: Hamilton AD2, Sebti SM3, Gibbs J4, Oliff A4, Cerniglia G’, Mick RI, Bernhard EJ’ University qf Pennsylvania, Philadelphia, PA, USA’; Yale University, New Haven, CT, USA’; University of South Florida, Tampa, FL, lJSA3; Merck Research Laboratories, West Point, PA, USA4 Purpose: Ras activity depends upon membrane binding mediated by post-translational farnesylation of ras which can be blocked by farnesyltransferase inhibitors (FTI). These inhibitors have been shown to radiosensitize H-ras transformed rodent cells and human tumor cells expressing activated H- or K-ras in tissue culture. Activated ras appears to be the target of
164
I. J. Radiation
Oncology
0 Biology
l
Physics
Volume
45. Number
3 Supplement
1999
FTI-inhibition that results in radiosensitization. To determine whether treatment with FTIs may be clinically useful, we have studied the effect of farnesyltransferase inhibitor treatment on the radiosensitivity of two human tumor xenografts in nude mice: T24 bladder carcinoma cells expressing a mutated H-ras and HT-29 colon carcinoma cells expressing wild-type ras. Materials and Methods: Ras prenylation was inhibited by intraperitoneal injection or osmotic pump administration of two different FTI (FTI-276 or L744;832). The effect of FTI on radiation survival was determined by two methods, clonogenic survival and comparison of the plating efficiency between tumors treated with drug or with carrier alone, and by regrowth delay experiments. Results: FTI treatment resulted in significant radiosensitization in T24 tumors (p
34 Beavis
A COMBINATION DELIVERY BASED ON SPECIFIC PATIENT AW’,‘:
Princess Royal CA, USA3
Whitton
VJ’,‘,
Hospital,
Hull,
of radiosensitizing human tumors in viva. Our studies also show that damage to cells expressing wild-type ras. These results suggest that of human tumors expressing mutated ras.
MODE FOR CASES
INTENSITY
MODULATED
RADIATION
THERAPY
L’
United
Kingdom’;
University
of Hull,
Hull,
United
Kingdom
‘. , Stanford
University,
Stanford,
Purpose/Objective: Currently there are two methods available for the delivery of Intensity Modulated Radiation Therapy (IMRT) beams by Multileaf Collimators (MLC): 1) Step and Shoot method and 2) Dynamic method. The aim of this study was to critically examine the role of these methods and identify clinically relevant scenarios within which each has its limitations, A method has been developed for delivery which utilises a combination of these two approaches while attempting to maintain a higher level of accuracy and efficiency. Materials and Methods: In IMRT highly optimised beams are computed by the Inverse Planning system based on the required dose coverage of the planning target volume and the tolerance of the sensitive structures. This criterion is designed by the radiation oncologist. The delivery of these beams requires the superposition of a large number of shaped sub-beams and is always an estimation of the ideal beam. The simplest method of achieving this superposition is known as the ‘Step and Shoot’ method where each sub-field is defined and delivered in sequence. The main disadvantage of this method is a sacrifice of accuracy in the delivery of desired dose distributions that have steep gradients. This may be significant in close proximity to critical organs, such as spinal cord, which the treatment is designed to avoid. The ‘Dynamic’ method delivers the required distribution by sweeping the leaf pair across the beam. In principle this is a more efficient method of delivery. However, since the leaves are continuously moving whilst the beam is on, accuracy in delivery will be compromised in that ‘flat’ profiles are rendered undeliverable. The IMRT plans for ten patients have been studied. It is seen from IMRT plans generated for clinical cases that this situation may often arise. We implemented an algorithm combing these two methods utilising the advantages of each. The scheme measures the slope of each segment of the intensity profile and chooses the suitable delivery component. Finally, the corresponding leaf sequence is generated. Results: We have shown how the required intensity profile and resulting leaf sequence may be divided, by our algorithm. into a combination of ‘Step and Shoot’ and ‘Dynamic’ components. We have calculated the magnitude of the discrepancies between the idealised intensity profile and that delivered by the two existing methods and the combination method for the ten patients. It is seen that the combination method can always improve the delivery in comparison to the Step and Shoot method. It was found that the improvement can be of the order of 6%. It was noticed that several intensity distributions could not be executed using dynamic technique alone, whereas the combination method provided a solution. Conclusions: In general the dynamic method is seen to be more accurate however, clinical situations exist where the dynamic leaf sequence fails to produce the required distribution because of the limitation of maximum leaf speed; Unless a sliding window is used. Our combination method is a viable approach for these cases. It is quite general, selecting the optimal appropriate delivery method. It reduces discrepancy between the idealised intensity profile and that actually delivered, which is significant where sensitive organs lie in close proximity to the tumour volume.
35
INVESTIGATION OF THE PATIENT HETEROGENEITIES
Pawlicki
T. Guerrero
Stanford
University
T, Jiang School
DOSE
PERTURBATIONS
SB, Deng J, Li J, Boyer
of Medicine,
Stanford,
AL;
IN IMRT
TREATMENT
PLANS
CAUSED
BY
Ma CM
CA, USA
Purpose: The use of IMRT is becoming a prevalent treatment option for cases where 3DCRT can not adequately cover the target volume or spare critical structures. However, the dose calculation algorithms in commercial inverse planning systems contain approximations that give uncertainty to the final calculated dose distribution. The purpose of this work was to investigate the perturbation of dose in heterogeneous regions of patient treatment plans that were calculated by a commercial inverse planning system. The resultant dose perturbations were investigated using Monte Carlo simulation. We considered several clinical cases where IMRT was chosen primarily as a method to spare critical structures.