- Email: [email protected]
Radiation response in 10 high-grade human soft tissue sarcoma xenografts to photons and fast neutrons
Proceedings
147
of the 31st Annual ASTRO Meeting
62 RADIORESPONSE OF HUMAN GLIAL TUMORS ACROSS GRADE AS A FUNCTION OF ACUTE HIGH DOSE RATE AND CHRONIC LOW DOSE RATE IRRADIATION: RESULTS OF AN ASTRO RESEARCH FELLOWSHIP 1988-89. Christopher J. Schultz, M.D. and Charles R. Geard, Ph.D. Department of Radiation Oncology, Radialogkal Research Laboratory, College of Physicians and Surgeons of Columbia University, New York, New York. Astrocytomas make up the largest group of primary brain tumors of giiai origin. Long term survival is rare with high grade tumors (grade III and IV) which recur despite subtotal resection, chemotherapy, and aggressive pOStOperatiVe radiation therapy. In contrast the 5 year survival for low grade astrocytomas (grade I and Ii) following subtotal resection and POStOperatiVe radiotherapy approaches 50%. Variable radiosensitivity across grade may contribute to the difference in the behavior of these Little informatbn is tumors. Several in vitro studies have shown high grade astrocytomas 10 be relattvely radioresistant. available on the in vitro radiosensitivity of low grade astrocytomas. Cell lines derived from normal glial tissue, a low grade astrocytoma (grade I), and 2 different high grade astrocytomas (glioblastoma multiforme, grade IV) were established in culture. The tumor lines were found to be aneuploid, the low grade line hyperdiploidand the high grade lines hypotetraploid. Population doubling time for the normal gliailine and bw grade linewas >lOOhrs while for each of the high grade lines it was 40hrs. The cell lines were irradiated with Cesium 137 gamma rays at a
dose rate of 1.3 Gy/min in single acute exposures of 0 lo 12 Gy. Continuous irradiation with Cesium 137 gamma rays was also performed at dose rates ranging from 14 cGy/hr to 79 cGy/hr lo total doses of 0 to 20 Gy. Survival curves were determined for each ceil line using a cional assay. The high grade lines were found to be less radioresponsive than the low grade or normal glial line when irradiated acutely a1 high dose rate. Dose rate effects were seen with continuous irradiation at the lowest dose rate though no dose rate effect was apparent at the higher dose rates. These studies demonstrate a difference In radbresponsiveness of giial tumors across grade . The results of the continuous irradiation studies provide “a lowest effective dose rate” and demonstrate that continuous irradiation at dose rates from 26cGylhr to 79 cGy/hr would result in survival similar to a course of fractionated irradiation at 200 cGy per fraction.
63 ALTERATIONS Michael
OF THE RETINOBLASTOMA
A. Beckett,
Department
RECESSIVE
Alan M. Diamond,
of Radiation
and Cellular
IN THE ETIOLOGY
ONCOGENE
Janet M. Cowan, Oncology,
and Ralph
University
OF OSTEO AND SOFT TISSUE
SARCOMA
R. Weichselbaum
of Chicago,
Chicago,
IL
Patients with heredity retinoblastoma who are cured of their eye tumor have a high incidence of developing osteo and soft tissue sarcomas. Recently several groups have isolated a gene (Rb) which when To test the possibility that sarcomas have alterations altered or deleted predisposes to retinoblastoma. in the Rb gene, we studied DNA and RNA from osteo and soft tissue sarcomas derived from patients without retinoblastoma. Nucleic acid was obtained from fresh tissue and/or cell lines. In 13 of 14 tumors, the DNA appeared structurally intact using the 3' 3.8 kb fragment of the Rb cDNA as the probe. In 4 of 12 tumors from which RNA was extracted, the Rb transcript was deleted or an altered transcript was observed. An absent or abnormal Rb gene product may contribute to the development of some spontaneously occurring osteosarcomas and soft tissue sarcomas. Ionizing radiation causes single base mutations, inversions and deletions that may lead to loss of function mutations in the Rb gene, that may contribute to radiation induced sarcomas. Retinoblastoma and osteo and soft tissue sarcomas may have a common etiology. Supported by grant CA-41068 from the National Cancer Institute.
64 RADIATION
RESPONSE
IN
10
HIGH-GRADE PHOTONS
HUMAN
AND
FAST
SOFT
TISSUE
SARCOF4A
XENOGRAPTS
TO
NEUTRONS.
Volker Budach, M.D., Martin Stuschke, M.D., Wilfried Budach, M.D., Michael Molls, M.D., Horst Sack, M.D. Dept. of Radiation
Oncology,
West German
Tumor
Center,
University
of Essen,
Essen,
FR-Germany
The inherent radiosensitivity of human tumors is considered to be a major factor in the outcome of radiation therapy in clinical practice. In order to determine preclinically the range of radiation response of human highgrade soft tissue sarcoma xenografts (STX), a number of 10 tumor lines was selected for single dose experiments from a pool of 44 established STX stored in liquid nitrogen. All tumors were characterized by means of volume doubling time CDT), histomorphology, DNA-index and the LDH- and GPD-isozyme pattern and shown to be human from these measurements. The histologic subgroups consisted mainly of leiomyosarcomas (EMX, n=5), malignant fibrous histiocytomas (EFX, n=3), a neurofibrosarcoma (ENX) and a spindle cell sarcoma (ESX). The DT’s ranged from 2.8 to 13.8 days. The DNA-content was aneuploid in 6 and euploid in 4 cases, respectively. An NMRI-background outbred nu/nu-mouse strain was provided for the investigations. Two days prior to xeno-
148
Radiation
Oncology,
Biology,
Physics
October
1989, Volume
17, Supplement
1
all animals obtained a single whole body irradiation of 5.0 Gy to suppress residual NK- and transplantation, macrophage-related immunity. Groups of 10 to 12 animals were stratified according to tumor size (median treatment volume: 200~1) and randomly allocated to 4 photonand 2 fast neutron single dose regimens including one control group for each treatment. The dose rates of the 6oCo source and the d(l4)+Be neutrons were 2.9 Gy/min and 0.5 Gy/min. respectively. The tumors were rendered acutely hypoxic by clamping the tumor bed 10 min prior to Total doses ranged from 8 to 40 Gy for photons and 3 to 10 Gy for fast neutrons, and during irradiation. respectively. The endpoint of the experiments was the specific growth delay (SGD). In terms of an isoeffective SGD of 2.0 DT’s, total photonand neutron doses in the range of 3.3 to 36.7 Gy and No significant correlation was found between the DT’s of the xeno1.3 to 8.9 Gy, respectively, were necessary. grafts and their radiosensitivities or values of the relative biological effectiveness (RBE). The ranking of the tumors according to the growthand SGD agreed with a correlation coefficient of 0.9. At SGD’s of 1.0 and 2.0 The RBE’s remained constant or decreased with DT’s, RBE’s ranged from 1.7 to 6.7 and 1.5 to 6.1, respectively. increasing effect level. The RBE values were corrected for the oxygen enhancement ratio (OER) at a SGD level of to be about 1.6 and 1.2 with respect to photons and neutrons in the EL5-line. 1 and 2 DT’s, which was determined The corrected RBE-values at a SGD level of 1.0 (2.0) DT’s were in the range of 1.1 (1.0) to 4.5 (4.11, respectia significant gain for at least 4 vely. Thus assuming an RBE of 3.0 as to be adequate for normal soft tissues, out of 10 tumor lines was observed. These results indicate a potential usefulness of fast neutrons for clinical radiation therapy of soft tissue tumors as well as the considerable heterogeneity of the radiation response despite homogeneous hypoxia.
65 SYNERGISM OF MYC WITH RAS IN THE INDUCTION OF RADIORESISTANCE Marisa C. Weiss’, Ruth J. Muschel*, Vincent J. Bakanauskas l, Brian Endlich3, Clifford Ling3, Martha Sack2 and W. Gillies McKennal. Depts of Radiation Oncology1 and Pathology and Laboratory Medicine 2, University of Pennsylvania, Philadelphia, PA and Dept of Radiation Oncology3, UCSF School of Medicine, San Francisco, CA.
Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may fail to be cured. Much is now known about the molecular mechanisms that underlie drug resistance but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells (RX) have been chosen as a model system in which the effects on radiation resistance of the Hras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any pre-existing mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The primary cells and cells immortalized spontaneously or with the myc oncogene were compared to a series of independent celI lines transformed by ras alone or ras cotransfected with myc. The results show that the activated Hras oncogene is associated with radiation resistance in primary rat cells after transformation. The effect of the oncogene by itself is small but is significant at p c 0.02 when compared by Student’s t test. However, the oncogene myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with Hras (p c 0.01). There appear to be differences in the phenotype of radiation resistance associated with with these two forms of transfectants. Thus, radiation resistance seen with I-has by itself is characterized by a change in the B component of the radiation survival curve. Little or no change is seen within the shoulder region of the radiation survival curve. Radiation resistance seen in Hras plus myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect on the a component within the shoulder region of the radiation survival curve. These studies led to the following conclusions; (1) the radioresistant phenotype is not due to pre-existing genetic heterogeneity in the cells prior to transfection; (2) the radiation resistant phenotype of cells transformed by Hras is seen to a greater degree in cells which also contain the myc oncogene; (3) the myc oncogene may play an important role in the phenotype of radiation resistance at low doses which is within the range most critical for clinical practice. The most important questions raised by these studies is whether the transfected oncogenes actually induce a change in the cells’ phenotype by their expression or whether these radioresistant cells represent different epigenetic or differentiation states of the cells. These could pm-exist transfection and be selected for in the transformation procedure or they could occur after oncogene transfection. To attempt to answer these questions we have now performed a series of sequential transfections. That is by first transfecting one oncogene, verifying the phenotype obtained, then hansfecting a second oncogene we can ask whether there is now a second change in cellular phenotype. The results of this analysis will be presented. Preliminary data suggest that karyotypic changes in the cells, which must have happened after transfection since we start with diploid cells, are important features which have to be considered in addition to the oncogene composition.
66 EXPRESSION
OF THE SRC ONCOGENE
DOES
Anna B. Hill, Ti Lin, Dina Millikin, Departments of Radiation Tucson, AZ, 85724
Oncology
NOT ALTER RADIOSENSITIVITY Sharon
L. Olson,
and Microbiology
David S. Shimm
and Immunology,
University
of Arizona
Cancer
Center,
Recent work in mouse NIH 3T3 and Chinese hamster cell lines has implicated the activated rd~ oncogene, whose gene product is a G-protein located in the plasma membrane, in contributing to radioresistance. Another
147
of the 31st Annual ASTRO Meeting
62 RADIORESPONSE OF HUMAN GLIAL TUMORS ACROSS GRADE AS A FUNCTION OF ACUTE HIGH DOSE RATE AND CHRONIC LOW DOSE RATE IRRADIATION: RESULTS OF AN ASTRO RESEARCH FELLOWSHIP 1988-89. Christopher J. Schultz, M.D. and Charles R. Geard, Ph.D. Department of Radiation Oncology, Radialogkal Research Laboratory, College of Physicians and Surgeons of Columbia University, New York, New York. Astrocytomas make up the largest group of primary brain tumors of giiai origin. Long term survival is rare with high grade tumors (grade III and IV) which recur despite subtotal resection, chemotherapy, and aggressive pOStOperatiVe radiation therapy. In contrast the 5 year survival for low grade astrocytomas (grade I and Ii) following subtotal resection and POStOperatiVe radiotherapy approaches 50%. Variable radiosensitivity across grade may contribute to the difference in the behavior of these Little informatbn is tumors. Several in vitro studies have shown high grade astrocytomas 10 be relattvely radioresistant. available on the in vitro radiosensitivity of low grade astrocytomas. Cell lines derived from normal glial tissue, a low grade astrocytoma (grade I), and 2 different high grade astrocytomas (glioblastoma multiforme, grade IV) were established in culture. The tumor lines were found to be aneuploid, the low grade line hyperdiploidand the high grade lines hypotetraploid. Population doubling time for the normal gliailine and bw grade linewas >lOOhrs while for each of the high grade lines it was 40hrs. The cell lines were irradiated with Cesium 137 gamma rays at a
dose rate of 1.3 Gy/min in single acute exposures of 0 lo 12 Gy. Continuous irradiation with Cesium 137 gamma rays was also performed at dose rates ranging from 14 cGy/hr to 79 cGy/hr lo total doses of 0 to 20 Gy. Survival curves were determined for each ceil line using a cional assay. The high grade lines were found to be less radioresponsive than the low grade or normal glial line when irradiated acutely a1 high dose rate. Dose rate effects were seen with continuous irradiation at the lowest dose rate though no dose rate effect was apparent at the higher dose rates. These studies demonstrate a difference In radbresponsiveness of giial tumors across grade . The results of the continuous irradiation studies provide “a lowest effective dose rate” and demonstrate that continuous irradiation at dose rates from 26cGylhr to 79 cGy/hr would result in survival similar to a course of fractionated irradiation at 200 cGy per fraction.
63 ALTERATIONS Michael
OF THE RETINOBLASTOMA
A. Beckett,
Department
RECESSIVE
Alan M. Diamond,
of Radiation
and Cellular
IN THE ETIOLOGY
ONCOGENE
Janet M. Cowan, Oncology,
and Ralph
University
OF OSTEO AND SOFT TISSUE
SARCOMA
R. Weichselbaum
of Chicago,
Chicago,
IL
Patients with heredity retinoblastoma who are cured of their eye tumor have a high incidence of developing osteo and soft tissue sarcomas. Recently several groups have isolated a gene (Rb) which when To test the possibility that sarcomas have alterations altered or deleted predisposes to retinoblastoma. in the Rb gene, we studied DNA and RNA from osteo and soft tissue sarcomas derived from patients without retinoblastoma. Nucleic acid was obtained from fresh tissue and/or cell lines. In 13 of 14 tumors, the DNA appeared structurally intact using the 3' 3.8 kb fragment of the Rb cDNA as the probe. In 4 of 12 tumors from which RNA was extracted, the Rb transcript was deleted or an altered transcript was observed. An absent or abnormal Rb gene product may contribute to the development of some spontaneously occurring osteosarcomas and soft tissue sarcomas. Ionizing radiation causes single base mutations, inversions and deletions that may lead to loss of function mutations in the Rb gene, that may contribute to radiation induced sarcomas. Retinoblastoma and osteo and soft tissue sarcomas may have a common etiology. Supported by grant CA-41068 from the National Cancer Institute.
64 RADIATION
RESPONSE
IN
10
HIGH-GRADE PHOTONS
HUMAN
AND
FAST
SOFT
TISSUE
SARCOF4A
XENOGRAPTS
TO
NEUTRONS.
Volker Budach, M.D., Martin Stuschke, M.D., Wilfried Budach, M.D., Michael Molls, M.D., Horst Sack, M.D. Dept. of Radiation
Oncology,
West German
Tumor
Center,
University
of Essen,
Essen,
FR-Germany
The inherent radiosensitivity of human tumors is considered to be a major factor in the outcome of radiation therapy in clinical practice. In order to determine preclinically the range of radiation response of human highgrade soft tissue sarcoma xenografts (STX), a number of 10 tumor lines was selected for single dose experiments from a pool of 44 established STX stored in liquid nitrogen. All tumors were characterized by means of volume doubling time CDT), histomorphology, DNA-index and the LDH- and GPD-isozyme pattern and shown to be human from these measurements. The histologic subgroups consisted mainly of leiomyosarcomas (EMX, n=5), malignant fibrous histiocytomas (EFX, n=3), a neurofibrosarcoma (ENX) and a spindle cell sarcoma (ESX). The DT’s ranged from 2.8 to 13.8 days. The DNA-content was aneuploid in 6 and euploid in 4 cases, respectively. An NMRI-background outbred nu/nu-mouse strain was provided for the investigations. Two days prior to xeno-
148
Radiation
Oncology,
Biology,
Physics
October
1989, Volume
17, Supplement
1
all animals obtained a single whole body irradiation of 5.0 Gy to suppress residual NK- and transplantation, macrophage-related immunity. Groups of 10 to 12 animals were stratified according to tumor size (median treatment volume: 200~1) and randomly allocated to 4 photonand 2 fast neutron single dose regimens including one control group for each treatment. The dose rates of the 6oCo source and the d(l4)+Be neutrons were 2.9 Gy/min and 0.5 Gy/min. respectively. The tumors were rendered acutely hypoxic by clamping the tumor bed 10 min prior to Total doses ranged from 8 to 40 Gy for photons and 3 to 10 Gy for fast neutrons, and during irradiation. respectively. The endpoint of the experiments was the specific growth delay (SGD). In terms of an isoeffective SGD of 2.0 DT’s, total photonand neutron doses in the range of 3.3 to 36.7 Gy and No significant correlation was found between the DT’s of the xeno1.3 to 8.9 Gy, respectively, were necessary. grafts and their radiosensitivities or values of the relative biological effectiveness (RBE). The ranking of the tumors according to the growthand SGD agreed with a correlation coefficient of 0.9. At SGD’s of 1.0 and 2.0 The RBE’s remained constant or decreased with DT’s, RBE’s ranged from 1.7 to 6.7 and 1.5 to 6.1, respectively. increasing effect level. The RBE values were corrected for the oxygen enhancement ratio (OER) at a SGD level of to be about 1.6 and 1.2 with respect to photons and neutrons in the EL5-line. 1 and 2 DT’s, which was determined The corrected RBE-values at a SGD level of 1.0 (2.0) DT’s were in the range of 1.1 (1.0) to 4.5 (4.11, respectia significant gain for at least 4 vely. Thus assuming an RBE of 3.0 as to be adequate for normal soft tissues, out of 10 tumor lines was observed. These results indicate a potential usefulness of fast neutrons for clinical radiation therapy of soft tissue tumors as well as the considerable heterogeneity of the radiation response despite homogeneous hypoxia.
65 SYNERGISM OF MYC WITH RAS IN THE INDUCTION OF RADIORESISTANCE Marisa C. Weiss’, Ruth J. Muschel*, Vincent J. Bakanauskas l, Brian Endlich3, Clifford Ling3, Martha Sack2 and W. Gillies McKennal. Depts of Radiation Oncology1 and Pathology and Laboratory Medicine 2, University of Pennsylvania, Philadelphia, PA and Dept of Radiation Oncology3, UCSF School of Medicine, San Francisco, CA.
Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may fail to be cured. Much is now known about the molecular mechanisms that underlie drug resistance but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells (RX) have been chosen as a model system in which the effects on radiation resistance of the Hras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any pre-existing mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The primary cells and cells immortalized spontaneously or with the myc oncogene were compared to a series of independent celI lines transformed by ras alone or ras cotransfected with myc. The results show that the activated Hras oncogene is associated with radiation resistance in primary rat cells after transformation. The effect of the oncogene by itself is small but is significant at p c 0.02 when compared by Student’s t test. However, the oncogene myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with Hras (p c 0.01). There appear to be differences in the phenotype of radiation resistance associated with with these two forms of transfectants. Thus, radiation resistance seen with I-has by itself is characterized by a change in the B component of the radiation survival curve. Little or no change is seen within the shoulder region of the radiation survival curve. Radiation resistance seen in Hras plus myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect on the a component within the shoulder region of the radiation survival curve. These studies led to the following conclusions; (1) the radioresistant phenotype is not due to pre-existing genetic heterogeneity in the cells prior to transfection; (2) the radiation resistant phenotype of cells transformed by Hras is seen to a greater degree in cells which also contain the myc oncogene; (3) the myc oncogene may play an important role in the phenotype of radiation resistance at low doses which is within the range most critical for clinical practice. The most important questions raised by these studies is whether the transfected oncogenes actually induce a change in the cells’ phenotype by their expression or whether these radioresistant cells represent different epigenetic or differentiation states of the cells. These could pm-exist transfection and be selected for in the transformation procedure or they could occur after oncogene transfection. To attempt to answer these questions we have now performed a series of sequential transfections. That is by first transfecting one oncogene, verifying the phenotype obtained, then hansfecting a second oncogene we can ask whether there is now a second change in cellular phenotype. The results of this analysis will be presented. Preliminary data suggest that karyotypic changes in the cells, which must have happened after transfection since we start with diploid cells, are important features which have to be considered in addition to the oncogene composition.
66 EXPRESSION
OF THE SRC ONCOGENE
DOES
Anna B. Hill, Ti Lin, Dina Millikin, Departments of Radiation Tucson, AZ, 85724
Oncology
NOT ALTER RADIOSENSITIVITY Sharon
L. Olson,
and Microbiology
David S. Shimm
and Immunology,
University
of Arizona
Cancer
Center,
Recent work in mouse NIH 3T3 and Chinese hamster cell lines has implicated the activated rd~ oncogene, whose gene product is a G-protein located in the plasma membrane, in contributing to radioresistance. Another