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Experimental studies of photo radiation therapy on neuroblastoma
Experimental Studies of Photo Radiation Therapy on N e u r o b l a s t o m a By Shun-ichi Makino, Toshio Nakojo, Yoshiaki Tsuchida, Kohei Hashizume, Masaharu Nakajima, Kazuhiko Atsumi, and John Tulip
Tokyo, Japan and Alberta, Canada 9 A combination of Photo Radiation Therapy (PRT) using Argon-Dye Laser with hematoporphyrin derivatives (HpD) was used experimentally on a cytogenetically highly malignant neuroblastoma xenograft, which exhibited a homogeneously staining region and caused DNA amplifications in chromosomes. The tumor tissue was treated with 500 joules/cm 2 of laser. The dosage of HpD was 50 mg per kg body weight. Necrosis of over 50% of the tumor was observed in half the specimens. Swollen cytoplasmic organelles and ruptured cell and nuclear membranes were observed by electron microscopy after PRT. PRT may be used with other treatment modalities for the removal of residual and metastatic tumors. 9 1986 by Grune & Stratton, Inc.
nu/nu
Fig 1.
mouse
Experimental arrangement for photo radiation therapy.
E C A U S E of its highly invasive characteristics and metastatic propensity, eradication of advanced neuroblastoma is difficult. The effects of chemotherapy and x-ray irradiation on this tumor, may be restricted if the patient is in a poor nutritional state or has bone marrow depletion or drug and x-ray resistance. Laser-stimulated photo therapy or Photo Radiation Therapy (PRT) has recently been shown to have a significant effect on some malignant tumors without producing side-effects on the patient. ~ We experimented with PRT on neuroblastoma using a human neuroblastoma xenograft.
Coherent 210 power meter after induction to quartz fiber. Hematoporphyrin derivatives (HpD) and protoporphyrins were injected intraperitoneally in doses of 50 mg/kg body weight, into the mice 72 hours before PRT. The accumulation of HpD on several tumors was confirmed by fluorescence with N2 laser. PRT was performed through the skin without any anesthesia, using the arrangement shown in Fig 1. The mice were divided into three groups: a control group, a group treated with PRT combined with HpD, and a group treated with PRT combined with protoporphyrins. The tumors of the test mice were measured every four days. Estimated tumor weight (W) in mg was calculated from the formula W = (a 2 x b)/2 where a is the width and b is the length in mm. s The effect of PRT on the tumor was assessed by macroscopic, light microscopic, and electron microscopic observations. It was also evaluated by the maximum rate of tumor regression (inhibition rate) as assessed by the formula: I R = [1 - (T,/T0) + (C./C0)] x 100 (%), where T,/T 0 is the change in mean tumor weight in the treatment group between the initial day (0) and day n from treatment, and C,/C 0 is that in the control group. Retardation of tumor growth is equivalent to an inhibition rate over 58%. An inhibition rate under 58% indicates inactivity.
MATERIALS A N D METHODS
RESULTS
The human neuroblastoma xenograft used originated from an abdominal neuroblastoma in a 15-month-old boy with Stage IV disease. This xenograft had a homogeneously staining region and exhibited N-myc DNA amplification, both of which are regarded as malignant characteristics.T M Tumor tissue was implanted subcutaneously on the backs of 26 mice, and allowed to grow to an average size of 9.3 mm in width and 12.4 mm in length: this required 4 to 5 weeks. Treatment using a Spectra Physics Argon-Dye Laser system was delivered by a 400 micron quartz fiber. Rhodamine 6100 was used for the dye to get a light spectrum of 630 nm. Strength of radiation was 500 joules/cm 2. The power density was measured by a
In t h e g r o u p w h o s e t r e a t m e n t w a s c o m b i n e d w i t h H p D , n e c r o s i s , as s h o w n b y d e s t r u c t i o n a n d p i k n o s i s ,
INDEX WORDS: Photo radiation therapy; neuroblastoma.
B
From the Department of Pediatric Surgery and Institute of Medical Electronics, University of Tokyo, Tokyo, Japan. and the Department of Electrical Engineering, University of Alberta, Alberta, Canada. Presented at the 32nd Annual Congress of the British Association of Paediatric Surgeons, Vienna, A ustria, July l 7-19. 1985. Address reprint requests to Shun-ichi Makino, MD, Dept of Pediatric Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. c~ 1986 by Grune & Stratton, Inc. 0022-3468/86/2103~9016503.00/0 240
was observed macroscopically and by light microscopy to b e t o t a l in t w o c a s e s , a f f e c t i n g m o r e t h a n 50% in four cases and
less t h a n
50%
in six c a s e s .
PRT
c o m b i n e d w i t h p r o t o p o r p h y r i n h a d no e f f e c t on t h e t u m o r ( F i g 2), S w e l l i n g o f t h e n e u r o b l a s t s w a s i d e n t i f i e d as t h e initial c h a n g e a f t e r P R T , a n d t h i s w a s f o l l o w e d b y -
No OF Cases
2
Appear. ante
Estimated % of Neceos,s
50%
~
NO of Cases
8
4
~
,50~176
~G
6
~
75",
12
Fig 2.
Appear ante
Est,mated ~-,~ of Necrosis
~
~ 50%
<50%
~
Localfindings after PRT.
Journal of Pediatric Surgery, Vol 21, No 3 (March), 1986: pp 240-243
PRT ON NEUROBLASTOMA
Fig 3.
Fig 4.
(A) Untreated implanted t u m o r under scanning electron microscope.
241
(B) Tumor one day after PRT.
(A) Untreated implanted t u m o r under transmission electron microscope.
(B) Tumor one day after PRT.
242
MAKINOETAL Tumor Volume (x/:~)
Tumor Volume ,(:rlx~)
22
22
'~
20
,8
/
16 14 12 10
/ Controls~/ (n=7) / /
1~ 16
Controls~j~
/
.7
14
]7~PRT 1 (protoporphyrin)
I~
#"
pD)
8 6 4
~0
,~ ~
12 1'6 2'8 i4 i8 3?aoy~
Fig 5. Tumor weight curves in PRT (combined with HpD) and control groups.
cloudiness of the cells, swelling of the mitochondria and other cytoplasmic organelles, and rupture of the cell and nuclear membranes (Figs 3, 4). Retardation of tumor growth was demonstrated by the tumor weight curve in the group treated with P R T combined with HpD. M a x i m u m inhibition rate for this group was 58.1%. There was no effect in the group in which P R T was combined with protoporphyrins (Figs 5, 6). DISCUSSION
The mechanism of tumor destruction by P R T is a photochemical reaction between visual light and a photosensitizer such as H p D or protoporphyrin. 6 Red light laser which has a wave length of 630 nm is usually used in such a system. The effect of P R T is governed by the strength of the laser, the accumulation of photosensitizer on the tumor, and the sensitivity of the tumor cells. The effects of P R T on C-1300 neuroblastoma in A / J mice were examined by the authors and Johnson et al 7 recently, and tumor regression was observed. In the experiments reported here, a human neuroblastoma xenograft taken from an advanced neuroblastoma was used. When examined in detail cytogenetically, this neuroblastoma was considered to be representative of the highly malignant variety described by Brodeur et a l l H p D is a mixture of several compounds of which hematoporphyrin, protoporphyrin, and hydroxyethyl-
2
j~,-.,-
o
~
~
1'2 1'6 2'o 2'4 2'8 3'2D.,,
Fig 6. Tumor weight curves in PRT (combined with protoporphyrin) and control groups.
vinyldeuterporphyrin account for about half. 6 The active fraction is an aggregate of varying sizes ranging from under 6,000 mol wt to 60,000. The accumulation of H p D on the tumor was observed by N2 laser, which gives off a red fluorescence# It was not uniform because of the varying size of the molecules: the large ones are unable to permeate the cell membranes. P R T combined with HpD had a considerable effect on the tumor, but protoporphyrin had no effect. There was no explanation for this result. P R T produced extensive necrosis at depths of 4 to 8 m m from the irradiated skin macroscopically. The changes consisted of enlargement and swelling of the neuroblasts and destruction of cell and nuclear membranes. The mechanism or subcellular target of P R T still remains unknown. Inhibition of tumor weight increase is seen in the group receiving P R T combined with HpD. The maximum inhibition rate was 58.1%. Maximum inhibition rate is used to judge the effect of chemotherapeutic agents, but it seems inappropriate for assessing the effect of PRT. A suitable index is not known at present. P R T might be used on residual tumor after surgery to eradicate remaining neuroblasts. It could also be used on metastatic lesions in bone, lymph node, or sites near the surface of the body. It is hoped that some methodology will be developed for the clinical application of laser phototherapy to neuroblastoma.
REFERENCES 1. Dougherty TJ, Kaufman JE, Goldfarb A, et al: Photoradiation 2. Tsuchida Y, Yokomori K, lwanaka T, et al: Nude mouse therapy for the treatment of malignant tumors. Cancer Res xenograftstudy for the treatment of neuroblastoma. J Pediatr Surg 38:2628-2635, 1978 19:72-76, 1984
PRT ON NEUROBLASTOMA
3. Brodeur GM, Green AA, Hayes A, et al: Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4678 4686, 1981 4. Kanda N, Schreck R, Alt F, et al: Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells. Proc Natl Acad Sci 80:4068-4073, 1983 5. Geran RI, Greenberg NH, Macdonald MM, et al: Protocols for screening chemical agents and natural products against animal
243
tumors and other biological systems. Cancer Chemother Rep 3:5161, 1972 6. Weishaupt KR, Gomer C J, Dougherty, T J: Identification of singlet oxygen as the cytotoxic agent in photo-inactivation of a murine tumor. Cancer Res 36:2326-2329, 1976 7. Johnson DG, et al: personal communication, October 1984 8. Lipson RL, Baldes EJ, Olsen AM: The use of a derivative of hematoporphyrin in tumor detection. J Natl Cancer Inst 26:1-8, 1961
Tokyo, Japan and Alberta, Canada 9 A combination of Photo Radiation Therapy (PRT) using Argon-Dye Laser with hematoporphyrin derivatives (HpD) was used experimentally on a cytogenetically highly malignant neuroblastoma xenograft, which exhibited a homogeneously staining region and caused DNA amplifications in chromosomes. The tumor tissue was treated with 500 joules/cm 2 of laser. The dosage of HpD was 50 mg per kg body weight. Necrosis of over 50% of the tumor was observed in half the specimens. Swollen cytoplasmic organelles and ruptured cell and nuclear membranes were observed by electron microscopy after PRT. PRT may be used with other treatment modalities for the removal of residual and metastatic tumors. 9 1986 by Grune & Stratton, Inc.
nu/nu
Fig 1.
mouse
Experimental arrangement for photo radiation therapy.
E C A U S E of its highly invasive characteristics and metastatic propensity, eradication of advanced neuroblastoma is difficult. The effects of chemotherapy and x-ray irradiation on this tumor, may be restricted if the patient is in a poor nutritional state or has bone marrow depletion or drug and x-ray resistance. Laser-stimulated photo therapy or Photo Radiation Therapy (PRT) has recently been shown to have a significant effect on some malignant tumors without producing side-effects on the patient. ~ We experimented with PRT on neuroblastoma using a human neuroblastoma xenograft.
Coherent 210 power meter after induction to quartz fiber. Hematoporphyrin derivatives (HpD) and protoporphyrins were injected intraperitoneally in doses of 50 mg/kg body weight, into the mice 72 hours before PRT. The accumulation of HpD on several tumors was confirmed by fluorescence with N2 laser. PRT was performed through the skin without any anesthesia, using the arrangement shown in Fig 1. The mice were divided into three groups: a control group, a group treated with PRT combined with HpD, and a group treated with PRT combined with protoporphyrins. The tumors of the test mice were measured every four days. Estimated tumor weight (W) in mg was calculated from the formula W = (a 2 x b)/2 where a is the width and b is the length in mm. s The effect of PRT on the tumor was assessed by macroscopic, light microscopic, and electron microscopic observations. It was also evaluated by the maximum rate of tumor regression (inhibition rate) as assessed by the formula: I R = [1 - (T,/T0) + (C./C0)] x 100 (%), where T,/T 0 is the change in mean tumor weight in the treatment group between the initial day (0) and day n from treatment, and C,/C 0 is that in the control group. Retardation of tumor growth is equivalent to an inhibition rate over 58%. An inhibition rate under 58% indicates inactivity.
MATERIALS A N D METHODS
RESULTS
The human neuroblastoma xenograft used originated from an abdominal neuroblastoma in a 15-month-old boy with Stage IV disease. This xenograft had a homogeneously staining region and exhibited N-myc DNA amplification, both of which are regarded as malignant characteristics.T M Tumor tissue was implanted subcutaneously on the backs of 26 mice, and allowed to grow to an average size of 9.3 mm in width and 12.4 mm in length: this required 4 to 5 weeks. Treatment using a Spectra Physics Argon-Dye Laser system was delivered by a 400 micron quartz fiber. Rhodamine 6100 was used for the dye to get a light spectrum of 630 nm. Strength of radiation was 500 joules/cm 2. The power density was measured by a
In t h e g r o u p w h o s e t r e a t m e n t w a s c o m b i n e d w i t h H p D , n e c r o s i s , as s h o w n b y d e s t r u c t i o n a n d p i k n o s i s ,
INDEX WORDS: Photo radiation therapy; neuroblastoma.
B
From the Department of Pediatric Surgery and Institute of Medical Electronics, University of Tokyo, Tokyo, Japan. and the Department of Electrical Engineering, University of Alberta, Alberta, Canada. Presented at the 32nd Annual Congress of the British Association of Paediatric Surgeons, Vienna, A ustria, July l 7-19. 1985. Address reprint requests to Shun-ichi Makino, MD, Dept of Pediatric Surgery, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan. c~ 1986 by Grune & Stratton, Inc. 0022-3468/86/2103~9016503.00/0 240
was observed macroscopically and by light microscopy to b e t o t a l in t w o c a s e s , a f f e c t i n g m o r e t h a n 50% in four cases and
less t h a n
50%
in six c a s e s .
PRT
c o m b i n e d w i t h p r o t o p o r p h y r i n h a d no e f f e c t on t h e t u m o r ( F i g 2), S w e l l i n g o f t h e n e u r o b l a s t s w a s i d e n t i f i e d as t h e initial c h a n g e a f t e r P R T , a n d t h i s w a s f o l l o w e d b y -
No OF Cases
2
Appear. ante
Estimated % of Neceos,s
50%
~
NO of Cases
8
4
~
,50~176
~G
6
~
75",
12
Fig 2.
Appear ante
Est,mated ~-,~ of Necrosis
~
~ 50%
<50%
~
Localfindings after PRT.
Journal of Pediatric Surgery, Vol 21, No 3 (March), 1986: pp 240-243
PRT ON NEUROBLASTOMA
Fig 3.
Fig 4.
(A) Untreated implanted t u m o r under scanning electron microscope.
241
(B) Tumor one day after PRT.
(A) Untreated implanted t u m o r under transmission electron microscope.
(B) Tumor one day after PRT.
242
MAKINOETAL Tumor Volume (x/:~)
Tumor Volume ,(:rlx~)
22
22
'~
20
,8
/
16 14 12 10
/ Controls~/ (n=7) / /
1~ 16
Controls~j~
/
.7
14
]7~PRT 1 (protoporphyrin)
I~
#"
pD)
8 6 4
~0
,~ ~
12 1'6 2'8 i4 i8 3?aoy~
Fig 5. Tumor weight curves in PRT (combined with HpD) and control groups.
cloudiness of the cells, swelling of the mitochondria and other cytoplasmic organelles, and rupture of the cell and nuclear membranes (Figs 3, 4). Retardation of tumor growth was demonstrated by the tumor weight curve in the group treated with P R T combined with HpD. M a x i m u m inhibition rate for this group was 58.1%. There was no effect in the group in which P R T was combined with protoporphyrins (Figs 5, 6). DISCUSSION
The mechanism of tumor destruction by P R T is a photochemical reaction between visual light and a photosensitizer such as H p D or protoporphyrin. 6 Red light laser which has a wave length of 630 nm is usually used in such a system. The effect of P R T is governed by the strength of the laser, the accumulation of photosensitizer on the tumor, and the sensitivity of the tumor cells. The effects of P R T on C-1300 neuroblastoma in A / J mice were examined by the authors and Johnson et al 7 recently, and tumor regression was observed. In the experiments reported here, a human neuroblastoma xenograft taken from an advanced neuroblastoma was used. When examined in detail cytogenetically, this neuroblastoma was considered to be representative of the highly malignant variety described by Brodeur et a l l H p D is a mixture of several compounds of which hematoporphyrin, protoporphyrin, and hydroxyethyl-
2
j~,-.,-
o
~
~
1'2 1'6 2'o 2'4 2'8 3'2D.,,
Fig 6. Tumor weight curves in PRT (combined with protoporphyrin) and control groups.
vinyldeuterporphyrin account for about half. 6 The active fraction is an aggregate of varying sizes ranging from under 6,000 mol wt to 60,000. The accumulation of H p D on the tumor was observed by N2 laser, which gives off a red fluorescence# It was not uniform because of the varying size of the molecules: the large ones are unable to permeate the cell membranes. P R T combined with HpD had a considerable effect on the tumor, but protoporphyrin had no effect. There was no explanation for this result. P R T produced extensive necrosis at depths of 4 to 8 m m from the irradiated skin macroscopically. The changes consisted of enlargement and swelling of the neuroblasts and destruction of cell and nuclear membranes. The mechanism or subcellular target of P R T still remains unknown. Inhibition of tumor weight increase is seen in the group receiving P R T combined with HpD. The maximum inhibition rate was 58.1%. Maximum inhibition rate is used to judge the effect of chemotherapeutic agents, but it seems inappropriate for assessing the effect of PRT. A suitable index is not known at present. P R T might be used on residual tumor after surgery to eradicate remaining neuroblasts. It could also be used on metastatic lesions in bone, lymph node, or sites near the surface of the body. It is hoped that some methodology will be developed for the clinical application of laser phototherapy to neuroblastoma.
REFERENCES 1. Dougherty TJ, Kaufman JE, Goldfarb A, et al: Photoradiation 2. Tsuchida Y, Yokomori K, lwanaka T, et al: Nude mouse therapy for the treatment of malignant tumors. Cancer Res xenograftstudy for the treatment of neuroblastoma. J Pediatr Surg 38:2628-2635, 1978 19:72-76, 1984
PRT ON NEUROBLASTOMA
3. Brodeur GM, Green AA, Hayes A, et al: Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4678 4686, 1981 4. Kanda N, Schreck R, Alt F, et al: Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells. Proc Natl Acad Sci 80:4068-4073, 1983 5. Geran RI, Greenberg NH, Macdonald MM, et al: Protocols for screening chemical agents and natural products against animal
243
tumors and other biological systems. Cancer Chemother Rep 3:5161, 1972 6. Weishaupt KR, Gomer C J, Dougherty, T J: Identification of singlet oxygen as the cytotoxic agent in photo-inactivation of a murine tumor. Cancer Res 36:2326-2329, 1976 7. Johnson DG, et al: personal communication, October 1984 8. Lipson RL, Baldes EJ, Olsen AM: The use of a derivative of hematoporphyrin in tumor detection. J Natl Cancer Inst 26:1-8, 1961