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Beneficial effects following carbon dioxide laser excision on experimental neuroblastoma
Beneficial Effects Following Carbon Dioxide Laser Excision on Experimental Neuroblastoma By Carter J. McCormack, John O. Naim, David W. Rogers, Mortiz M. Ziegler, and J. Raymond Hinshaw Rochester, New York and Philadelphia, Pennsylvania 9 T r e a t m e n t of neuroblastoma in children consists of primary excision with adjuvant radiation and chemotherapy. W h e n the tumor invades surrounding structures that cannot be safely excised or when distant metastasis is present, the patient has a poor prognosis. Because the CO 2 laser can he used to excise malignant tumors without seeding the surrounding tissue and because the defocused beam can vaporize malignant cells, w e compared partial scalpel excision and partial laser excision of C 1 3 0 0 murine neuroblastoma to the g r o w t h rate of residual tumor. In 25 mice, 7 5 % of the tumor was excised with a scalpel, and in another 25, the same percentage was excised with the CO 2 laser (10 W). COz laser excision significantly decreases the growth of residual neuroblastoma (P < .01 ). However, the effect appears to be a function of increased tumor immunogenicity after laser excision rather than the increased tumor kill. W e conclude that CO 2 laser excision of neuroblastoma may prove to be superior to scalpel excision for primary surgical t r e a t m e n t of neuroblastoma. 9 1989 by Grune & Stratton. Inc. INDEX W O R D S : Neuroblastoma; CO z laser.
' E U R O B L A S T O M A , the most common extracranial malignant solid tumor in children, represents about 10% of all childhood malignancies and 25% to 50% of all malignancies in children <30 months of age. 1 Approximately 500 to 600 new cases occur annually in the United States. The overall disease-free survival of 37% reflects extensive local disease and widespread metastasis usually apparent at the time of diagnosis. 1 Neuroblastoma can regress spontaneously, most often in children < 1 year of age? This phenomenon may represent an immunologic body response to the tumor. Surgical excision has remained the primary therapy for stage I, II, and III disease (Table 1). 1'3 Adjuvant radiotherapy and chemotherapy have not significantly improved long-term survival. The complications from surgical excision can be devastating and may be more common than generally believed. 4 The goal is surgical excision without damage to locally involved important blood vessels and nerves. Animal model mammary tumors can be excised more precisely with the CO2 laser than with a scalpel. 5'~ The laser also seals small blood vessels and lymphatics. Tumor recurrence rates have been shown to decrease with the use of CO2 laser excision. 5 In this study, we attempted to determine whether the CO2 laser is useful in the excision of neuroblastoma, and if laser excision has any effect on the hosttumor immunologic relationship. The murine C1300
N
Journal of Pediatric Surgery,Vo124, No 2 (February), 1989: pp 201-203
neuroblastoma has many properties similar to human neuroblastoma, including antigenicity, which makes it an ideal animal model for this study. MATERIALS AND METHODS Fifty A / J male mice (Jackson Laboratories, Bar Harbor, ME) were used to study the effect of CO2 laser excision of neuroblastoma. C1300 neuroblastoma that had been serially maintained by tissue culture was implanted subcutaneoulsy by injection (approximately 1 million viable neuroblastoma cells). Tumor growth was recorded daily until tumor nodules measured 0.5 to 0.1 cm in diameter (day 19), and two groups of mice with similar mean tumor diameters and ranges were separated. At this time, 75 percent of each tumor was excised by a CO2 laser (n = 25) or scalpel (n = 25). For the next 2 weeks, the growth of residual tumor was measured daily. Laser excision was accomplished with a Sharplan 733 CO2 laser at 10 W, continuous mode, in focus with a 125-ram focal length lens system that gives a 0.2-mm spot size and yields a power density of 26,000 W/cm 2 at the target site. To study the host-tumor immunologic effect of COs laser excision of neuroblastoma, 45 A / J male mice were divided into three groups of 15 each. Group 1 animals had scalpel excision and group 2 had CO 2 laser excision. Both groups received an initial inoculation of a 0.25-cm piece of C1300 neuroblastoma tumor subcutaneously through the trocar method into the flank. Group 3, the control mice, received sham inoculation. On day 14, total excision by the selected method (groups 1 and 2) were carried out under pentobarbital anesthesia. Total body irradiation of 425 tad from a cobalt 60 source was delivered to all 45 animals to eliminate the primary immune response (day 15). Therefore, any host immunologic effect would be delivered from previous immunization and previously differentiated host factors. On day 17, 1 x 106 viable C1300 neuroblastoma cells suspended in tissue culture media were reinoculated into the opposite flank of all three groups. The new site was charted for tumor growth daily for 2 weeks. Antigenic ratios were calculated with the following formula~: Antigenic ratio = Growth of tumor in control mice (group 3) Growth of tumor in immunized mice (groups I and 2) If the value is >one, the response to the tumor is considered to be antigenic.
RESULTS Excision T h e r e is a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e s i z e o f the residual C1300 neuroblastoma excised with a
From the Department of Surgery, Rochester General Hospital and University of Rochester Medical Center, New York; and the Department of Surgery, Children's Hospital of Philadelphia. Address reprint requests to Carter J. McCormack, MD, 187 McKenzie Dr, Pittsburgh, PA 15235. 9 1989 by Grune & Stratton, Inc. 0022-3468/89/2402-0015503.00/0 201
McCORMACK ET AL
202
Table 1, Staging and Current Therapy of Neuroblastoma 22
Stage
Extent
Therapy
I
Confined to structure of
Surgery
II
origin Beyond origin, not across midline, • nodes same
Surgery • radiation
III
side Beyond origin, across midline, +_ nodes bilateral
IV IVS
Remote disease Stage I or II with remote dis-
~-
$CAPELEXCISION
2
20
18 16 14 12
Chemotherapy, surgery, radia-
tion Chemotherapy • surgery . Chemotherapy
10 a ll4-
ease in liver/skin/bone
2-
marrow
0 : 10
20
3O
~M[ (days)
scalpel and that excised with the CO2 laser on days 25 and 32 of the study, following 3/4 excision (Fig 1). The mean tumor diameter on day 25 was 13.6 _+ 1.2 mm (value _ SEM) for scalpel excision, and 9.4 _ 1.0 mm for CO2 laser excision (P < .01). The mean tumor size on day 32 showed a similar difference: 21.6 _+ 1.4 mm for scalpel excision compared with 13.1 _+ 1.5 mm for CO2 laser excision (P < .01). Although there were no CO2 laser reduced regressions of C 1300 murine neuroblastoma, four tumors did not increase in size during the study period following laser excision. CO2 laser excision caused less blood loss than scalpel excision without increasing operating time.
Antigenic Ratio Antigenic ratios were calculated when 75% of the control animals reached a tumor diameter of >5 mm. In group 1 (scalpel excision), this occurred on day 14 when the mean tumor diameter reached 5.1 ___0.6 mm. On day 14, the tumor diameter for group 2 animals (laser excision) was 3.3 ___ 0.7 mm, and for group 3 (controls) it was 5.9 _ 0.5 mm. The calculated antigenic ratio was 1.78 for CO2 laser excision and 1.15 for scalpel excision. This demonstrates that CO2 laser excision renders the C1300 neuroblastoma more immunogenic to the host. DISCUSSION
Seventy percent of children with neuroblastoma have stage III or IV disease when diagnosed. Outcome is dismal, with the 2-year survival rate for these stages being 10% to 40%. In stage III and IV, surgical resection is always incomplete and the operation can damage blood vessels and nerves encased by the tumor. Radiation and multiagent chemotherapy has prolonged the length of survival, but has not altered the outcome) The nutritional status of the child is important. In the animal model, poor nutrition may adversely affect the host antitumor immunologic response. 8 The elucidation of the immunologic response to
Fig 1. Growth of residual neuroblastoma following partial excision with a scalpel and CO 2 laser; 75% of the tumor was excised on day 19.
neuroblastoma is still in its infancy. Lymphocytes from children with neuroblastoma do inhibit tumor growth in vitro. 9 Children with neuroblastoma who failed to respond to surgical and/or adjuvant therapy produce a "blocking antibody" that inhibits the action of cytotoxic lymphocytes. 1~This blocking antibody is actually an antigen-antibody (IgG) immune complex that requires the presence of neuroblastoma to be active. "Killer" T lymphocyte activity is decreased in children with neuroblastoma. 11 Neuroblastoma surface antigens have been identified with the use of monoclonal antibodies. 12 Stimulation of the immune system with BCG extract and Coley's toxins has been reported to improve length of remission. 19J4 Electrocautery has been shown in mice to induce immunity to neuroblastoma and to potentiate the host antitumor response. 7 Unfortunately, there is no clear improvement in overall survival of children after immune stimulation. CO2 laser excision offers advantages of surgical precision and hemostasis, and it apparently increases tumor immunogenicity. The depth of tissue destruction is minimal, the same as that of the scalpel) 5 Laser excision avoids the wide heat effect of electrocautery, but retains the desirable ability to seal small blood vessels to provide hemostasis. Our data indicate that partial excision of murine C1300 neuroblastoma with the CO2 laser does significantly reduce residual tumor growth when compared with scalpel excision. This is not an effect of increased tumor kill in the residual portion because of the minimal depth of tissue destruction of the focused CO2 laser beam) 5 It appears to be an effect of increased tumor immunogenicity as demonstrated by the higher antigenic ratio after CO2 laser excision. The increased tumor immunogenicity may be a result of the process in which neuroblastoma antigens
CARBON DIOXIDE LASER EXCISION
203
are present to the host defense mechanisms. CO2 laser excision will vaporize n e u r o b l a s t o m a cells in direct contact with the beam, but will literally explode surr o u n d i n g ceils by boiling intracellular water. T h e surface antigens of n e u r o b l a s t o m a cell fragments m a y not be d e n a t u r e d in this process, a n d thus m a y be readily available for i m m u n e system detection a n d reaction. A majority of C1300 n e u r o b l a s t o m a cells are in a "resting" phase during progressive t u m o r growth. After excision there is a rapid burst of cell proliferation within 12 to 24 hours. As shown on the growth curves of Fig 1, CO2 laser excision decreases postexcisional t u m o r growth. A decrease in the growth phase would
explain the cessation of growth of four residual tumors after CO2 laser excision. This m a y result from a n i m m u n e - m e d i a t e d p h e n o m e n o n through a decrease in production of the blocking antibody, or it m a y be simply a function of cell kinetics. Partial CO2 laser excision of C1300 m u r i n e neuroblastoma decreases the s u b s e q u e n t growth rate of the residual tumor. T h e t u m o r appears to become more antigenic a n d to activate the host i m m u n e system. Laser excision is precise a n d provides a d e q u a t e hemostasis, both of which are i m p o r t a n t in the surgical t r e a t m e n t of neuroblastoma. W e believe the CO2 laser is a useful i n s t r u m e n t for p r i m a r y excision of neuroblastoma.
REFERENCES
1. Grosfeld JL: Neuroblastoma, in Welch KJ, Randolph JG, Raviteh MM, et al (eds): Pediatric Surgery chap 33 (ed 4). Chicago, Year Book Medical, 1986, pp 283-293 2. Everson TC, Cole WH: Spontaneous Regression of Cancer. Philadelphia, Saunders, 1966, pp 88-163 3. D'Angio GJ, Evons AE, Koop CE: Special pattern of widespread neuroblastoma with favorable prognosis. Lancet 1:7708:1046-1049, 1971 4. Azizkhan RG, Shaw A, Chandler JG: Surgical complications of neuroblastoma resection. Surgery 97:514-517, 1985 5. Lanzafame RJ, Rogers DW, Nain JO, et al: The effect of CO2 laser excision on local tumor recurrence. Lasers Surg Med. 6:103105, 1986 6. Lanzafame RJ, McCormack CJ, Rogers DW, et al: Effect of laser sterilization on local recurrence in experimental mammary tumors. Surg Forum ACS 37:480-482, 1986 7. Ziegler MM, Vega A, Koop CE: Electrocoagulation-induced immunity--Anexplanation for regressionof neuroblastoma. J Pediatr Surg 15:34-37, 1980 8. Ziegler MM, Kirby J, McCarrick JW Ili, et al: Neuroblastoma and nutritional support: Influenceon the host-tumor relationship. J Pediatr Surg 21:236-239, 1986
9. Hellstrom I, Hellstrom KE, Pierce GE, et al: Demonstration of cell-boundand humoral immunity against neuroblastoma cells. Proc Natl Acad Sci USA 60:123/-1238, 1968 10. Bill AH: Immune aspects of neuroblastoma. Am J Surg 122:142-147, 1971 11. Gerson JM, Heberman RB: Systemic and in-situ natural killer activity in patients with neuroblastoma, in Evans AE (ed): Advances in Neuroblastoma Research. New York, Raven, 1980, pp 251-260 12. Momoi M, Kennett RH, Glick MC: A surface glycoproteinas a human neuroblastoma antigen detected by monoclonalantibodies, in Evans AE (ed): Advances in Neuroblastoma Research. New York, Raven, 1980, pp 177-182 13. Fowler GA, Nauts HC: The apparently beneficial effect of concurrent infections, inflammation or fever and of bacterial toxin therapy on neuroblastoma. NY Cancer Res lnst, monograph no. 11, 1970 14. Necheles TF, Ravsen AR, Kung FH, et al: Immunochemotherapy in advanced neuroblastoma. Cancer 41:1282-1288, 1978 15. Hall RR, Beach AO, Baker E, et al: Incision of tissue by carbon dioxide laser. Nature 232:131-132, 1971
' E U R O B L A S T O M A , the most common extracranial malignant solid tumor in children, represents about 10% of all childhood malignancies and 25% to 50% of all malignancies in children <30 months of age. 1 Approximately 500 to 600 new cases occur annually in the United States. The overall disease-free survival of 37% reflects extensive local disease and widespread metastasis usually apparent at the time of diagnosis. 1 Neuroblastoma can regress spontaneously, most often in children < 1 year of age? This phenomenon may represent an immunologic body response to the tumor. Surgical excision has remained the primary therapy for stage I, II, and III disease (Table 1). 1'3 Adjuvant radiotherapy and chemotherapy have not significantly improved long-term survival. The complications from surgical excision can be devastating and may be more common than generally believed. 4 The goal is surgical excision without damage to locally involved important blood vessels and nerves. Animal model mammary tumors can be excised more precisely with the CO2 laser than with a scalpel. 5'~ The laser also seals small blood vessels and lymphatics. Tumor recurrence rates have been shown to decrease with the use of CO2 laser excision. 5 In this study, we attempted to determine whether the CO2 laser is useful in the excision of neuroblastoma, and if laser excision has any effect on the hosttumor immunologic relationship. The murine C1300
N
Journal of Pediatric Surgery,Vo124, No 2 (February), 1989: pp 201-203
neuroblastoma has many properties similar to human neuroblastoma, including antigenicity, which makes it an ideal animal model for this study. MATERIALS AND METHODS Fifty A / J male mice (Jackson Laboratories, Bar Harbor, ME) were used to study the effect of CO2 laser excision of neuroblastoma. C1300 neuroblastoma that had been serially maintained by tissue culture was implanted subcutaneoulsy by injection (approximately 1 million viable neuroblastoma cells). Tumor growth was recorded daily until tumor nodules measured 0.5 to 0.1 cm in diameter (day 19), and two groups of mice with similar mean tumor diameters and ranges were separated. At this time, 75 percent of each tumor was excised by a CO2 laser (n = 25) or scalpel (n = 25). For the next 2 weeks, the growth of residual tumor was measured daily. Laser excision was accomplished with a Sharplan 733 CO2 laser at 10 W, continuous mode, in focus with a 125-ram focal length lens system that gives a 0.2-mm spot size and yields a power density of 26,000 W/cm 2 at the target site. To study the host-tumor immunologic effect of COs laser excision of neuroblastoma, 45 A / J male mice were divided into three groups of 15 each. Group 1 animals had scalpel excision and group 2 had CO 2 laser excision. Both groups received an initial inoculation of a 0.25-cm piece of C1300 neuroblastoma tumor subcutaneously through the trocar method into the flank. Group 3, the control mice, received sham inoculation. On day 14, total excision by the selected method (groups 1 and 2) were carried out under pentobarbital anesthesia. Total body irradiation of 425 tad from a cobalt 60 source was delivered to all 45 animals to eliminate the primary immune response (day 15). Therefore, any host immunologic effect would be delivered from previous immunization and previously differentiated host factors. On day 17, 1 x 106 viable C1300 neuroblastoma cells suspended in tissue culture media were reinoculated into the opposite flank of all three groups. The new site was charted for tumor growth daily for 2 weeks. Antigenic ratios were calculated with the following formula~: Antigenic ratio = Growth of tumor in control mice (group 3) Growth of tumor in immunized mice (groups I and 2) If the value is >one, the response to the tumor is considered to be antigenic.
RESULTS Excision T h e r e is a s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e s i z e o f the residual C1300 neuroblastoma excised with a
From the Department of Surgery, Rochester General Hospital and University of Rochester Medical Center, New York; and the Department of Surgery, Children's Hospital of Philadelphia. Address reprint requests to Carter J. McCormack, MD, 187 McKenzie Dr, Pittsburgh, PA 15235. 9 1989 by Grune & Stratton, Inc. 0022-3468/89/2402-0015503.00/0 201
McCORMACK ET AL
202
Table 1, Staging and Current Therapy of Neuroblastoma 22
Stage
Extent
Therapy
I
Confined to structure of
Surgery
II
origin Beyond origin, not across midline, • nodes same
Surgery • radiation
III
side Beyond origin, across midline, +_ nodes bilateral
IV IVS
Remote disease Stage I or II with remote dis-
~-
$CAPELEXCISION
2
20
18 16 14 12
Chemotherapy, surgery, radia-
tion Chemotherapy • surgery . Chemotherapy
10 a ll4-
ease in liver/skin/bone
2-
marrow
0 : 10
20
3O
~M[ (days)
scalpel and that excised with the CO2 laser on days 25 and 32 of the study, following 3/4 excision (Fig 1). The mean tumor diameter on day 25 was 13.6 _+ 1.2 mm (value _ SEM) for scalpel excision, and 9.4 _ 1.0 mm for CO2 laser excision (P < .01). The mean tumor size on day 32 showed a similar difference: 21.6 _+ 1.4 mm for scalpel excision compared with 13.1 _+ 1.5 mm for CO2 laser excision (P < .01). Although there were no CO2 laser reduced regressions of C 1300 murine neuroblastoma, four tumors did not increase in size during the study period following laser excision. CO2 laser excision caused less blood loss than scalpel excision without increasing operating time.
Antigenic Ratio Antigenic ratios were calculated when 75% of the control animals reached a tumor diameter of >5 mm. In group 1 (scalpel excision), this occurred on day 14 when the mean tumor diameter reached 5.1 ___0.6 mm. On day 14, the tumor diameter for group 2 animals (laser excision) was 3.3 ___ 0.7 mm, and for group 3 (controls) it was 5.9 _ 0.5 mm. The calculated antigenic ratio was 1.78 for CO2 laser excision and 1.15 for scalpel excision. This demonstrates that CO2 laser excision renders the C1300 neuroblastoma more immunogenic to the host. DISCUSSION
Seventy percent of children with neuroblastoma have stage III or IV disease when diagnosed. Outcome is dismal, with the 2-year survival rate for these stages being 10% to 40%. In stage III and IV, surgical resection is always incomplete and the operation can damage blood vessels and nerves encased by the tumor. Radiation and multiagent chemotherapy has prolonged the length of survival, but has not altered the outcome) The nutritional status of the child is important. In the animal model, poor nutrition may adversely affect the host antitumor immunologic response. 8 The elucidation of the immunologic response to
Fig 1. Growth of residual neuroblastoma following partial excision with a scalpel and CO 2 laser; 75% of the tumor was excised on day 19.
neuroblastoma is still in its infancy. Lymphocytes from children with neuroblastoma do inhibit tumor growth in vitro. 9 Children with neuroblastoma who failed to respond to surgical and/or adjuvant therapy produce a "blocking antibody" that inhibits the action of cytotoxic lymphocytes. 1~This blocking antibody is actually an antigen-antibody (IgG) immune complex that requires the presence of neuroblastoma to be active. "Killer" T lymphocyte activity is decreased in children with neuroblastoma. 11 Neuroblastoma surface antigens have been identified with the use of monoclonal antibodies. 12 Stimulation of the immune system with BCG extract and Coley's toxins has been reported to improve length of remission. 19J4 Electrocautery has been shown in mice to induce immunity to neuroblastoma and to potentiate the host antitumor response. 7 Unfortunately, there is no clear improvement in overall survival of children after immune stimulation. CO2 laser excision offers advantages of surgical precision and hemostasis, and it apparently increases tumor immunogenicity. The depth of tissue destruction is minimal, the same as that of the scalpel) 5 Laser excision avoids the wide heat effect of electrocautery, but retains the desirable ability to seal small blood vessels to provide hemostasis. Our data indicate that partial excision of murine C1300 neuroblastoma with the CO2 laser does significantly reduce residual tumor growth when compared with scalpel excision. This is not an effect of increased tumor kill in the residual portion because of the minimal depth of tissue destruction of the focused CO2 laser beam) 5 It appears to be an effect of increased tumor immunogenicity as demonstrated by the higher antigenic ratio after CO2 laser excision. The increased tumor immunogenicity may be a result of the process in which neuroblastoma antigens
CARBON DIOXIDE LASER EXCISION
203
are present to the host defense mechanisms. CO2 laser excision will vaporize n e u r o b l a s t o m a cells in direct contact with the beam, but will literally explode surr o u n d i n g ceils by boiling intracellular water. T h e surface antigens of n e u r o b l a s t o m a cell fragments m a y not be d e n a t u r e d in this process, a n d thus m a y be readily available for i m m u n e system detection a n d reaction. A majority of C1300 n e u r o b l a s t o m a cells are in a "resting" phase during progressive t u m o r growth. After excision there is a rapid burst of cell proliferation within 12 to 24 hours. As shown on the growth curves of Fig 1, CO2 laser excision decreases postexcisional t u m o r growth. A decrease in the growth phase would
explain the cessation of growth of four residual tumors after CO2 laser excision. This m a y result from a n i m m u n e - m e d i a t e d p h e n o m e n o n through a decrease in production of the blocking antibody, or it m a y be simply a function of cell kinetics. Partial CO2 laser excision of C1300 m u r i n e neuroblastoma decreases the s u b s e q u e n t growth rate of the residual tumor. T h e t u m o r appears to become more antigenic a n d to activate the host i m m u n e system. Laser excision is precise a n d provides a d e q u a t e hemostasis, both of which are i m p o r t a n t in the surgical t r e a t m e n t of neuroblastoma. W e believe the CO2 laser is a useful i n s t r u m e n t for p r i m a r y excision of neuroblastoma.
REFERENCES
1. Grosfeld JL: Neuroblastoma, in Welch KJ, Randolph JG, Raviteh MM, et al (eds): Pediatric Surgery chap 33 (ed 4). Chicago, Year Book Medical, 1986, pp 283-293 2. Everson TC, Cole WH: Spontaneous Regression of Cancer. Philadelphia, Saunders, 1966, pp 88-163 3. D'Angio GJ, Evons AE, Koop CE: Special pattern of widespread neuroblastoma with favorable prognosis. Lancet 1:7708:1046-1049, 1971 4. Azizkhan RG, Shaw A, Chandler JG: Surgical complications of neuroblastoma resection. Surgery 97:514-517, 1985 5. Lanzafame RJ, Rogers DW, Nain JO, et al: The effect of CO2 laser excision on local tumor recurrence. Lasers Surg Med. 6:103105, 1986 6. Lanzafame RJ, McCormack CJ, Rogers DW, et al: Effect of laser sterilization on local recurrence in experimental mammary tumors. Surg Forum ACS 37:480-482, 1986 7. Ziegler MM, Vega A, Koop CE: Electrocoagulation-induced immunity--Anexplanation for regressionof neuroblastoma. J Pediatr Surg 15:34-37, 1980 8. Ziegler MM, Kirby J, McCarrick JW Ili, et al: Neuroblastoma and nutritional support: Influenceon the host-tumor relationship. J Pediatr Surg 21:236-239, 1986
9. Hellstrom I, Hellstrom KE, Pierce GE, et al: Demonstration of cell-boundand humoral immunity against neuroblastoma cells. Proc Natl Acad Sci USA 60:123/-1238, 1968 10. Bill AH: Immune aspects of neuroblastoma. Am J Surg 122:142-147, 1971 11. Gerson JM, Heberman RB: Systemic and in-situ natural killer activity in patients with neuroblastoma, in Evans AE (ed): Advances in Neuroblastoma Research. New York, Raven, 1980, pp 251-260 12. Momoi M, Kennett RH, Glick MC: A surface glycoproteinas a human neuroblastoma antigen detected by monoclonalantibodies, in Evans AE (ed): Advances in Neuroblastoma Research. New York, Raven, 1980, pp 177-182 13. Fowler GA, Nauts HC: The apparently beneficial effect of concurrent infections, inflammation or fever and of bacterial toxin therapy on neuroblastoma. NY Cancer Res lnst, monograph no. 11, 1970 14. Necheles TF, Ravsen AR, Kung FH, et al: Immunochemotherapy in advanced neuroblastoma. Cancer 41:1282-1288, 1978 15. Hall RR, Beach AO, Baker E, et al: Incision of tissue by carbon dioxide laser. Nature 232:131-132, 1971