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Direct prenatal chromosome diagnosis of a malignancy
Direct Prenatal Chromosome Diagnosis of a Malignancy Frederick Hecht, Arthur Grix, Jr., Barbara K. Hecht, Carol Berger, Helen Bixenman, Susan Szucs, Daniel O'Keeffe, and Harris J. Finberg
ABSTRACT: A fetal tumor was suspected at 31 weeks of gestation. The occurrence of polyhydramnios led to an ultrasound examination, which revealed deformation of the fetal head, face, eye, and neck. This was confirmed by computerized tomography. Amniocentesis yielded cells with an inverted duplication of chromosome #1. This abnormality of chromosome #1 marked the malignant teratoma cells in the amniotic fluid. Cytogenetic analysis of tumor tissue and of normal tissue obtained postnatally confirmed that the abnormality of chromosome #1 observed in amniotic fluid cells was confined to the tumor. The constitutional karyotype was normal. To our knowledge, this is the first report of the direct chromosomal detection of malignancy before birth.
INTRODUCTION Certain constitutional c h r o m o s o m e abnormalities predispose the affected individual to a malignancy. For example, trisomy #21 (Dawn's syndrome) carries with it a markedly increased risk of leukemia, so that prenatal diagnosis of trisomy #21 implies a heightened risk of leukemia. Similarly, prenatal diagnosis of a partial deletion of the short (p) arm of c h r o m o s o m e #11 or of the long (q) arm of chromosome #13 dispose to an elevated risk of Wilm's tumor and retinoblastoma, respectively. However, the antenatal diagnosis of such constitutional c h r o m o s o m e anomalies does not constitute a direct finding of the malignancy itself, merely of the increased risk. We report the prenatal cytogenetic diagnosis of an intracranial teratoma that had undergone malignant transformation, as e v i d en ced by a rearrangement of chromosome #1 within the tumor cells. This rearrangement was found in amniotic fluid cells derived by desquamation of teratoma cells into amniotic fluid.
CASE R E P O R T
A 28-year-old w o m a n (Genetics Center no. 14441) was gravida 3, para 0, spontaneous abortions 2. At 28 weeks of gestation, p o l y h y d r a m n i o s was noted, and at 31 weeks she was referred for evaluation. From the Genetics and Cancer Centers, Southwest Biomedical Research Institute, Tempe, AZ and Perinatal Medicine and Radiology, Good Samaritan Hospital, Phoenix, AZ. Address requests for reprints to Dr. Frederick Hecht, Genetics Center of Southwest Biomedical Research Institute, 123 East University Drive, Tempe, AZ 85281. Received January 14, 1983; accepted March 17, 1983.
107 ~ 1984 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 11, 107 111 (1984) 0165-4608/84/$03.00
108
F. Hecht et al.
The w o m a n had 13 siblings, including a set of identical twins, and her mate had 10 siblings, all of w h o m were living and well. There was no consanguinity. The wo m an was white and her mate was black. At 31 weeks gestation, ultrasound confirmed a marked excess of amniotic fluid. The fetal biparietal diameter was 87 ram, w h i c h is abnormally large for this point in gestation, while the m a x i m u m transverse diameter of the ab d o m en was 75 ram. resulting a head d i a m e t e r : a b d o m i n a l ratio of 1:1.15, well above the normal range. The fetal facial features were abnormal, with a round soft tissue mass containing solid and cystic components extending from the face and neck. X-rays of supine and oblique projections of the mother's abdomen showed an extensive bone defect in the fetal skull, involving the orbit and frontal parietal regions. Computerized tomography was utilized to obtain a limited series of contiguous 1-cm thick images of the fetal cranium. No bone was recognized in the area of the left orbit. The osseous defect also involved the left lateral face. A soft tissue mass was observed. This mass displaced part of the sphenoid bone into the brain. Amniocentesis was performed, and 580 cc bloody fluid was removed. The laboratory studies of the fluid and cells in the fluid are described below. A baby girl was delivered at 31.5 weeks of gestation. Apgar scores were 1 at 1 min and 1 at 5 min. The baby expired soon after birth.
LABORATORY STUDIES Alpha-fetoprotein (AFP) in amniotic fluid was 2740 IU/ml. This level is w i t h i n normal limits, up to 3310 IU/ml in our laboratory for this gestational age (see Discussion). Gel electrophoresis of amniotic fluid was performed for acetylcholinesterase (ACHE) [1]. Normally, AChE is not detectable in amniotic fluid, and no AChE was detected in this case. Amniotic fluid cells were cultured in situ on coverstips, with each coverslip in a separate culttire dish [2]. Fifteen colonies were examined. All cells were found to contain a partial duplication of c h r o m o s o m e #1 (Fig. 1). The duplication involved part of lq. The segment of l q extending from l q t e r to lq21 was duplicated, inverted, and attached to l p at band 1p35, so that the resultant c h r o m o s o m e formula was: 46,XX,inv dup(1)(qter--+q21 :: p35--+qter). A skin biopsy from over the fetal sternum was obtained after the demise of the patient. The biopsy was cultured and cells harvested after 17 days. Thirty cells were examined, and all had a normal 46,XX karyotype. Tumor tissue was sectioned with a Stadie-Riggs microtome, and a single cell suspension was obtained by enzymatic disaggregation with DNAase I and collagenase [3]. Cells were cultured in suspension for 48 hr and yielded 16 metaphases: 8 with a normal 46,XX karyotype and 8 with the partial duplication of c h r o m o s o m e #1. Cells cultured in monolayer for 72 hr yielded 12 metaphases: 11 normal and 1 with partial duplication of c h r o m o s o m e #1. Additional tumor tissue was minced into small pieces and explants established in culture. Cells harvested after 12 days yielded 45 metaphases, all of which were chromosomally normal. AUTOPSY The baby measured 24 cm (crown to rump) and 36 cm (rump to heel). Head circumference was 31.5 cm. The tumor deformed the left head, face, and neck, and the left eye and eyelid were involved. The cranial sutures were widely separated and the cerebral hemispheres were
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displaced to the right by the tumor, which occupied the basal portion of the cranial fossa in the m i d l i n e extending into the left eye and face. The oral cavity was obstructed by the tumor. The tumor was a teratoma with elements derived from all three germ layers. Immature areas were seen containing n u m e r o u s mitotic figures and cells resembling neuroblasts. The tumor measured 10 cm in diameter and weighed 210 g. By comparison, the baby (minus the tumor) weighed 1365 g. DISCUSSION This intracranial teratoma was large. It was equal in weight to 15% of the baby's total tumorless weight (210 g/1365 g = 15%). The malignant teratoma cells had an inverted partial duplication of chromosome #1. The amniotic fluid cells with this chromosome #1 abnormality reflected desquamation of malignant cells, probably via the left orbit and/or oral cavity, into amniotic fluid. Analysis of tumor tissue in vitro after 48 hr showed a 1 : 1 mix of chromosomally n o r m a l : a b n o r m a l cells. After 72 hr there was a marked p r e d o m i n a n c e of normal cells and, after 12 days in culture, only cells with normal chromosomes were ohserved. Thus, in vitro, the cells with normal chromosomes had a selective growth advantage. The amniotic fluid AFP level appeared normal. However, there was a great excess of amniotic fluid. Assuming, conservatively, that the amniotic fluid volume was twice normal, the AFP was clearly elevated. This is in keeping with reports of sacrococcygeal teratomas [4, 5] and an intracranial teratoma [6] associated with high amniotic fluid AFP levels. In all probability, AChE might also have been detectable by gel electrophoresis in this case, were it not for dilution by polyhydramnios. This is not the first case with an intracranial teratoma to be suspected antenatally. Other cases [6-8] have been suspected by ultrasound. The case observed by Saul [6] is particularly reminiscent of ours in that the intracranial teratoma proved to involve the oral cavity of the fetus and to contain p r e d o m i n a n t l y neural elements. At 24-weeks gestation, the tumor was still benign and had normal chromosomes [6]. It is surely an u n c o m m o n event for fetal tumor cells to desquamate into the anmiotic fluid. Most tumors and leukemias spread and shed internally. They are not, therefore, amenable to detection by cytogenetic analysis of amniotic fluid cells. Possible exceptions might i n c l u d e (a) sacrococcygeal teratomas, (b) embryonal yolk sac tumors, and (c) Wilms's tumors of the kidney. Sacrococcygeal teratomas and embryonal yolk sac tumors are located at the surface of the fetus so cells from these cancers may desquamate into amniotic fluid. Wilms's tumors may invade the renal pelvis and shed tumor cells into fetal urine and thence into amniotic fluid. The fetus in this case had a normal constitutional karyotype. The normal 46,XX karyotype was observed in certain cells cultured from the tumor postnatally and in all cells cultured from a fetal skin biopsy. The chromosome #1 abnormality signaled the teratoma cells in amniotic fluid prenatally and in tumor tissue postnatally. The chromosome anomaly itself is a partial duplication of lq. This actually represents two abnormalities: partial duplication (partial trisomy) of lq (1qter--~1q21), and partial deletion (partial monosomy) of l p (lpter--~1p35). Clearly, partial duplication of l q is found in a wide variety of malignant diseases [9]. It is a n o n r a n d o m chromosome abnormality in h u m a n cancer cells, associated
Prenatal Chromosome Diagnosis
111
w i t h c l o n a l e v o l u t i o n of t h e k a r y o t y p e a n d p r o g r e s s i v e m a l i g n a n t d i s e a s e i n m a n y
cases. Partial deletion of l p is less common in cancer cells. It has been identified as the most consistent finding to date on a chromosome level in human neuroblastomas [10]. In light of this, it is interesting that the tumor in this case had "numerous mitotic figures and cells resembling neuroblasts." Neural elements are common in teratomas. The key cytogenetic event associated with this malignant transformation was partial deletion of lp, partial duplication of lq, and juxtaposition of band lq21 with band ip35. The rearrangement brought together segments of chromosome # I that are normally separated from one another. This is the first case of which fetal cancer cells have been cytogenetically detected in amniotic fluid. Predictably, it will not be the last, thanks to cytogenetic analysis of amniocytic fluid cells; clearly, other cases will be discovered and be of comparable or even greater interest. Supported in part by NCI Grant CA-25055 for research on Cytogenetics of Clonal Neoplasias. We thank the family who suffered this tragedy for their assistance and invaluable cooperation.
REFERENCES 1. Smith AD, Wald NJ, Cuckle HS, Stirrat GM, Bobrow M, Lagercrantz H (1979): Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy. Lancet 1,685-688. 2. Hecht F, Peakman D, Kaiser-McCaw B (1981): Amniocyte clones for prenatal cytogenetics. Am I Med Genet 10, 51-54. 3. Wake N, Slocum H, Rustum Y, Matsui S, Sandberg AA (1981): Chromosomes and causation of h u m a n cancer and leukemia. XLIV. A method for chromosome analysis of solid tumors. Cancer Genet Cytogenet 3, 1-10. 4. Schmid W, Muhlethaler JP (1975): High amniotic fluid alpah-l-fetoprotein in a case of fetal sacrococcygeal teratoma. Humangenetik 26, 353-354. 5. Hecht F, Hecht BK, O'Keeffe D (1982): Sacrococcygeal teratoma: Prenatal diagnosis with elevated alpha-fetoprotein and acetylcholinesterase in amniotic fluid. Prenatal Diag 2, 229-231. 6. Saul RA (1982): Prenatal documentation of craniofacial teratomas. Proc Greenwood Genetic Center 1, 34-35. 7. Hoff NR, Mackay IM (1980): Prenatal ultrasound diagnosis of intracranial teratoma. J Clin Ultrasound 8, 247-248. 8. Paes BA, deSA DJ, Hunter DJS, Pirani M (1982): Benign intracranial teratoma. Prenatal diagnosis influencing early delivery. Am J Obstet Gynecol 143, 600-601. 9. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier NorthHolland, New York. 10. Brodeur GM, Green AA, Hayes FA (1980): Cytogenetic studies in primary h u m a n neuroblastomas. In: Advances in Neuroblastoma Research, A Evans, ed. Raven, New York, pp. 73-89.
ABSTRACT: A fetal tumor was suspected at 31 weeks of gestation. The occurrence of polyhydramnios led to an ultrasound examination, which revealed deformation of the fetal head, face, eye, and neck. This was confirmed by computerized tomography. Amniocentesis yielded cells with an inverted duplication of chromosome #1. This abnormality of chromosome #1 marked the malignant teratoma cells in the amniotic fluid. Cytogenetic analysis of tumor tissue and of normal tissue obtained postnatally confirmed that the abnormality of chromosome #1 observed in amniotic fluid cells was confined to the tumor. The constitutional karyotype was normal. To our knowledge, this is the first report of the direct chromosomal detection of malignancy before birth.
INTRODUCTION Certain constitutional c h r o m o s o m e abnormalities predispose the affected individual to a malignancy. For example, trisomy #21 (Dawn's syndrome) carries with it a markedly increased risk of leukemia, so that prenatal diagnosis of trisomy #21 implies a heightened risk of leukemia. Similarly, prenatal diagnosis of a partial deletion of the short (p) arm of c h r o m o s o m e #11 or of the long (q) arm of chromosome #13 dispose to an elevated risk of Wilm's tumor and retinoblastoma, respectively. However, the antenatal diagnosis of such constitutional c h r o m o s o m e anomalies does not constitute a direct finding of the malignancy itself, merely of the increased risk. We report the prenatal cytogenetic diagnosis of an intracranial teratoma that had undergone malignant transformation, as e v i d en ced by a rearrangement of chromosome #1 within the tumor cells. This rearrangement was found in amniotic fluid cells derived by desquamation of teratoma cells into amniotic fluid.
CASE R E P O R T
A 28-year-old w o m a n (Genetics Center no. 14441) was gravida 3, para 0, spontaneous abortions 2. At 28 weeks of gestation, p o l y h y d r a m n i o s was noted, and at 31 weeks she was referred for evaluation. From the Genetics and Cancer Centers, Southwest Biomedical Research Institute, Tempe, AZ and Perinatal Medicine and Radiology, Good Samaritan Hospital, Phoenix, AZ. Address requests for reprints to Dr. Frederick Hecht, Genetics Center of Southwest Biomedical Research Institute, 123 East University Drive, Tempe, AZ 85281. Received January 14, 1983; accepted March 17, 1983.
107 ~ 1984 by Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 10017
Cancer Genetics and Cytogenetics 11, 107 111 (1984) 0165-4608/84/$03.00
108
F. Hecht et al.
The w o m a n had 13 siblings, including a set of identical twins, and her mate had 10 siblings, all of w h o m were living and well. There was no consanguinity. The wo m an was white and her mate was black. At 31 weeks gestation, ultrasound confirmed a marked excess of amniotic fluid. The fetal biparietal diameter was 87 ram, w h i c h is abnormally large for this point in gestation, while the m a x i m u m transverse diameter of the ab d o m en was 75 ram. resulting a head d i a m e t e r : a b d o m i n a l ratio of 1:1.15, well above the normal range. The fetal facial features were abnormal, with a round soft tissue mass containing solid and cystic components extending from the face and neck. X-rays of supine and oblique projections of the mother's abdomen showed an extensive bone defect in the fetal skull, involving the orbit and frontal parietal regions. Computerized tomography was utilized to obtain a limited series of contiguous 1-cm thick images of the fetal cranium. No bone was recognized in the area of the left orbit. The osseous defect also involved the left lateral face. A soft tissue mass was observed. This mass displaced part of the sphenoid bone into the brain. Amniocentesis was performed, and 580 cc bloody fluid was removed. The laboratory studies of the fluid and cells in the fluid are described below. A baby girl was delivered at 31.5 weeks of gestation. Apgar scores were 1 at 1 min and 1 at 5 min. The baby expired soon after birth.
LABORATORY STUDIES Alpha-fetoprotein (AFP) in amniotic fluid was 2740 IU/ml. This level is w i t h i n normal limits, up to 3310 IU/ml in our laboratory for this gestational age (see Discussion). Gel electrophoresis of amniotic fluid was performed for acetylcholinesterase (ACHE) [1]. Normally, AChE is not detectable in amniotic fluid, and no AChE was detected in this case. Amniotic fluid cells were cultured in situ on coverstips, with each coverslip in a separate culttire dish [2]. Fifteen colonies were examined. All cells were found to contain a partial duplication of c h r o m o s o m e #1 (Fig. 1). The duplication involved part of lq. The segment of l q extending from l q t e r to lq21 was duplicated, inverted, and attached to l p at band 1p35, so that the resultant c h r o m o s o m e formula was: 46,XX,inv dup(1)(qter--+q21 :: p35--+qter). A skin biopsy from over the fetal sternum was obtained after the demise of the patient. The biopsy was cultured and cells harvested after 17 days. Thirty cells were examined, and all had a normal 46,XX karyotype. Tumor tissue was sectioned with a Stadie-Riggs microtome, and a single cell suspension was obtained by enzymatic disaggregation with DNAase I and collagenase [3]. Cells were cultured in suspension for 48 hr and yielded 16 metaphases: 8 with a normal 46,XX karyotype and 8 with the partial duplication of c h r o m o s o m e #1. Cells cultured in monolayer for 72 hr yielded 12 metaphases: 11 normal and 1 with partial duplication of c h r o m o s o m e #1. Additional tumor tissue was minced into small pieces and explants established in culture. Cells harvested after 12 days yielded 45 metaphases, all of which were chromosomally normal. AUTOPSY The baby measured 24 cm (crown to rump) and 36 cm (rump to heel). Head circumference was 31.5 cm. The tumor deformed the left head, face, and neck, and the left eye and eyelid were involved. The cranial sutures were widely separated and the cerebral hemispheres were
x
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0
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,o
0
o®
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O,
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gL~
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~e
0
r~
o ~ .~>
~0
O, •~
~
110
F. Hecht et el.
displaced to the right by the tumor, which occupied the basal portion of the cranial fossa in the m i d l i n e extending into the left eye and face. The oral cavity was obstructed by the tumor. The tumor was a teratoma with elements derived from all three germ layers. Immature areas were seen containing n u m e r o u s mitotic figures and cells resembling neuroblasts. The tumor measured 10 cm in diameter and weighed 210 g. By comparison, the baby (minus the tumor) weighed 1365 g. DISCUSSION This intracranial teratoma was large. It was equal in weight to 15% of the baby's total tumorless weight (210 g/1365 g = 15%). The malignant teratoma cells had an inverted partial duplication of chromosome #1. The amniotic fluid cells with this chromosome #1 abnormality reflected desquamation of malignant cells, probably via the left orbit and/or oral cavity, into amniotic fluid. Analysis of tumor tissue in vitro after 48 hr showed a 1 : 1 mix of chromosomally n o r m a l : a b n o r m a l cells. After 72 hr there was a marked p r e d o m i n a n c e of normal cells and, after 12 days in culture, only cells with normal chromosomes were ohserved. Thus, in vitro, the cells with normal chromosomes had a selective growth advantage. The amniotic fluid AFP level appeared normal. However, there was a great excess of amniotic fluid. Assuming, conservatively, that the amniotic fluid volume was twice normal, the AFP was clearly elevated. This is in keeping with reports of sacrococcygeal teratomas [4, 5] and an intracranial teratoma [6] associated with high amniotic fluid AFP levels. In all probability, AChE might also have been detectable by gel electrophoresis in this case, were it not for dilution by polyhydramnios. This is not the first case with an intracranial teratoma to be suspected antenatally. Other cases [6-8] have been suspected by ultrasound. The case observed by Saul [6] is particularly reminiscent of ours in that the intracranial teratoma proved to involve the oral cavity of the fetus and to contain p r e d o m i n a n t l y neural elements. At 24-weeks gestation, the tumor was still benign and had normal chromosomes [6]. It is surely an u n c o m m o n event for fetal tumor cells to desquamate into the anmiotic fluid. Most tumors and leukemias spread and shed internally. They are not, therefore, amenable to detection by cytogenetic analysis of amniotic fluid cells. Possible exceptions might i n c l u d e (a) sacrococcygeal teratomas, (b) embryonal yolk sac tumors, and (c) Wilms's tumors of the kidney. Sacrococcygeal teratomas and embryonal yolk sac tumors are located at the surface of the fetus so cells from these cancers may desquamate into amniotic fluid. Wilms's tumors may invade the renal pelvis and shed tumor cells into fetal urine and thence into amniotic fluid. The fetus in this case had a normal constitutional karyotype. The normal 46,XX karyotype was observed in certain cells cultured from the tumor postnatally and in all cells cultured from a fetal skin biopsy. The chromosome #1 abnormality signaled the teratoma cells in amniotic fluid prenatally and in tumor tissue postnatally. The chromosome anomaly itself is a partial duplication of lq. This actually represents two abnormalities: partial duplication (partial trisomy) of lq (1qter--~1q21), and partial deletion (partial monosomy) of l p (lpter--~1p35). Clearly, partial duplication of l q is found in a wide variety of malignant diseases [9]. It is a n o n r a n d o m chromosome abnormality in h u m a n cancer cells, associated
Prenatal Chromosome Diagnosis
111
w i t h c l o n a l e v o l u t i o n of t h e k a r y o t y p e a n d p r o g r e s s i v e m a l i g n a n t d i s e a s e i n m a n y
cases. Partial deletion of l p is less common in cancer cells. It has been identified as the most consistent finding to date on a chromosome level in human neuroblastomas [10]. In light of this, it is interesting that the tumor in this case had "numerous mitotic figures and cells resembling neuroblasts." Neural elements are common in teratomas. The key cytogenetic event associated with this malignant transformation was partial deletion of lp, partial duplication of lq, and juxtaposition of band lq21 with band ip35. The rearrangement brought together segments of chromosome # I that are normally separated from one another. This is the first case of which fetal cancer cells have been cytogenetically detected in amniotic fluid. Predictably, it will not be the last, thanks to cytogenetic analysis of amniocytic fluid cells; clearly, other cases will be discovered and be of comparable or even greater interest. Supported in part by NCI Grant CA-25055 for research on Cytogenetics of Clonal Neoplasias. We thank the family who suffered this tragedy for their assistance and invaluable cooperation.
REFERENCES 1. Smith AD, Wald NJ, Cuckle HS, Stirrat GM, Bobrow M, Lagercrantz H (1979): Amniotic fluid acetylcholinesterase as a possible diagnostic test for neural tube defects in early pregnancy. Lancet 1,685-688. 2. Hecht F, Peakman D, Kaiser-McCaw B (1981): Amniocyte clones for prenatal cytogenetics. Am I Med Genet 10, 51-54. 3. Wake N, Slocum H, Rustum Y, Matsui S, Sandberg AA (1981): Chromosomes and causation of h u m a n cancer and leukemia. XLIV. A method for chromosome analysis of solid tumors. Cancer Genet Cytogenet 3, 1-10. 4. Schmid W, Muhlethaler JP (1975): High amniotic fluid alpah-l-fetoprotein in a case of fetal sacrococcygeal teratoma. Humangenetik 26, 353-354. 5. Hecht F, Hecht BK, O'Keeffe D (1982): Sacrococcygeal teratoma: Prenatal diagnosis with elevated alpha-fetoprotein and acetylcholinesterase in amniotic fluid. Prenatal Diag 2, 229-231. 6. Saul RA (1982): Prenatal documentation of craniofacial teratomas. Proc Greenwood Genetic Center 1, 34-35. 7. Hoff NR, Mackay IM (1980): Prenatal ultrasound diagnosis of intracranial teratoma. J Clin Ultrasound 8, 247-248. 8. Paes BA, deSA DJ, Hunter DJS, Pirani M (1982): Benign intracranial teratoma. Prenatal diagnosis influencing early delivery. Am J Obstet Gynecol 143, 600-601. 9. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier NorthHolland, New York. 10. Brodeur GM, Green AA, Hayes FA (1980): Cytogenetic studies in primary h u m a n neuroblastomas. In: Advances in Neuroblastoma Research, A Evans, ed. Raven, New York, pp. 73-89.