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Translocation (19;?) in two stage II neuroblastomas
Translocation (19;?) in Two Stage II Neuroblastomas Folker Franke, B irbel Rudolph, and Fritz Lampert
ABSTRACT: Chromosomes of two abdominal neuroblastomas (stage II) from a lO-month-old male infant and a 6-year-old girl were analyzed. A modal chromosome number of 70 and 46, respectively, was found. Neither double minute chromosomes (DM) or homogeneously staining regions (HSR), nor visible structural abnormalities of chromosome #1 could be detected. However, a common aberration was found to be a translocation on the short arm of chromosome #19. After tumor resection, both children are alive without any evidence of tumor disease.
INTRODUCTION Chromosome abnormalities of h u m a n tumor cells, more or less n o n r a n d o m and specific, have been found in increasing n u m b e r s of malignancies [1] and can help in classifying tumor subgroups or in defining clinical prognosis [2, 3]. In h u m a n neuroblastoma, double minutes (DM), homogeneously staining regions (HSR), and, most frequently, a deletion and/or translocation of the short arm of chromosome # 1 have been described, but almost uniformly in tissue cultures or samples taken from metastases or primary tumors of stage III or IV [4-6]. These unresectable or metastatic neuroblastomas have a very poor prognosis in patients over 1 year of age, with almost no chance of survival despite an aggressive chemotherapy. In contrast, cytogenetically, little is k n o w n about neuroblastomas of stages I and II, which are localized and resectable. Using a simple procedure to accumulate a sufficient a m o u n t of mitotic cells, we succeeded analyzing two samples of these localized tumors. PATIENTS Case 1
T. T., a 10-month-old male infant, was found to have a right-sided abdominal tumor during a routine examination. Laboratory values at diagnosis (10/27/83) were hemoglobin 12 g/dl, thrombocytes 616.0/nl, and leukocytes 15.6/nl. Urinary vanil
From the OncocytogeneticLaboratory,Kinderpoliklinik,Universityof Giessen,West Germany. Address requests for reprints to Dr. Fritz Lampert, Universitiits Kinderpoliklinik, Feulgenstra[~e 12, D-6300 Giessen, West Germany. This article is part of F. Franke's doctoral thesis. Received January 28, 1985; accepted April 15, 1985.
129 © 1986 Elsevier Science PublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet20:129-135 (1986) 0165-4608/86/$03.50
130
F. Franke et al.
mandelic acid, homovanillic acid, and dopamine were elevated. Bone marrow aspiration was normal. The fist-sized tumor with three adjacent lymph nodes was excised in toto at the Children's Hospital Dortmund (we thank Dr. Strehl for kindly providing us with tumor material). Histologically, neuroblastoma tissue, including neurofibrillar elements, pseudorosettes, and some differentiation in rare parts were seen (Hughes grade 2). Two of the lymph nodes were infiltrated with tumor tissue. In touch preparations, the neuroblastoma cell population was seen to be dispersed with small lymphocytes. Because of regional lymph node metastases, this patient with abdominal neuroblastoma stage II (Evans) was treated postoperatively with three chemotherapy cycles (one every 3 weeks), according to the NB 79 protocol of the German Society of Pediatric Oncology [7]. Cytostatic blocks consisted of adriamycin 35 mg/m 2, day 1, and cyclophosphamide 150 mg/m 2, days 1-7. Except for some vomiting, the patient tolerated this mild and short-term chemotherapy well and remains in good health with no evidence of tumor disease, now more than 2 years after operation. Case 2
R. Z., a 6-year-old girl, complained of occasional abdominal pain of a few months duration. During a routine medical check-up at school, a fist-sized, left-sided abdominal tumor was palpated. Laboratory values on admission (4/27/83) were hemoglobin 13.5 g/dl, thrombocytes 194.0/nl, leukocytes 7.0/nl, serum lactate dehydrogenase 309IU/L; and serum ferritin 26 ng/ml. Urinary catecholamines were slightly elevated: vanil mandelic acid 22.4 ~mol/day (12.6 nmol/~mol Kreatinin) and homovanillic acid 33.4 o~mol/ day (18.8 nmol/~mol Kreatinin). Bone marrow aspiration was normal. Upon surgery (4/28/83), the tumor, measuring 10 cm in diameter, and three adjacent lymph nodes were excised in total. Histologically undifferentiated neuroblastoma tissue with focal differentiation (Hughes grade 2) was seen. One of the excised lymph nodes was also infiltrated. In touch preparations, typical neuroblastoma cells were seen, as were pseudorosettes and a few dispersed lymphocytes. Because of regional lymph node infiltration, this patient with stage II abdominal neuroblastoma was treated with three chemotherapy cycles postoperatively, as described in case 1. The patient tolerated this mild and short-term chemotherapy very well and remains in good health with no evidence of tumor disease up to the present day (2 years after operation). MATERIALS AND METHODS
Attempts to collect analyzable tumor metaphases were successful in two of five patients with neuroblastoma of stage I or II. Tissue samples were taken from tumors immediately after their surgical excision, These samples were cleaned, washed in Hanks' balanced salt solution (BSS), transsected into small pieces, and transferred into Falcon culture flasks (25 cm3). As culture medium, RPMI 1640 plus 15% fetal calf serum plus 50 U/ml penicillin and 50 ~g/ml streptomycin was used. Different culture times at 37°C (3 hr; 3, 5, and 8 days) and different methods for chromosome preparation were applied. A 5-day culture in combination with a subsequent 17-hr cell synchronization with 10-7M methotrexate yielded the best results regarding mitotic index, as well as quality of metaphases. Two washing procedures were followed by a further 6 hr "releasing" of the tumor cells by addition of 10-5M thymidine. During the last 2 hr
~'igure 1
tB I!
19
rT
J
4/149
20
14
7
15
21
8
2
22
9
1
3
16
10
?
Near-triploid metaphase of patient TT-018 showing two marker chromosomes (arrows).
13
3
6
I,,ll,
J
7
iiii
11
K
18
12
13 2
F. Franke et al. of this period, colcemide was a d d e d in a concentration of 0.01 ~g/ml. Further preparation c o r r e s p o n d e d to previously p u b l i s h e d methods [6]. In addition, the chromosomes of p e r i p h e r a l blood l y m p h o c y t e s of both patients were analyzed using a modified Yunis technique [8].
RESULTS The majority of the tumor cells of patient T. T. revealed a near-triploid chromosome set (Fig. 1). Besides a small marker chromosome, del(4)(q2)?, a translocation of material of u n k n o w n origin was found as a constant aberration on the short arm (very likely) of a chromosome #19 (Fig. 1). The aberration of c h r o m o s o m e #19 was also consistently found in the tumor metaphases of patient R. Z. and appears to be very similar in structure to that of T. T. The involvement of the short arm of c h r o m o s o m e # 1 9 in a translocation with breakpoint p13.1 is illustrated in Figure 2,A and B. Neither the origin of the translocated chromosome material nor the remaining segment 19p13.1--~pter could be located in the preparations examined. On the other hand, the translocation (19;?) in six complete karyotypable p s e u d o d i p l o i d metaphases was the only striking aberration. Eight of 61 analyzed tumor metaphases showed a secondary translocation on the short arm of c h r o m o s o m e #7, w h i c h was most likely a t(7;21) (Fig. 3). Table 1 summarizes the cytogenetic data of both cases. It is noteworthy that we did not find a n y of the abnormalities hitherto described to be typical for metastasizing neuroblastomas [9]. Neither abnormal changes of c h r o m o s o m e s l p , lq, or #17, nor DM or HSR could be detected. Although an increased portion of fibroblast mitoses could be expected in a cul-
A)
F
20
19
? -.4v---.- p13.1
B)
19 Figure 2 Patient RZ-032: translocation (19;?)(p13.1;?) in comparison with chromosomes #20 (A) and with schematic diagram (B).
RZ
1
%
J~
-II 9
3
*
20
14
7
2
15
8
3
9
t6
10
4
18
12
!
21
22
I '-~ & ~ a
17
tl
5
Y
X
Figure 3 Aside from t(19;?), this metaphase of the dipoloid range shows a secondary translocation of the short arm of chromosome #7, most likely a t(7;21). (Second chromosome #11 is crossed by chromosome #1.1
24/156
ID
K~7;213(p22; p13")
l(19 i ? )(p13; ? )
1.4,XX. - 2 0 , - 2 1
~IB I!
t
e4
g
c.o
134 Table 1
F. Franke et al.
Cytogenetic analysis of two resectable abdominal neuroblastomas
Patient
Modal chromosome number
Analyzed tumor metaphases
Banded karyotypes
Chromosomal aberrations
TT-018 RZ-032
70(45-71) 46(39-78)
40 61
5 9
t(19;?)(p13;?), mar t(19;?)(p13;?), t(7;21)(p22;p13)
ture of tumor material cultivated for up to 8 days, the n u m b e r of normal diploid mitoses in both cases stayed distinctly u n d e r 10%. Analysis of blood lymphocyte chromosomes in hr-banding of both patients also revealed no conspicuous findings.
DISCUSSION Clinically, besides the patient's age, neuroblastoma can be divided roughly into two groups: (a) localized tumors (stages I and II) with a good prognosis and a cure rate of over 80%, and (b) unresectable or early metastasizing tumors (stages III and IV), which have a very poor prognosis in children over 1 year of age [7]. The transition of a primary localized abdominal neuroblastoma into a metastasizing tumor is observed very rarely and was seen in only 1 of 66 cases [10]. Up to now, almost all cytogenetic data have been collected from metastasizing neuroblastoma. Over 70% of all examined tumors, i n c l u d i n g cell lines, had abnormalities of chromosome l p , 66% had DM and/or HSR, and more than 20% contained additional l q or 17q material [9]. In contrast, n o n e of these markers could be found in our preparations. Moreover, the tumor cells of the two patients examined revealed consistently a novel translocation on the short arm of chromosome #19. Its sole appearance in pseudodiploid metaphases likewise indicates that this cytogenetic abnormality may occur as a n o n r a n d o m and specific one in abdominal neuroblastomas with good prognosis. From these data we assume that localized neuroblastoma, analogous to its clinically different course, also differs from the metastasizing tumor cytogenetically. A decreased proliferation tendency, as well as a low drug resistance of the transformed neuroblasts of localized tumors, may reflect an i n v o l v e m e n t of genes that are different from those responsible for metastatic disease. This study was supported by the Deutsche Forschungsgemeinschaft and by the Parent Initiative Giessen. Secretarial assistance of Mrs. M. Mourek, as well as translation work of Mrs. A. Tonsen and Mr. A. Walther, are gratefully appreciated.
REFERENCES 1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. New York, Elsevier/North Holland. 2. Yunis JJ, Brunning RD, Howe RB, Lobell M (1984): High resolution chromosomes as an independent prognostic indicator in adult acute nonlymphocytic leukemia. N Engl J Med 311:812-818. 3. Secker-Walker LM (1984): The prognostic implications of chromosomal findings in acute lymphoblastic leukemia. Cancer Genet Cytogenet 11:233-248. 4. Brodeur GM, Green AA, Hayes FA, Williams KJ, Williams DL, Tsiatis AA (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4678-4686.
t(19;?) i n Stage II N e u r o b l a s t o m a s
135
5. Gilbert F, Balaban G, Moorhead P, Bianchi D, Schlesinger H (1982]: Abnormalities of chromosome l p in h u m a n neuroblastoma tumors and cell lines. Cancer Genet Cytogenet 7:3342. 6. Franke F, Lampert F (1982): Chromosomenuntersuchungen beim metastasierten Neuroblastom. Onkologie 5:268-272. 7. Berthold F, Kaatsch P, Evers G, Harms D, J(irgens H, Niethammer D, Ritter J, Wahlen W, Treuner J, Lampert F (1984): Intensive Kombinationschemotherapie und B-Interferon zur Behandlung von Kindern mit metastasiertem Neuroblastom: Studie GPO-NB 79/82. Klin Padiat 196:143-149. 8. Yunis JJ (1976): High resolution of h u m a n chromosomes. Science 191:1268-1270. 9. Gilbert F, Feder M, Balaban G, Brangman D, Lurie DK, Podolsky R, Rinaldt V, Vinikoor N, Weisband J (1984): Human neuroblastomas and abnormalities of chromosomes 1 and 17. Cancer Res 44:5444-5449. 10. Berthold F (1985): Personal communication: Neuroblastomstudie 79/82 and 82/85 as of November 15, 1984.
ABSTRACT: Chromosomes of two abdominal neuroblastomas (stage II) from a lO-month-old male infant and a 6-year-old girl were analyzed. A modal chromosome number of 70 and 46, respectively, was found. Neither double minute chromosomes (DM) or homogeneously staining regions (HSR), nor visible structural abnormalities of chromosome #1 could be detected. However, a common aberration was found to be a translocation on the short arm of chromosome #19. After tumor resection, both children are alive without any evidence of tumor disease.
INTRODUCTION Chromosome abnormalities of h u m a n tumor cells, more or less n o n r a n d o m and specific, have been found in increasing n u m b e r s of malignancies [1] and can help in classifying tumor subgroups or in defining clinical prognosis [2, 3]. In h u m a n neuroblastoma, double minutes (DM), homogeneously staining regions (HSR), and, most frequently, a deletion and/or translocation of the short arm of chromosome # 1 have been described, but almost uniformly in tissue cultures or samples taken from metastases or primary tumors of stage III or IV [4-6]. These unresectable or metastatic neuroblastomas have a very poor prognosis in patients over 1 year of age, with almost no chance of survival despite an aggressive chemotherapy. In contrast, cytogenetically, little is k n o w n about neuroblastomas of stages I and II, which are localized and resectable. Using a simple procedure to accumulate a sufficient a m o u n t of mitotic cells, we succeeded analyzing two samples of these localized tumors. PATIENTS Case 1
T. T., a 10-month-old male infant, was found to have a right-sided abdominal tumor during a routine examination. Laboratory values at diagnosis (10/27/83) were hemoglobin 12 g/dl, thrombocytes 616.0/nl, and leukocytes 15.6/nl. Urinary vanil
From the OncocytogeneticLaboratory,Kinderpoliklinik,Universityof Giessen,West Germany. Address requests for reprints to Dr. Fritz Lampert, Universitiits Kinderpoliklinik, Feulgenstra[~e 12, D-6300 Giessen, West Germany. This article is part of F. Franke's doctoral thesis. Received January 28, 1985; accepted April 15, 1985.
129 © 1986 Elsevier Science PublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet20:129-135 (1986) 0165-4608/86/$03.50
130
F. Franke et al.
mandelic acid, homovanillic acid, and dopamine were elevated. Bone marrow aspiration was normal. The fist-sized tumor with three adjacent lymph nodes was excised in toto at the Children's Hospital Dortmund (we thank Dr. Strehl for kindly providing us with tumor material). Histologically, neuroblastoma tissue, including neurofibrillar elements, pseudorosettes, and some differentiation in rare parts were seen (Hughes grade 2). Two of the lymph nodes were infiltrated with tumor tissue. In touch preparations, the neuroblastoma cell population was seen to be dispersed with small lymphocytes. Because of regional lymph node metastases, this patient with abdominal neuroblastoma stage II (Evans) was treated postoperatively with three chemotherapy cycles (one every 3 weeks), according to the NB 79 protocol of the German Society of Pediatric Oncology [7]. Cytostatic blocks consisted of adriamycin 35 mg/m 2, day 1, and cyclophosphamide 150 mg/m 2, days 1-7. Except for some vomiting, the patient tolerated this mild and short-term chemotherapy well and remains in good health with no evidence of tumor disease, now more than 2 years after operation. Case 2
R. Z., a 6-year-old girl, complained of occasional abdominal pain of a few months duration. During a routine medical check-up at school, a fist-sized, left-sided abdominal tumor was palpated. Laboratory values on admission (4/27/83) were hemoglobin 13.5 g/dl, thrombocytes 194.0/nl, leukocytes 7.0/nl, serum lactate dehydrogenase 309IU/L; and serum ferritin 26 ng/ml. Urinary catecholamines were slightly elevated: vanil mandelic acid 22.4 ~mol/day (12.6 nmol/~mol Kreatinin) and homovanillic acid 33.4 o~mol/ day (18.8 nmol/~mol Kreatinin). Bone marrow aspiration was normal. Upon surgery (4/28/83), the tumor, measuring 10 cm in diameter, and three adjacent lymph nodes were excised in total. Histologically undifferentiated neuroblastoma tissue with focal differentiation (Hughes grade 2) was seen. One of the excised lymph nodes was also infiltrated. In touch preparations, typical neuroblastoma cells were seen, as were pseudorosettes and a few dispersed lymphocytes. Because of regional lymph node infiltration, this patient with stage II abdominal neuroblastoma was treated with three chemotherapy cycles postoperatively, as described in case 1. The patient tolerated this mild and short-term chemotherapy very well and remains in good health with no evidence of tumor disease up to the present day (2 years after operation). MATERIALS AND METHODS
Attempts to collect analyzable tumor metaphases were successful in two of five patients with neuroblastoma of stage I or II. Tissue samples were taken from tumors immediately after their surgical excision, These samples were cleaned, washed in Hanks' balanced salt solution (BSS), transsected into small pieces, and transferred into Falcon culture flasks (25 cm3). As culture medium, RPMI 1640 plus 15% fetal calf serum plus 50 U/ml penicillin and 50 ~g/ml streptomycin was used. Different culture times at 37°C (3 hr; 3, 5, and 8 days) and different methods for chromosome preparation were applied. A 5-day culture in combination with a subsequent 17-hr cell synchronization with 10-7M methotrexate yielded the best results regarding mitotic index, as well as quality of metaphases. Two washing procedures were followed by a further 6 hr "releasing" of the tumor cells by addition of 10-5M thymidine. During the last 2 hr
~'igure 1
tB I!
19
rT
J
4/149
20
14
7
15
21
8
2
22
9
1
3
16
10
?
Near-triploid metaphase of patient TT-018 showing two marker chromosomes (arrows).
13
3
6
I,,ll,
J
7
iiii
11
K
18
12
13 2
F. Franke et al. of this period, colcemide was a d d e d in a concentration of 0.01 ~g/ml. Further preparation c o r r e s p o n d e d to previously p u b l i s h e d methods [6]. In addition, the chromosomes of p e r i p h e r a l blood l y m p h o c y t e s of both patients were analyzed using a modified Yunis technique [8].
RESULTS The majority of the tumor cells of patient T. T. revealed a near-triploid chromosome set (Fig. 1). Besides a small marker chromosome, del(4)(q2)?, a translocation of material of u n k n o w n origin was found as a constant aberration on the short arm (very likely) of a chromosome #19 (Fig. 1). The aberration of c h r o m o s o m e #19 was also consistently found in the tumor metaphases of patient R. Z. and appears to be very similar in structure to that of T. T. The involvement of the short arm of c h r o m o s o m e # 1 9 in a translocation with breakpoint p13.1 is illustrated in Figure 2,A and B. Neither the origin of the translocated chromosome material nor the remaining segment 19p13.1--~pter could be located in the preparations examined. On the other hand, the translocation (19;?) in six complete karyotypable p s e u d o d i p l o i d metaphases was the only striking aberration. Eight of 61 analyzed tumor metaphases showed a secondary translocation on the short arm of c h r o m o s o m e #7, w h i c h was most likely a t(7;21) (Fig. 3). Table 1 summarizes the cytogenetic data of both cases. It is noteworthy that we did not find a n y of the abnormalities hitherto described to be typical for metastasizing neuroblastomas [9]. Neither abnormal changes of c h r o m o s o m e s l p , lq, or #17, nor DM or HSR could be detected. Although an increased portion of fibroblast mitoses could be expected in a cul-
A)
F
20
19
? -.4v---.- p13.1
B)
19 Figure 2 Patient RZ-032: translocation (19;?)(p13.1;?) in comparison with chromosomes #20 (A) and with schematic diagram (B).
RZ
1
%
J~
-II 9
3
*
20
14
7
2
15
8
3
9
t6
10
4
18
12
!
21
22
I '-~ & ~ a
17
tl
5
Y
X
Figure 3 Aside from t(19;?), this metaphase of the dipoloid range shows a secondary translocation of the short arm of chromosome #7, most likely a t(7;21). (Second chromosome #11 is crossed by chromosome #1.1
24/156
ID
K~7;213(p22; p13")
l(19 i ? )(p13; ? )
1.4,XX. - 2 0 , - 2 1
~IB I!
t
e4
g
c.o
134 Table 1
F. Franke et al.
Cytogenetic analysis of two resectable abdominal neuroblastomas
Patient
Modal chromosome number
Analyzed tumor metaphases
Banded karyotypes
Chromosomal aberrations
TT-018 RZ-032
70(45-71) 46(39-78)
40 61
5 9
t(19;?)(p13;?), mar t(19;?)(p13;?), t(7;21)(p22;p13)
ture of tumor material cultivated for up to 8 days, the n u m b e r of normal diploid mitoses in both cases stayed distinctly u n d e r 10%. Analysis of blood lymphocyte chromosomes in hr-banding of both patients also revealed no conspicuous findings.
DISCUSSION Clinically, besides the patient's age, neuroblastoma can be divided roughly into two groups: (a) localized tumors (stages I and II) with a good prognosis and a cure rate of over 80%, and (b) unresectable or early metastasizing tumors (stages III and IV), which have a very poor prognosis in children over 1 year of age [7]. The transition of a primary localized abdominal neuroblastoma into a metastasizing tumor is observed very rarely and was seen in only 1 of 66 cases [10]. Up to now, almost all cytogenetic data have been collected from metastasizing neuroblastoma. Over 70% of all examined tumors, i n c l u d i n g cell lines, had abnormalities of chromosome l p , 66% had DM and/or HSR, and more than 20% contained additional l q or 17q material [9]. In contrast, n o n e of these markers could be found in our preparations. Moreover, the tumor cells of the two patients examined revealed consistently a novel translocation on the short arm of chromosome #19. Its sole appearance in pseudodiploid metaphases likewise indicates that this cytogenetic abnormality may occur as a n o n r a n d o m and specific one in abdominal neuroblastomas with good prognosis. From these data we assume that localized neuroblastoma, analogous to its clinically different course, also differs from the metastasizing tumor cytogenetically. A decreased proliferation tendency, as well as a low drug resistance of the transformed neuroblasts of localized tumors, may reflect an i n v o l v e m e n t of genes that are different from those responsible for metastatic disease. This study was supported by the Deutsche Forschungsgemeinschaft and by the Parent Initiative Giessen. Secretarial assistance of Mrs. M. Mourek, as well as translation work of Mrs. A. Tonsen and Mr. A. Walther, are gratefully appreciated.
REFERENCES 1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. New York, Elsevier/North Holland. 2. Yunis JJ, Brunning RD, Howe RB, Lobell M (1984): High resolution chromosomes as an independent prognostic indicator in adult acute nonlymphocytic leukemia. N Engl J Med 311:812-818. 3. Secker-Walker LM (1984): The prognostic implications of chromosomal findings in acute lymphoblastic leukemia. Cancer Genet Cytogenet 11:233-248. 4. Brodeur GM, Green AA, Hayes FA, Williams KJ, Williams DL, Tsiatis AA (1981): Cytogenetic features of human neuroblastomas and cell lines. Cancer Res 41:4678-4686.
t(19;?) i n Stage II N e u r o b l a s t o m a s
135
5. Gilbert F, Balaban G, Moorhead P, Bianchi D, Schlesinger H (1982]: Abnormalities of chromosome l p in h u m a n neuroblastoma tumors and cell lines. Cancer Genet Cytogenet 7:3342. 6. Franke F, Lampert F (1982): Chromosomenuntersuchungen beim metastasierten Neuroblastom. Onkologie 5:268-272. 7. Berthold F, Kaatsch P, Evers G, Harms D, J(irgens H, Niethammer D, Ritter J, Wahlen W, Treuner J, Lampert F (1984): Intensive Kombinationschemotherapie und B-Interferon zur Behandlung von Kindern mit metastasiertem Neuroblastom: Studie GPO-NB 79/82. Klin Padiat 196:143-149. 8. Yunis JJ (1976): High resolution of h u m a n chromosomes. Science 191:1268-1270. 9. Gilbert F, Feder M, Balaban G, Brangman D, Lurie DK, Podolsky R, Rinaldt V, Vinikoor N, Weisband J (1984): Human neuroblastomas and abnormalities of chromosomes 1 and 17. Cancer Res 44:5444-5449. 10. Berthold F (1985): Personal communication: Neuroblastomstudie 79/82 and 82/85 as of November 15, 1984.