Advances in Neuroblastoma Research. New York, Alan R. ... Deftos LJ, Linnoila RI, Carney DN, Burton DW, Leong SS,. O'Connor DT, Murray SS, Gazdar AF: ...
American Journal ofPathology, Vol. 136, No. 2, Februa?y 1990 Copyright © American Association ofPathologists
The Neuroendocrine and Neural Profiles of Neuroblastomas, Ganglioneuroblastomas, and Ganglioneuromas Willemina M. Molenaar,* David L. Baker,t David Pleasure,t Virginia M.-Y. Lee,* and John Q. Trojanowski* From the Department ofPathology and Laboratory Medicine,* Division ofNeuropathology, University ofPennsylvania, Philadelphia, and the Division of Oncology,J and Neurology Researchb, Children's Hospital ofPhiladelphia, Pennsylvania
To establish the neuroendocrine and neural features ofperipheral neuroblastic tumors, aprospectively collected group of 12 neuroblastomas (NB), 2 ganglioneuroblastomas (GNB), and 4 ganglioneuromas (GN) was probed with apanel of monoclonal antibodies (MAbs) to neuroendocrine and neural antigens. All tumors expressed the pan-neuroendocrine markers synaptophysin and chromogranin A. They also showed extensive expression of neuronal antigens, ie, of each of the neurofilament (NF) tripletproteins and ofthe microtubule-associatedproteins (MAPs) MAP2 and tau-protein. However, only in the GNBs and GNs was the pattern of NFphosphoisoforms relatively mature. In the latter tumors glial fibrillary acidic protein (GFAP) and myelin basicprotein (MBP) could be demonstrated as well, suggesting the presence ofnonmyelinating and myelinating Schwann cells, respectively. The glial markers did not colocalize with the neural markers. On the basis of these data, it was concluded that all peripheral neuroblastic tumors manifest molecular characteristics of neuroendocrine cells and of neurons. The latter were most developed in GNBs and GNs, in which they were accompanied by Schwann cell differentiation in a separate population of cells. The above-outlined neuronalprofile ofperipheral neuroblastic tumors, including NBs, distinguishes this group of tumors from the much-less neuronally differentiatedprimitive neuroectodermal tumors of the central nervous system. (AmjPathol 1990, 136:3 75-382)
Tumors arising in the adrenal medulla or sympathetic trunk in childhood, ie, neuroblastomas (NBs), ganglioneu-
roblastomas (GNBs), and ganglioneuromas (GN), are derived from the neural crest, an origin that places them in the broad group of neuroendocrine tumors.1-3 It has been suggested4 that within the family of neuroendocrine tumors two main branches can be distinguished, ie, the neural types and the epithelial types. The neural neuroendocrine or neuroblastic tumors, which are the subject of this report, may show the full range of neuronal differentiation from undifferentiated, primitive tumors, ie, NBs, to highly differentiated tumors composed of mature-looking ganglion cells and nerve bundles, ie, GNs. The neuroendocrine aspects of such tumors were underscored by the demonstration of the pan-neuroendocrine markers synaptophysin4-9 and chromogranin A9-10 in the majority of studied cases. Morphologically, neuronal features can be recognized in highly differentiated tumors and the availability of immunohistologic markers makes it possible to demonstrate them also in the morphologically less welldifferentiated tumors11-16 and cell lines.17 18 Although the expression of neurofilament (NF) proteins by these tumors certainly demonstrates their neuronal character at the molecular level, it is as yet unknown, whether their NF profile is mature, immature or aberrant as compared to normal and developing neurons. In particular, the phosphorylation state of the medium (M) and high (H) molecular weight NF proteins, which increases during normal neuronal maturation,19-21 has not been studied previously in these tumors. The current study describes the findings in 18 prospectively collected frozen tumor samples. First we addressed the neuroendocrine aspects of these tumors by probing them with monoclonal antibodies (MAbs) to the neuroendocrine markers synaptophysin and chromoAccepted for publication October 5, 1989. This work was supported in part by NIH-grants CA-36245, CA-47983, NS-08075, and NS-18616. W. M. Molenaar, a Fulbright Scholar, is on leave of absence from the University of Groningen, Groningen, The Netherlands. Address correspondence and reprint requests to John 0. Trojanowski, MD, PhD, Department of Pathology and Laboratory Medicine, Division of Neuropathology, Hospital of the University of Pennsylvania, Basement Maloney, Room A009, 34th and Spruce Streets, Philadelphia, PA 19104.
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Table 1. Patient Data
Case NB
Age at Dx
Sex F M M
Stage at Dx IV IV IV III IVs IV II Il II II
Ferritin (ng/dl)
84 41 NT 150 23 506 5 24 15 62
6 7 8 9 10
18 mo 13 mo 25 yrs 3 yrs 4 mo 19 mo 19 mo 2 yrs 20 mo 2 mo
11 12
7 mo 2 yrs
F M
II II
17 67
1 2
7 yrs 4 yrs
F F
II
30 20
1 2 3 4
6 yrs 9 yrs 2 yrs 6 yrs
M M M F
II II IV III
1
2 3 4 5
GNB
GN
F M F M F F M
29 NT >800 53
Primary site
Outcome
Abdomen Cervical ganglia Adrenal* Abdomen Abdomen Adrenal Abdomen Pelvis Mediastinum Mediastinum chest wall Cervical ganglia Adrenal
D/9 mo A/10 mo A/27 mo A/2 mo D/18 mo D/10 mo A/23 mo A/4 mo A/2 mo A/7 mo
Mediastinum
Paraspinal Adrenal Mediastinal
A/29 mo A/12 mo
A/13 mo A/24 mo
A/21 mo A/4 mo D/22 mo A/3 mo
Abdomen* Abdomen Summary of the main patient characteristics and clinical parameters at the time of diagnosis (Dx) and outcome. The patients are grouped according to the histologic diagnosis: NB: neuroblastoma, GNB: ganglioneuroblastoma, GN: ganglioneuroma; M/F: male/female; mo: months, yrs: years; stage is given according to Evans' criteria26; levels of ferritin are given as nanograms/deciliter; normal value H RMdO 20 P+ NF-M only RMO 281 P+ NF-M only HO 14 P+ NF-H > M TA 50 P+ NF-H only TA 51 P+ NF-H only HO 57 p++ NF-H only RMO 217 NF-H only RMO 24 pid 2.2B10 GFAP pind Tau-protein T 14 pind MAP-2 M 12 NT Myelin basic protein MBP Summary of the major characteristics of the Mabs specific for neuro-
nal and glial proteins. * As initially defined by Lee et al.22 the designations Pd, -, and +, + +++ indicate that an antibody recognizes a NF subunit independent of its phosphorylation state, only in its nonphosphorylated form or in its poorly, moderately, and heavily phosphorylated states, respectively. Antibodies that bind determinants in the alpha-helical filament forming core domain of NF proteins are identified by a t; all of the other anti-NF antibodies bind epitopes in the peripheral domain of these subunits. All except the antibody to NF-L are monoclonal. NT, not tested.
protein,30 as well as MAbs to glial proteins, ie, and MBP.32
GFAP3'
Immunohistology Immunohistologic procedures were performed on frozen, air-dried sections using the avidin-biotin complex peroxidase technique (Vectastin Labs kits, Burlingame, CA) or the peroxidase-antiperoxidase technique. lmmunofluorescence double labeling was performed in three cases using as primary antibodies rat 2.2B1 0 and mouse RMdO2O. Anti-rat fluorescein and anti-mouse rhodamine were used as secondary antibodies. The immunohistochemical procedures used in this study have been previously described.2022,23
Results Histology The major histologic characteristics of the tumors are summarized in Table 3. To allow correlations between morphology and immunophenotype, the features indicated are those that were seen in the frozen tissue samples that were used for the immunohistologic studies. These were comparable to the paraffin sections of the same biopsy specimens. In case GN 3 only the residual tumor obtained after 12 months of therapy was available
for this study, and this sample was distinctly more differentiated than the initial tumor.
Immunohistology Neuroendocrine Antigens (Table 4). Both synaptophysin and chromogranin could be demonstrated in all tumors. However, in NBs staining for synaptophysin was always more extensive than that for chromogranin A positivity. In GNBs and GNs synaptophysin was demonstrated in both fiber bundles and ganglion cells, but chromogranin A was more prominent in ganglion cells than in fibers. Indeed, chromogranin staining was often helpful in detecting such cells.
Neuronal and Glial Markers (Tables 5 and 6) NF proteins could be demonstrated in all tumors and, with the exception of NBs 1, 4, and 5, they expressed NFL, -M, and -H (Table 5). NBs 1, 4, and 5 expressed NF-L and -M, but not -H. Staining for NF-M was obtained with all MAbs, ie, those recognizing phosphorylated or nonphosphorylated epitopes or epitopes in peripheral or core domains independent of phosphorylation. In NBs staining for NF-H was largely restricted to MAbs recognizing poorly phosphorylated epitopes (H+), whereas in GNBs and GNs the more heavily phosphorylated epitopes (H++ and H+..) could also be demonstrated. In GNBs and GNs virtually all MAbs stained both fiber bundles and ganglion cells, although ganglion cells were especially prominent with MAbs to nonphosphorylated epitopes. In NBs different staining patterns were observed, ie, 1) relatively diffuse staining of neuropil in cellular or noncellular areas and in the center of Homer-Wright rosettes (Figure 1 A), 2) distinct cytoplasmic staining, and 3) staining of short, abortive fibers, clearly related to tumor cells (Figure 1 B). The latter type of staining was observed with all MAbs, except that recognizing unphosphorylated NF-M and -H
(RMdO20). MAP2 and/or tau, could be demonstrated in all GNBs and GNs and in 8 of the 11 NBs (Table 6). In the latter, positivity was largely confined to neuropil. GFAP was demonstrated in all GNBs and GNs, whereas MBP could be demonstrated in one GNB and three GNs. Both antigens were clearly associated with fibers (Figure 1 C, D). Double immunofluorescence with MAbs to GFAP and NF-M/H in one GNB and two GNs showed positivity for both epitopes, but no colocalization in the same cells.
Discussion The purpose of the current study was to explore the extent of neuroendocrine and neural maturation in pediatric
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Table 3. Histology Case NB
GNB GN
U
Cells
1 2 3 4 5 6 7 8
+++ +++ +++ +++ +++ ++ ++ ++
+ +
9
++ + +
G
Ros.
Neuropil
-
-
-
-
-
-
-
-
+ +
+ ++
-
++ -
+ ++
-
-
+ +++ ++
10 11 12
++ ++
-
+
1 2
+
++ +
+ +
_ _ -
-
+
+
-
Calc. -
+
-
-
Necrosis
-++
-
-
-
-
Fiber bundles
+
-
_ _ ++
_ _ _ _
+
_ -
_
_
_
+
+
+ + +
-
_
_
-
++ +++
+
+++ + 1 +++ ++ 2 -+++ ++ 3 +++ + 4 of the frozen material was used for immunohistology. The first three columns that Summary of the histologic parameters observed in H&E sections
indicate in a semiquantitative manner the relative contribution of undifferentiated cells (U), cells with more cytoplasm and vesicular nuclei, ie, intermediately differentiated cells (I), and ganglion cells (G) to the total cellular content of the tumors. The last five columns estimate the abundance of Homer-Wright rosettes (ros.), neuropil, fiber bundles, necrosis and calcifications (calc.), which are scored as - when absent, + when minimal, ++ when more extensive, and +++ when very extensive. Neuropil is characterized by fine fibrillary background between tumor cell nuclei. Fiber bundles refer to structures resembling mature peripheral nerve bundles.
peripheral neuroblastic tumors by probing a prospectively collected group of such tumors with a large panel of highly specific MAbs. Their neuroendocrine character was determined with the aid of MAbs to synaptophysin and chromogranin A, both of which have been shown to be panneuroendocrine markers.4 61033-35 Synaptophysin is an Table 4. Neuroendocrine Antigens Case Syn. NB 1 ++c 2 +++c +++cp 3 4 ++CP ++CP 5 ++c 6 +++cp 7 +++cP 8 +++cp 9 +++cP 10 +++c 11 ++++c 12 ++__FC GNB 1 GN
Chrom. +CP +c +cP +c +cP +c +c
++cP ++cP
++c +c
+++c ++C
2
+++FG
+++G
1
++++FG
+
2 3 4
++++FG ++++FG ++FG
++G ++GF ++G
+GF
Summary of the findings obtained with MAbs to the pan neuroendocrine markers synaptophysin (Syn) and chromogranin A (Chrom). The extent of the immunoreactivity is scored semiquantitatively and the structures that are staining are indicated: +, ++, +++, ++++ positivity approximately up to 25%, 25% to 50%, 50% to 75%, and 75% to 100%, respectively. The staining pattern is also indicated: C, cytoplasmic; P, neuropil; F, fiber bundles; G, ganglion cells. Staining intensity is not taken into consideration.
integral membrane glycoprotein occurring in presynaptic vesicles of central and peripheral neurons and in the adrenal medulla.5 In rat cerebellar cortex it was found to increase with maturation, but it could be demonstrated before the establishment of synapses.' Synaptophysin has previously been described in ganglion cells in virtually all GNBs and GNs in both paraffin and frozen tissue,569 but in NBs the relative number of positive cases was less in paraffin than in frozen tissue. 6,869 This may be explained by a correlation between antigen expression and differentiation, as in embryogenesis,36 which makes the levels in poorly differentiated cells so low as to be detectable only in frozen tissue.37 The finding of positivity in all of the current cases studied in frozen sections is in keeping with this notion. The same may be true for chromogranin A, which was demonstrated, albeit to a limited extent, in all of the current cases, but in only 23 of the 33 paraffinembedded NBs reported by Hachitanda et al.9 In the current cases synaptophysin staining was stronger and more extensive than chromogranin A staining and synaptophysin, more than chromogranin A, stained fiber bundles as well as ganglion cells in GNBs and GNs. NF proteins have been probed in neuroblastic tumors in several previous studies.56116 In paraffin-embedded material, they were more frequently demonstrated in GNBs and GNs than in NBs.11-13 In two studies,14,15 each of the NF triplet proteins was probed separately and shown to be expressed in all GNBs and GNs, whereas in NBs the three subunits were more readily demonstrated in frozen than in paraffin-embedded tissue.15 These findings
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Table 5. Neurofilament Proteins NF-M ind NF-L
Case NB
1 2 3 4 5 6 7
8 9 10
GNB GN
NF-M/H-
per. core
+f
+cp
+++
+
_ _
+++p +PC
++p
+P
++p
+cf
+f
+P
cp
++
++
_ _ ++-
+++p +f
++p
++f
_
+P
+f
++p
++f
+
++
++
++
++P
+ ++P
++P
+++Pf
+++cp
+++P
+++p +++PC
+++Pf +++PIC
++PI +++Pf
+++f
++Pf
+++PC, +++P
+++CP ++P
+p
+++p
11
+ +Pf
+++P +++P
++Pf
+++p
+++p
+++p ++P
12
++
+++cp
+++p
++PC
++c'
+++C
+++f +++PI +++c
++++FP +++FG
++++FP +++FP
++++PF +++F
++++FP +++FG
++++FG
++FP
2
++++FPG
+++FG
1
++++FG
++++FG
++++FG
++++FG
++++FG
2 3 4
++++FG ++++FG ++++F
++++FG ++++FG ++++F
++++FG ++++FG ++++FG
++++FG ++++FG ++++F
++++FG ++++FG ++++F
1
+++cp
NF-H... _ -
+P
_
+1
+++p +++PI
NF-H++
NF-H/M+
++f +P
NF-H+
NF-M+ _
_
_
-
_
+P _
+P
+++p
+
+++FP
++F
++FPG
+++FPG
++F
_
++++F
++F
++++FG
++FG
++++FG ++++FG ++++F
++++FG ++++FG ++++F
+++FG ++F +++F
++++F +++F +++FG
Summary of the findings with antibodies to each of the NF-trplet proteins and phosphoisoforms of NF-M and NF-H. Antibodies that recognize the same NF epitopes are grouped together (Table 2). "d, -, +, ++, +++: indicate the phosphorylation state of the epitopes, ie, independent of phosphorylation or recognizing non-phosphorylated, poorly, moderately, or highly phosphorylated, respectively (see Table 2). For an explanation of the scoring and a description of the different staining patterns, See Table 4. f: cell-related abortive fibers (see text), as distinct from well-formed fiber bundles.
suggest that the level of NF expression is higher in the better-differentiated tumors and may be too low to be detected in poorly differentiated tumors after paraffin embedding. This is supported by the findings in the current study, using snap-frozen material, in which the neuronal character of NBs, GNBs, and GNs was convincingly demonstrated by their widespread expression of NF proteins, which included all three NF triplet proteins in all but three NBs. Even in poorly differentiated NBs NF proteins could Table 6. Neuronal MAPs and Glial Proteins Case NB
MAPs Tau
1 2
-
-
-
-
3
+
+
4 5 6
-
++PC
-
++
+
-
++P
+++P +++P
-
-
-
-
+++P
-
-
7 8 9
+P ++P
10
GNB GN
Glial proteins
Neuronal MAP2
GFAP _ _ _ _ _
MBP
_ _ _ _
-++P
11
++P
+++P
-
-
12
-
-
-
-
1
++PG
++PF
++F
+F
2
+++FG
+++FG
++F
_
1
+++FG
+F
+++GF ++GF
+++FG +++FG
++F
2
+F
+F
++GF ++F
++F
_ +F
3 4
+G
+F
Summary of the findings obtained with MAbs to microtubule associated proteins (MAP2 and tau protein) and MAbs to glial proteins (GFAP and MBP). For an explanation of the scoring and a description of the type of staining, see Table 4.
be demonstrated and there appeared to be an increase in the amount of NF immunoreactivity with increasing differentiation. Moreover, only highly differentiated tumors, ie, GNBs and GNs, were found to express the heavily phosphorylated isoforms of NF-H. The significance of this finding derives from the fact that these NFH phosphoisoforms are the last to appear in normal mammalian development and their induction may signify the endpoint of neuronal maturation.19-21 The spatial distribution of NF-isoforms as seen in normal neurons, ie, nonphosphorylated epitopes predominantly in the cell bodies and phosphorylated epitopes predominantly in the axons and dendrites,21'22 was not replicated with fidelity in the tumors. Thus, although the MAb recognizing NF-M/Hwas especially prominent in ganglion cells and did not stain abortive, cell-associated fibers, it did stain fiber bundles in GNBs and GNs. Conversely, MAbs recognizing phosphorylated epitopes were stronger in fiber bundles but also recognized ganglion cells. In addition to NF-proteins, neuron-specific MAPs were demonstrated in most of the tumors, as previously described for MAPs by Artlieb et al.13 As with the NF proteins, we found an increasing level of expression of MAP2 and tau with increasing differentiation. The above findings indicate that there is a progressive level of neuronal differentiation from NBs through GNBs to GNs, which reaches a virtually mature pattern in GNs. A very high level of differentiation was also indicated by the presence of GFAP with or without MBP in fiber bundles in GNBs and GNs. This suggests the presence of GFAP-positive nonmyelinating Schwann cells and GFAP-negative myelinating Schwann cells, respectively.38-3 GFAP-positivity was
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Figure 1. Immunoperoxidase stains demonstrating NF triplet proteins (A, B) and glialproteins (C, D). A: Staining for NF-M/I-F in the center ofHomer-Wright rosettes (arrows) in NB 6 (X 150); B: stainingforNF-H- in the cytoplasm oftumor cells and in small abortive fibers (arrows) in NB 7 (X 300); C: staining for GFAP in fiber bundles in case GNB 2 (X 150); and D: staining for MBP (arrows) associated withfiber bundles in case GN3 (X 300).
described in only one other report,12 where it was found in undifferentiated cells (for discussion, see below). It is interesting to compare the current peripheral neuroectodermal tumors to central neuroectodermal tumors, which have been grouped together in the family of primi-
tive neuroectodermal tumors (PNETs'42). Similar to the current peripheral tumors, the central PNETs display neuroendocrine features such as expression of synaptophysin,43 (Gould et al, manuscript in preparation). However, morphologic features of differentiation are relatively com-
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mon in the peripheral tumors but are rare in the central tumors. Furthermore, even poorly differentiated tumors in the current group of tumors expressed different NF triplet proteins, whereas in a large group of central PNETs4 NF protein expression was absent in almost one half of the cases. Even in the positive central tumors NF expression was limited, both in extent and in the number of NF triplet proteins and phosphoisoforms. It thus appears that even the least neuronally differentiated peripheral tumors are more differentiated than the most neuronally differentiated central PNETs. Glial differentiation was observed in the highly differentiated tumors of the current group of peripheral tumors in a tumor cell population separate from the population expressing NF proteins. In contrast, GFAP was demonstrated in primitive cells of 3 of 17 undifferentiated or partly differentiated peripheral tumors described by Carlei et al,'2 as well as in those of about one half of our own central PNETs.43 These observations suggest that, in addition to highly differentiated GFAP-expressing cells, multipotential primitive cells may be present in both groups of tumors. In the central PNETs this notion was supported by the localization of GFAP and NF proteins in the same cells. The neuroendocrine features of all currently studied neuroblastic tumors were confirmed by their expression of pan-neuroendocrine markers. The neural aspects also could be established in all tumors by the demonstration of NF proteins and MAPs, even in morphologically undifferentiated tumors. NF proteins were increasingly more prominent with increasing levels of morphologic differentiation. This finding further extends the morphologic interpretation of maturation by demonstrating that also the complement of NF phosphoisoforms is more mature in GNBs and GNs than in NBs. Together these findings suggest that neuroendocrine antigens are more primitive and less-specific markers than neural antigens. Finally, in comparison with the PNETs of the central nervous system, neuroectodermal tumors of the adrenal medulla and sympathetic trunk appear much more neuroblastic and far less primitive.
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7:3474-3488 23. Schmidt ML, Carden MJ, Lee VM-Y, Trojanowski JQ: Phosphate dependent and independent neurofilament epitopes in the axonal swellings of patients with motor neuron disease and controls. Lab Invest 1987, 56:282-294 24. Black MM, Lee VM-Y: Phosphorylation of neurofilament proteins in intact neurons: Demonstration of phosphorylation in cell bodies and axons. J Neurosci 1988,8:3296-3305 25. Lee VM-Y, Otvos L Jr, Schmidt ML, Trojanowski JQ: Alzheimer's disease tangles share immunological similarities with multiphosphorylation repeats in the two large neurofilament proteins. Proc Natl Ac Sci USA 1988, 85:7384-7388 26. Evans AE, D'Angio GJ, Randolph J: A proposed staging for children with neuroblastoma. Cancer 1971, 21:324-328 27. Evans AE, D'Angio GJ, Propert K, Anderson J, Hann MW: Prognostic factors in neuroblastoma. Cancer 1987, 59: 1853-1859 28. Trojanowski JQ, Kelsten ML, Lee VM-Y: Phosphate dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283) cells. Am J Pathol 1989,135:747-758. 29. Trojanowski JQ, Schuck T, Schmidt ML, Lee VM-Y: Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues. J Neurosci Meth 1989, 29:171 180. 30. Trojanowski JO, Schuck T, Schmidt ML, Lee VM-Y: Distribution of tau proteins in the normal human central and peripheral nervous system. J Histochem Cytochem 1989, 37:209215 31. Lee VM-Y, Page C, Wu HL, Schlaepfer WW: Monoclonal antibodies against gel excised glial filament proteins and their reactivity with other intermediate filament proteins. J Neurochem 1984, 42:25-32 32. Hickey WF, Lee V, Trojanowski JQ, McMillan LJ, McKearn TJ, Gonatas J, Gonatas NK: Immunohistochemical applications of monoclonal antibodies against myelin basic protein and neurofilament triplet proteins: Advantages over antisera
33.
34.
35.
36.
37.
38.
39. 40. 41.
42.
43.
44.
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Acknowledgments The authors thank Paul Newman and Ann O'Brien for their invaluable help in the conduct of these studies and Drs. A. E. Evans (Children's Hospital of Philadelphia) and R. C. Seeger (Neuroblastoma Registry, Los Angeles, California) for making tissue available to us.
