Medical and Pediatric Oncology 18:491496 (1990)
Histological, Immunohistochemical, and Chromosomal Studies on Non-Adrenal Neuroblastomas Without Increased Excretion of Catecholamines and Their Metabolites in Urine H. Sakatoku, MD, Y. Komada, MD, Y. Hamazaki, MD, H. Kamiya, MD, and M. Sakurai, MD We reviewed our records for the last 10 years and found three of the36 patients with neuroblastoma did not excrete significantly increased quantities of catecholamine metabolites in urine. All of these tumors were histologically characterized by small round cells and possessed a few dense core granules on the electron microscopic examination. All of them were reacted with antineuron-specific enolase (NSE) antibodies,
OKB2, P1153/3 monoclonal antibodies (MoAbs). The HNK-1, BA-I, and SJ-9A4MoAbs reacted with two out of three. One of them demonstrated a reciprocal translocation involving the short arm deletion of chrornosome 1. This multidisciplinary study has been helpful in making more accurate diagnoses for neuroblastoma without classical clinical characters.
Key words: neuroblastoma, catecholamine metabolites, urine
INTRODUCTION
It has been widely accepted that quantitation of catecholamine metabolites in urine is essential and critically important for the diagnosis of neuroblastoma. Only 5% of children with neuroblastoma, however, fail to excrete increased quantities of these substances [l]. In such cases, immunohistochemical and electron microscopic studies may be helpful in obtaining the accurate diagnosis [2,3]. In addition, several MoAbs were recently described to be reactive with neuroblastoma cells and could be used for the differential diagnosis of neuroblastoma from other small round cell tumors, including malignant lymphoma, acute lymphoblastic leukemia (ALL), rhabdomyosarcoma, and Ewing’s sarcoma. The purpose of this study is to report our experience with three children with neuroblastoma without significant increased excretion of catecholamine metabolites and to show the usefulness of the multidisciplinary studies including immunohistochemical analysis, electron microscopic examination, and chromosomal studies on the tumor cells in obtaining a definite diagnosis.
films of the chest demonstrated a large mass in the right mediastinum. She was referred to Mie University Hospital for definite diagnosis and treatment of the mediastinal tumor. A computed tomographic (CT) scan of the chest confirmed the presence of a solid tumor with speckled calcification lying on the right side of the thoracic vertebra. Abdominal CT scan and intravenous pyelography showed that bilateral adrenals were intact. Laboratory data was almost normal including the urinary excretion of vanillyl mandelic acid (VMA), homovanillic acid (HVA), and dopamine (8.0 mg/day, 8.6 mg/day, and 207.9 pg/day respectively). Followed by complete removal of the tumor, adjuvant chemotherapy was initiated and the patient remained disease-free for 17 months. Case 2. A 5-month-old girl was admitted because of a right cervical mass which was found 1 month before admission without tenderness or change of skin color. A CT scan of the neck revealed a solid mass without calcification and showed compression of trachea, right ca-
From the Department of Pediatrics, M i e University, School of Medicine, Mie (H.S., Y .K.,H.K., M.S.), and Department of Pathology, Shizuoka Children’s Hospital, Shizuoka (Y.H.), Japan.
MATERIALS AND METHODS Case Reports
Received February 18, 1988; accepted August 15, 1988.
Case 1. A 5-month-old girl was presented to her family doctor with a history of a persistent cough. X-ray
Address reprint requests to H. Sakatoku, Dept. of Pediatrics, Mie University, School of Medicine, Mie, Japan 514.
0 1990 Wiley-Liss, Inc.
Sakatoku et al. rotid artery, and sternocleidomastoideal muscle. Bilateral adrenals were intact. The urinary excretion of VMA, HVA, and dopamine was normal (4.7 mgiday, 7.0 mg/ day, and 125.0 pg/day respectively). The primary tumor of the right neck and its regional metastatic lymph node were completely resected. Postoperative adjuvant chemotherapy was administered and she remained diseasefree for 17 months. Case 3. A 6-year-old boy was admitted because of a mediastinal mass. He was well until 1 month earlier, when a cough and chest pain developed. An X-ray film of the chest disclosed a large mediastinal tumor with calcification. The myelography revealed that the spinal cord was compressed by the tumor at the level of the seventh thoracic vertebra. Bilateral adrenals were of normal size. The urinary catecholamines and their metabolites were at normal levels (VMA 8.0 mg/day; HVA 2.9 mg/day; and dopamine 327 pg/day). Surgical resection was performed and the primary mediastinal mass was almost completely removed. After the procedure, aggressive chemotherapy was applied to the patient and he remained disease-free for 17 months. 492
Tissue Specimens
Tissue specimens for the present study were obtained from the primary tumors of the three patients described above. For indirect immunofluorescenceassay (IFA) and chromosomal examination, tissue specimens were immediately placed in RPMI 1640 culture medium with 10% fetal bovine serum (FBS), finely minced with scissors, and made into single cell suspension. For immunohistochemical study and electron microscopic examination tissue specimens were treated according to the conventional procedures.
TABLE I. Monoclonal Antibodies Used in the Present Study Designation Anti-T cell group OKT6 Anti-B cell group SmIg OKB2 Anti-NWK cell group HNK- 1 Anti-HLA-DR group OKIa 1 Anti-myeloid-monocyte group LeuM 1 Anti-leukemia associated group P1153/3 SJ-9A4 BA- 1
Expression Thymocyte Surface immunoglobulin B cell and granulocyte NWK cell HLA-DR antigens Myeloid-monocyte associated Leukemia-neuroblastoma associated Leukemia associated Leukemia associated
lmmunohistochemical Study
Five-micron-thick sections from paraffin blocks were soaked in methanol containing hydrogen peroxide to eliminate endogenous peroxidase activity. Next the sections were incubated with rabbit antibody against an a or p subunit of S-100 protein or neuron-specific enolase. After washing with PBS, these sections were stained by the PAP technique. Chromosomal Analysis
The cells prepared from tumor specimens were cultured in ES medium with 15% FBS. They were harvested and analyzed by means of regular Giemsa staining and the quinacrine banding technique. RESULTS Histological and Ultrastructural Findings
The primary tumors of the three patients were histologically very cellular in nature and composed of small Indirect lmrnunofluorescenceAssay (IFA) round cells, which were divided into small lobules by Cell samples (2 x lo5 cells) were incubated with Mo- fibrovascular stroma containing mats of neurofibrils and Abs listed in Table I at 4°C for 30 min in microtiter wells tangled networks of cytoplasm (Fig. 1). The tumor cells (Dynatech, Alexandria, Virginia). The cells were then seemed to be poorly differentiated and similar to lymwashed once with phosphate-buffered saline (PBS) con- phocytes or lymphoblasts. It is quite difficult to distintaining 5% FBS and subsequently incubated with fluo- guish them from ALL cells, lymphoma cells, Ewing’s rescein-conjugated goat anti-mouse immunoglobulin di- sarcoma cells, and rhabdomyosarcoma cells by light miluted 1:20 with PBS containing 0.08% sodium azide pH croscopic findings alone. In all cases, PAS staining was 7.4 for 30 min at 4°C. The cells were again washed with negative and rosette formations were not present. The PBS containing 5% FBS and then resuspended in PBS electron microscopic examination demonstrated that the containing 60% glycerol. All samples were examined for tumor cells of all three cases possessed a few dense core fluorescence by two investigators and at least 200 cells granules, having features of neurosecretory granules in per sample were examined. Results were expressed as the cytoplasm. In case 1, tumor cells had many interlacing processes containing some dense core granules (Fig. 2). percentage of fluorescence-positive cells.
Non-Adrenal Neuroblastomas lmmunohistochemical and lmmunofluorescence Study of Tumor Cells
Immunohistochemical analysis using the PAP technique revealed that tumor cells of all three cases reacted with anti-NSE antibody (Fig. 3). However none of them reacted with the antiserum against the a type of S-100 protein. Some tumor cells of case 1 showed weak reactivity with the antiserum against the p type of S-100 protein, but the tumor cells of cases 2 and 3 were not reactive at all. IFA using a panel of MoAbs was performed to characterize the surface phenotype of the tumor cells. The OKB2 and PI153/3 MoAbs showed strong reactivity with all three cases. The HNK-1, BA-1, and SJ-9A4 MoAbs reacted with two out of three cases. The rest of the MoAbs used in this study including OKT6, Leu M1, and OKIa1 did not show any significant reactivities with three tumor cells (Table II). Chromosomal Analysis (Table 111).
The tumor of case 1 showed normal karyotype without any structural abnormalities. The modal chromosomal number of case 2 was 68 without abnormality of chromosome 1, double minutes (DM), or homogeneously staining regions (HSR). In case 3, a reciprocal translocation involving the short arm of chromosome 1 was seen, but DM or HSR could not be identified (Fig. 4). The detailed chromosomal findings of cases 2 and 3 were reported elsewhere [8].
DISCUSSION
It is fairly difficult to diagnose neuroblastoma without increased urinary excretion of catecholamine metabolites. In a recent study, only 5% of patients with proven neuroblastomas failed to demonstrate increased urinary excretion of catecholamines and their metabolites [ 11. The etiologic possibilities include: 1) lack of activities for enzymes unique to catecholamine secreting neurons, 2) cholinergic tumors, which demonstrate neurosecretory granules on electron microscopic examination [4], and 3) immaturity of tumor cells. The diagnosis of small round cell tumor on the basis of morphology alone may be extremely difficult. Some investigators have attempted to identify tissue-specific analysis for each of these tumors that could be detected easily by immunohistochemical method [ 5 ] . Neuron-specific enolase is an enzyme of the yy type of glycolytic enzyme enolase, which has been shown to be fairly spe-
493
cific for neurons and neuroendocrine cells except for liver cells and differentiated rhabdomyosarcoma cells. Antiserum against NSE demonstrated the most specific reactivity against all three cases and was helpful for the definitive diagnosis. S-100 protein is a calcium-binding protein, which consists of a and p subunits. The aa type is specific for monocyte and macrophage. The ap type is specific for glial cell and myoepithelium. The pp type is revealed to be highly specific for Schwann cells and Langerhans cells. Although it has been reported that some of the differentiating neuroblastoma cells demonstrate s-100 positive reaction, none of the present cases revealed distinct reactivity, suggesting that these tumor cells might be poorly differentiated. Previous studies demonstrated that several MoAbs primarily developed against hematopoietic cells cross-reacted with neuroblastoma cells [6] and other solid tumor cells in childhood. The analysis of surface membrane antigen may be utilized for clinical diagnosis and subclassification of this tumor. The tumor cells in our three patients strongly reacted with HNK- 1 , OKB2, PI 153/3, SJ-9A4, and BA-1 MoAbs. The surface phenotypes of the presented cases were quite compatible with those of neuroblastoma cell lines. A recent report showed that the established neuroblastoma cell lines in vitro consisted of two morphologically distinct types-a neuroblast-like type and an epithelial-like type-and Leu l l b MoAb reacts with only epithelial-like cells, which lack tyrosine hydroxylase and dopamine hydroxylase activities, unique to the catecholamine secreting neuron [7]. Analysis of karyotypes has been useful for accurate diagnosis in some malignancies: for example, Philadelphia chromosome in chronic myelogenous leukemia, deletion of part of the short arm of chromosome l l in Wilms’ tumor, and a specific segmental deletion of the long arm of chromosome 13 in retinoblastoma. The rearrangements of the short arm of chromosome 1 and the presence of DM or HSR appeared to be relatively unique for advanced neuroblastoma [8]. Chromosomal analysis in case 3 showed a reciprocal translocation involving the short arm deletion of chromosome 1. It is suggested that chromosomal studies allow accurate diagnosis and prognosis. In conclusion, we tried the multidisciplinary study for the three patients with small round cell tumors, which had normal urinary VMA, HVA, and/or dopamine levels. These tumors were subsequently diagnosed accurately and treated with adequate therapeutic regimens after the surgical treatment. All of the three patients remain disease-free for 17 months. These results would suggest that the multidisciplinary study was helpful to make more accurate diagnosis for neuroblastomas.
Fig. 1 . The tumor of case 1 is very cellular and composed of small round cells with scant cytoplasm. Rosette formation is not present. Neurofibrils indicative of neuroblastoma are shown among the tumor cells ( x 700).
Fig. 2. Electron microscopic findings of case 1. There are many interlacing processes containing dense core granules ( x 37,500).
Non-Adrenal Neuroblastomas
Fig. 3. Immunohistochemical staining with anti-neuron-specific enolase serum in case 1. The majority of the tumor cells and intercellular fibers reveal positive staining ( X 700).
Fig. 4. The kaqotype of case 3. The arrows demonstrate the translocation between the short arm of chromosome 1 and the long arm of chromosome 11.
495
496
Sakatoku et al. TABLE 11. Reactivity With a Panel of Monoclonal Antibodies Against the Tumor Cells in Three Cases Monoclonal antibodies Patient Case 1 Case 2 Case 3
HNK-1 95.2 96.2 0.9
OKB2
BA-I
SJ-9A4
PI153/3
OKT6
Smlg
LeuMl
OKIal
92.7 96.2 65.7
91.9 11.8 92.0
8.9 46.9 43.0
80.9 44.3 68.3
0.6 0.0 0.6
3.5 0.6 1.4
0.0 0.0 6.2
7.1 2.8 2.4
TABLE 111. Chromosomal Findings of Three Cases Modal chromosomal Patient No. IP Case 1 46 Case 2 68 * Case 3 51 * = deletion of the short arm of chromosome 1; DM = double minute; HSR region.
ACKNOWLEDGMENTS
The authors thank Prof. Kazuaki Misugi and Yasuhiko Kaneko, MD, for their help with this manuscript.
4. 5.
REFERENCES 1. Siege1 SE, Walter EL, et al.: Pattern of urinary catecholamine metabolite excretion in neuroblastoma. In Evans AE (ed): “Advances in Neuroblastoma Research.” New York: Raven Press, 1980, pp. 25-32. 2. Tsokos M, Linnoila RI, et al.: Neuron-specific enolase in the diagnosis of neuroblastoma and other small, round-cell tumors in children. Hum Pathol 15575-584, 1984. 3. Berthold F, Kracht J, et al.: Ultrastructural, biochemical, and cellculture studies of a presumed extraskeletal Ewing’s sarcoma with
6.
7.
8.
DM
=
HSR -
-
homogeneously staining
special reference to differential diagnosis from neuroblastoma. J Cancer Res Clin Oncol 103:293-304, 1982. Kedar NP, et al.: Demonstration of cholinergic cells in human neuroblastoma and ganglioneuroma. J Pediatr 82:677-679, 1973. Misugi K, et al.: Nervous tissue specific proteins (S-100 & NSE) in pediatric tumors. Jpn J Pediatr Surg 18:19-25, 1986. Komada Y, Peiper SC, et al.: A monoclonal antibody (SJ-9A4) to P24 present on common ALLs, neuroblastomas and platelets-I. Characterization and development of a unique radioimmunometric assay. Leuk Res 7:487-498, 1983. Komada Y, Azuma E, Kamiya H, Sakurai M: Phenotypic profile of human neuroblastoma cell lines: Association with morphological characteristics. Br J Cancer 54:711-715, 1986. Kaneko Y, et al.: Different karyotypic patterns in early and advanced stage neuroblastomas. Cancer Res 47:311-318, 1987.
Histological, Immunohistochemical, and Chromosomal Studies on Non-Adrenal Neuroblastomas Without Increased Excretion of Catecholamines and Their Metabolites in Urine H. Sakatoku, MD, Y. Komada, MD, Y. Hamazaki, MD, H. Kamiya, MD, and M. Sakurai, MD We reviewed our records for the last 10 years and found three of the36 patients with neuroblastoma did not excrete significantly increased quantities of catecholamine metabolites in urine. All of these tumors were histologically characterized by small round cells and possessed a few dense core granules on the electron microscopic examination. All of them were reacted with antineuron-specific enolase (NSE) antibodies,
OKB2, P1153/3 monoclonal antibodies (MoAbs). The HNK-1, BA-I, and SJ-9A4MoAbs reacted with two out of three. One of them demonstrated a reciprocal translocation involving the short arm deletion of chrornosome 1. This multidisciplinary study has been helpful in making more accurate diagnoses for neuroblastoma without classical clinical characters.
Key words: neuroblastoma, catecholamine metabolites, urine
INTRODUCTION
It has been widely accepted that quantitation of catecholamine metabolites in urine is essential and critically important for the diagnosis of neuroblastoma. Only 5% of children with neuroblastoma, however, fail to excrete increased quantities of these substances [l]. In such cases, immunohistochemical and electron microscopic studies may be helpful in obtaining the accurate diagnosis [2,3]. In addition, several MoAbs were recently described to be reactive with neuroblastoma cells and could be used for the differential diagnosis of neuroblastoma from other small round cell tumors, including malignant lymphoma, acute lymphoblastic leukemia (ALL), rhabdomyosarcoma, and Ewing’s sarcoma. The purpose of this study is to report our experience with three children with neuroblastoma without significant increased excretion of catecholamine metabolites and to show the usefulness of the multidisciplinary studies including immunohistochemical analysis, electron microscopic examination, and chromosomal studies on the tumor cells in obtaining a definite diagnosis.
films of the chest demonstrated a large mass in the right mediastinum. She was referred to Mie University Hospital for definite diagnosis and treatment of the mediastinal tumor. A computed tomographic (CT) scan of the chest confirmed the presence of a solid tumor with speckled calcification lying on the right side of the thoracic vertebra. Abdominal CT scan and intravenous pyelography showed that bilateral adrenals were intact. Laboratory data was almost normal including the urinary excretion of vanillyl mandelic acid (VMA), homovanillic acid (HVA), and dopamine (8.0 mg/day, 8.6 mg/day, and 207.9 pg/day respectively). Followed by complete removal of the tumor, adjuvant chemotherapy was initiated and the patient remained disease-free for 17 months. Case 2. A 5-month-old girl was admitted because of a right cervical mass which was found 1 month before admission without tenderness or change of skin color. A CT scan of the neck revealed a solid mass without calcification and showed compression of trachea, right ca-
From the Department of Pediatrics, M i e University, School of Medicine, Mie (H.S., Y .K.,H.K., M.S.), and Department of Pathology, Shizuoka Children’s Hospital, Shizuoka (Y.H.), Japan.
MATERIALS AND METHODS Case Reports
Received February 18, 1988; accepted August 15, 1988.
Case 1. A 5-month-old girl was presented to her family doctor with a history of a persistent cough. X-ray
Address reprint requests to H. Sakatoku, Dept. of Pediatrics, Mie University, School of Medicine, Mie, Japan 514.
0 1990 Wiley-Liss, Inc.
Sakatoku et al. rotid artery, and sternocleidomastoideal muscle. Bilateral adrenals were intact. The urinary excretion of VMA, HVA, and dopamine was normal (4.7 mgiday, 7.0 mg/ day, and 125.0 pg/day respectively). The primary tumor of the right neck and its regional metastatic lymph node were completely resected. Postoperative adjuvant chemotherapy was administered and she remained diseasefree for 17 months. Case 3. A 6-year-old boy was admitted because of a mediastinal mass. He was well until 1 month earlier, when a cough and chest pain developed. An X-ray film of the chest disclosed a large mediastinal tumor with calcification. The myelography revealed that the spinal cord was compressed by the tumor at the level of the seventh thoracic vertebra. Bilateral adrenals were of normal size. The urinary catecholamines and their metabolites were at normal levels (VMA 8.0 mg/day; HVA 2.9 mg/day; and dopamine 327 pg/day). Surgical resection was performed and the primary mediastinal mass was almost completely removed. After the procedure, aggressive chemotherapy was applied to the patient and he remained disease-free for 17 months. 492
Tissue Specimens
Tissue specimens for the present study were obtained from the primary tumors of the three patients described above. For indirect immunofluorescenceassay (IFA) and chromosomal examination, tissue specimens were immediately placed in RPMI 1640 culture medium with 10% fetal bovine serum (FBS), finely minced with scissors, and made into single cell suspension. For immunohistochemical study and electron microscopic examination tissue specimens were treated according to the conventional procedures.
TABLE I. Monoclonal Antibodies Used in the Present Study Designation Anti-T cell group OKT6 Anti-B cell group SmIg OKB2 Anti-NWK cell group HNK- 1 Anti-HLA-DR group OKIa 1 Anti-myeloid-monocyte group LeuM 1 Anti-leukemia associated group P1153/3 SJ-9A4 BA- 1
Expression Thymocyte Surface immunoglobulin B cell and granulocyte NWK cell HLA-DR antigens Myeloid-monocyte associated Leukemia-neuroblastoma associated Leukemia associated Leukemia associated
lmmunohistochemical Study
Five-micron-thick sections from paraffin blocks were soaked in methanol containing hydrogen peroxide to eliminate endogenous peroxidase activity. Next the sections were incubated with rabbit antibody against an a or p subunit of S-100 protein or neuron-specific enolase. After washing with PBS, these sections were stained by the PAP technique. Chromosomal Analysis
The cells prepared from tumor specimens were cultured in ES medium with 15% FBS. They were harvested and analyzed by means of regular Giemsa staining and the quinacrine banding technique. RESULTS Histological and Ultrastructural Findings
The primary tumors of the three patients were histologically very cellular in nature and composed of small Indirect lmrnunofluorescenceAssay (IFA) round cells, which were divided into small lobules by Cell samples (2 x lo5 cells) were incubated with Mo- fibrovascular stroma containing mats of neurofibrils and Abs listed in Table I at 4°C for 30 min in microtiter wells tangled networks of cytoplasm (Fig. 1). The tumor cells (Dynatech, Alexandria, Virginia). The cells were then seemed to be poorly differentiated and similar to lymwashed once with phosphate-buffered saline (PBS) con- phocytes or lymphoblasts. It is quite difficult to distintaining 5% FBS and subsequently incubated with fluo- guish them from ALL cells, lymphoma cells, Ewing’s rescein-conjugated goat anti-mouse immunoglobulin di- sarcoma cells, and rhabdomyosarcoma cells by light miluted 1:20 with PBS containing 0.08% sodium azide pH croscopic findings alone. In all cases, PAS staining was 7.4 for 30 min at 4°C. The cells were again washed with negative and rosette formations were not present. The PBS containing 5% FBS and then resuspended in PBS electron microscopic examination demonstrated that the containing 60% glycerol. All samples were examined for tumor cells of all three cases possessed a few dense core fluorescence by two investigators and at least 200 cells granules, having features of neurosecretory granules in per sample were examined. Results were expressed as the cytoplasm. In case 1, tumor cells had many interlacing processes containing some dense core granules (Fig. 2). percentage of fluorescence-positive cells.
Non-Adrenal Neuroblastomas lmmunohistochemical and lmmunofluorescence Study of Tumor Cells
Immunohistochemical analysis using the PAP technique revealed that tumor cells of all three cases reacted with anti-NSE antibody (Fig. 3). However none of them reacted with the antiserum against the a type of S-100 protein. Some tumor cells of case 1 showed weak reactivity with the antiserum against the p type of S-100 protein, but the tumor cells of cases 2 and 3 were not reactive at all. IFA using a panel of MoAbs was performed to characterize the surface phenotype of the tumor cells. The OKB2 and PI153/3 MoAbs showed strong reactivity with all three cases. The HNK-1, BA-1, and SJ-9A4 MoAbs reacted with two out of three cases. The rest of the MoAbs used in this study including OKT6, Leu M1, and OKIa1 did not show any significant reactivities with three tumor cells (Table II). Chromosomal Analysis (Table 111).
The tumor of case 1 showed normal karyotype without any structural abnormalities. The modal chromosomal number of case 2 was 68 without abnormality of chromosome 1, double minutes (DM), or homogeneously staining regions (HSR). In case 3, a reciprocal translocation involving the short arm of chromosome 1 was seen, but DM or HSR could not be identified (Fig. 4). The detailed chromosomal findings of cases 2 and 3 were reported elsewhere [8].
DISCUSSION
It is fairly difficult to diagnose neuroblastoma without increased urinary excretion of catecholamine metabolites. In a recent study, only 5% of patients with proven neuroblastomas failed to demonstrate increased urinary excretion of catecholamines and their metabolites [ 11. The etiologic possibilities include: 1) lack of activities for enzymes unique to catecholamine secreting neurons, 2) cholinergic tumors, which demonstrate neurosecretory granules on electron microscopic examination [4], and 3) immaturity of tumor cells. The diagnosis of small round cell tumor on the basis of morphology alone may be extremely difficult. Some investigators have attempted to identify tissue-specific analysis for each of these tumors that could be detected easily by immunohistochemical method [ 5 ] . Neuron-specific enolase is an enzyme of the yy type of glycolytic enzyme enolase, which has been shown to be fairly spe-
493
cific for neurons and neuroendocrine cells except for liver cells and differentiated rhabdomyosarcoma cells. Antiserum against NSE demonstrated the most specific reactivity against all three cases and was helpful for the definitive diagnosis. S-100 protein is a calcium-binding protein, which consists of a and p subunits. The aa type is specific for monocyte and macrophage. The ap type is specific for glial cell and myoepithelium. The pp type is revealed to be highly specific for Schwann cells and Langerhans cells. Although it has been reported that some of the differentiating neuroblastoma cells demonstrate s-100 positive reaction, none of the present cases revealed distinct reactivity, suggesting that these tumor cells might be poorly differentiated. Previous studies demonstrated that several MoAbs primarily developed against hematopoietic cells cross-reacted with neuroblastoma cells [6] and other solid tumor cells in childhood. The analysis of surface membrane antigen may be utilized for clinical diagnosis and subclassification of this tumor. The tumor cells in our three patients strongly reacted with HNK- 1 , OKB2, PI 153/3, SJ-9A4, and BA-1 MoAbs. The surface phenotypes of the presented cases were quite compatible with those of neuroblastoma cell lines. A recent report showed that the established neuroblastoma cell lines in vitro consisted of two morphologically distinct types-a neuroblast-like type and an epithelial-like type-and Leu l l b MoAb reacts with only epithelial-like cells, which lack tyrosine hydroxylase and dopamine hydroxylase activities, unique to the catecholamine secreting neuron [7]. Analysis of karyotypes has been useful for accurate diagnosis in some malignancies: for example, Philadelphia chromosome in chronic myelogenous leukemia, deletion of part of the short arm of chromosome l l in Wilms’ tumor, and a specific segmental deletion of the long arm of chromosome 13 in retinoblastoma. The rearrangements of the short arm of chromosome 1 and the presence of DM or HSR appeared to be relatively unique for advanced neuroblastoma [8]. Chromosomal analysis in case 3 showed a reciprocal translocation involving the short arm deletion of chromosome 1. It is suggested that chromosomal studies allow accurate diagnosis and prognosis. In conclusion, we tried the multidisciplinary study for the three patients with small round cell tumors, which had normal urinary VMA, HVA, and/or dopamine levels. These tumors were subsequently diagnosed accurately and treated with adequate therapeutic regimens after the surgical treatment. All of the three patients remain disease-free for 17 months. These results would suggest that the multidisciplinary study was helpful to make more accurate diagnosis for neuroblastomas.
Fig. 1 . The tumor of case 1 is very cellular and composed of small round cells with scant cytoplasm. Rosette formation is not present. Neurofibrils indicative of neuroblastoma are shown among the tumor cells ( x 700).
Fig. 2. Electron microscopic findings of case 1. There are many interlacing processes containing dense core granules ( x 37,500).
Non-Adrenal Neuroblastomas
Fig. 3. Immunohistochemical staining with anti-neuron-specific enolase serum in case 1. The majority of the tumor cells and intercellular fibers reveal positive staining ( X 700).
Fig. 4. The kaqotype of case 3. The arrows demonstrate the translocation between the short arm of chromosome 1 and the long arm of chromosome 11.
495
496
Sakatoku et al. TABLE 11. Reactivity With a Panel of Monoclonal Antibodies Against the Tumor Cells in Three Cases Monoclonal antibodies Patient Case 1 Case 2 Case 3
HNK-1 95.2 96.2 0.9
OKB2
BA-I
SJ-9A4
PI153/3
OKT6
Smlg
LeuMl
OKIal
92.7 96.2 65.7
91.9 11.8 92.0
8.9 46.9 43.0
80.9 44.3 68.3
0.6 0.0 0.6
3.5 0.6 1.4
0.0 0.0 6.2
7.1 2.8 2.4
TABLE 111. Chromosomal Findings of Three Cases Modal chromosomal Patient No. IP Case 1 46 Case 2 68 * Case 3 51 * = deletion of the short arm of chromosome 1; DM = double minute; HSR region.
ACKNOWLEDGMENTS
The authors thank Prof. Kazuaki Misugi and Yasuhiko Kaneko, MD, for their help with this manuscript.
4. 5.
REFERENCES 1. Siege1 SE, Walter EL, et al.: Pattern of urinary catecholamine metabolite excretion in neuroblastoma. In Evans AE (ed): “Advances in Neuroblastoma Research.” New York: Raven Press, 1980, pp. 25-32. 2. Tsokos M, Linnoila RI, et al.: Neuron-specific enolase in the diagnosis of neuroblastoma and other small, round-cell tumors in children. Hum Pathol 15575-584, 1984. 3. Berthold F, Kracht J, et al.: Ultrastructural, biochemical, and cellculture studies of a presumed extraskeletal Ewing’s sarcoma with
6.
7.
8.
DM
=
HSR -
-
homogeneously staining
special reference to differential diagnosis from neuroblastoma. J Cancer Res Clin Oncol 103:293-304, 1982. Kedar NP, et al.: Demonstration of cholinergic cells in human neuroblastoma and ganglioneuroma. J Pediatr 82:677-679, 1973. Misugi K, et al.: Nervous tissue specific proteins (S-100 & NSE) in pediatric tumors. Jpn J Pediatr Surg 18:19-25, 1986. Komada Y, Peiper SC, et al.: A monoclonal antibody (SJ-9A4) to P24 present on common ALLs, neuroblastomas and platelets-I. Characterization and development of a unique radioimmunometric assay. Leuk Res 7:487-498, 1983. Komada Y, Azuma E, Kamiya H, Sakurai M: Phenotypic profile of human neuroblastoma cell lines: Association with morphological characteristics. Br J Cancer 54:711-715, 1986. Kaneko Y, et al.: Different karyotypic patterns in early and advanced stage neuroblastomas. Cancer Res 47:311-318, 1987.
