174 Enlarged and Prominent Nucleoli May Be Indicative of MYCN Amplification A Study of Neuroblastoma (Schwannian Stroma Poor), Undifferentiated/Poorly Differentiated Subtype with High Mitosis-Karyorrhexis Index Chie Kobayashi, M.D. 1 Hector L. Monforte-Munoz, M.D. 1 Robert B. Gerbing, M.A. 2 Daniel O. Stram, Ph.D. 3 Katherine K. Matthay, M.D. 4 John N. Lukens, M.D. 5 Robert C. Seeger, M.D. 6 Hiroyuki Shimada, M.D., Ph.D. 1 1 Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles/Keck School of Medicine, University of Southern California, Los Angeles, California. 2 Operations Office, Children s Oncology Group, Arcadia, California. 3 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California. 4 Department of Pediatrics, University of California San Francisco School of Medicine, San Francisco, California. 5 Division of Pediatric Hematology/Oncology, Vanderbilt Children s Hospital, Nashville, Tennessee. 6 Division of Hematology/Oncology, Childrens Hospital Los Angeles/Keck School of Medicine, University of Southern California, Los Angeles, California. Supported in part by Grant CA 13539 from the Division of Cancer Treatment, National Cancer Institute. The authors thank Dr. Inge M. Ambros (Children s Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria) for her generous support and encouragement throughout the course of the current study. The authors also thank Mrs. Judy Kelly for her assistance with article preparation. Address for reprints: Hiroyuki Shimada, M.D., Ph.D., Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, 4650 Sunset Blvd., M.S. #43, Los Angeles, CA 90027; Fax: (323) 667-1123; E-mail: hshimada@chla.usc.edu Received May 20, 2004; revision received September 1, 2004; accepted September 7, 2004. BACKGROUND. According to the International Neuroblastoma Pathology Classification, neuroblastomas exhibiting MYCN amplification (A-MYCN) have unique histologic features namely, undifferentiated/poorly differentiated subtype with a high mitosis-karyorrhexis index (U/PD-H). Nonetheless, certain tumors possessing these histologic characteristics contain a nonamplified MYCN gene (NA-MYCN). METHODS. The clinical characteristics of patients from the Children s Cancer Group (CCG) 3881 and 3891 studies who had neuroblastoma, U/PD-H, exhibiting A-MYCN (n 68) or NA-MYCN (n 33) were investigated. The histologic and cytologic features of tumors (A-MYCN, n 62; NA-MYCN, n 28) filed at the Pathology Reference Laboratory, Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, were reviewed, and nucleolar areas in undifferentiated neuroblastic cells were evaluated using image analysis methods. RESULTS. All 68 patients whose tumors exhibited A-MYCN had disease that was in an advanced clinical stage (Stage III or IV); 89.7% of these patients were diagnosed between ages 0.5 and 3.5 years, and 67 of the 68 had been treated with the high-risk protocol in the CCG-3891 study. Children whose tumors exhibited NA-MYCN were evenly distributed across all age groups; 30 of these 33 children had advanced-stage disease, and 26 had been treated with a high-risk protocol. The prognosis associated with A-MYCN (event free survival [EFS], 15.7%; overall survival [OS], 22.2%) was significantly poorer than the prognosis associated with NA-MYCN (EFS, 56.1%; OS, 69.3%). The lone histologic/cytologic difference between tumors exhibiting A-MYCN and tumors exhibiting NA-MYCN involved nucleolar appearance. Neuroblastic cells in tumors exhibiting A-MYCN were characterized by the presence of 1 or more large, prominent nucleoli, and the mean nucleolar area was significantly greater in the 18 tumors exhibiting A-MYCN that were assessed (7.63 m 2 ) than in the 16 tumors exhibiting NA-MYCN that were assessed (5.53 m 2 ; P 0.004). CONCLUSIONS. Neuroblastomas, U/PD-H, were found to vary in terms of molecular background and clinical behavior. The results of the current study indicate that nucleolar enlargement in neuroblastic cells may be a sign of MYCN amplification. Cancer 2005;103:174 80. 2004 American Cancer Society. KEYWORDS: neuroblastoma, International Neuroblastoma Pathology Classification, MYCN status, histology, cytology, nucleoli, morphometry, clinical characteristics, prognosis. Peripheral neuroblastic tumors (pnts [neuroblastomas, ganglioneuroblastomas, and ganglioneuromas]) make up a unique group of pediatric solid tumors. These tumors, which are derived from primordial neural crest cells, can be found in the adrenal medulla or in structures anatomically related to the sympathetic nervous system. In the past, pnts have been characterized as enigmatic neoplasms, due to their unpredict- 2004 American Cancer Society DOI 10.1002/cncr.20717 Published online 17 November 2004 in Wiley InterScience (www.interscience.wiley.com).
Large Nucleoli in MYCN-Amplified NB/Kobayashi et al. 175 able clinical behavior; possible clinical courses include involution, spontaneous regression, maturation, and aggressive progression. 1 In 1984, Shimada et al. 2 proposed an age-linked histologic classification system for pnts that included the following four categories: neuroblastoma; ganglioneuroblastoma, intermixed; ganglioneuroma; and ganglioneuroblastoma, nodular. Those investigators also described the possible clinical courses associated with tumors (especially neuroblastomas) possessing a given set of morphologic features. In 1999, the Shimada system was adopted as the International Neuroblastoma Pathology Classification, with neuroblastomas being separated into age-linked prognostic categories on the basis of grade of neuroblastic differentiation (undifferentiated, poorly differentiated, or differentiating) and mitosis-karyorrhexis index (MKI; low, 100 per 5000 cells; intermediate, 100 200 per 5000 cells; or high, 200 per 5000 cells). 3,4 These categories repeatedly proved to be significantly predictive of clinical outcome for patients with neuroblastoma. 5 MYCN gene amplification is known to be strongly predictive of poor clinical outcome for patients with pnts. 6,7 In addition, biologically and prognostically significant associations between histopathologic characteristics and MYCN status have been documented in pnts. 8 MYCN amplification, with the subsequent overexpression of MYCN protein, is considered to be a powerful driving force with regard to the prevention of neuroblastic differentiation and the up-regulation of mitotic and karyorrhectic activity. 9 In the Children s Cancer Group (CCG) 3881 and 3891 studies, 93.9% of all MYCN-amplified neuroblastomas (108 of 115) exhibited no neuroblastic differentiation, and 86.1% had either a high (71 of 115 [61.7%]) or intermediate MKI (28 of 115 [24.3%]). Thus, an MYCN-amplified tumor can most commonly be described as a neuroblastoma (Schwannian stroma poor) of undifferentiated or poorly differentiated subtype with high MKI (NBL, U/PD-H). 8 Despite these findings, some pnts without MYCN amplification also exhibit no neuroblastic differentiation and increased MKI. In the current study, we compared the clinical and morphologic characteristics of MYCN-amplified and MYCN-nonamplified tumors that were originally classified as NBL, U/PD-H, by the central pathologic review boards for the CCG-3881 and CCG-3891 studies. MATERIALS AND METHODS A total of 911 patients with pnts were enrolled in the CCG-3881 and CCG-3891 studies between August 1, 1991, and August 1, 1995. The CCG-3881 study was designed for patients in the low-risk and intermediaterisk categories; low-risk patients received surgery alone, 10 whereas intermediate-risk patients received surgery plus a moderate dose of chemotherapy. 11,12 In contrast, the CCG-3891 protocol, which was designed for high-risk patients, consisted of intensive chemotherapy with or without autologus bone marrow transplantation. 13 Appropriate informed consent procedures were followed, with consent being obtained from patients parents or guardians. Between these two studies, a total of 628 tumors were histopathologically evaluated and tested for MYCN status. Three-hundred thirty-nine tumors were found to have favorable histology (FH) and nonamplified MYCN (NA-MYCN), 8 were found to have FH and amplified MYCN (A-MYCN), 172 were found to have unfavorable histology (UH) and NA-MYCN, and 109 were found to have UH and A-MYCN. 14 Central histopathologic evaluation was performed at the Pathology Reference Laboratory, Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles (Los Angeles, CA), using the International Neuroblastoma Pathology Classification. Between 1989 and 1993, MYCN status was assessed via Southern blot analysis of gene copy numbers. Since 1993, immunoperoxidase staining has been used to detect MYCN protein expression, and semiquantitative polymerase chain reaction analysis has been used to evaluate MYCN gene copy numbers. MYCN was considered to be amplified if more than 10 copies of the gene were detected. 6,7 MYCN testing was performed at the CCG Neuroblastoma Reference Laboratory, Childrens Hospital Los Angeles. Of the cases that were evaluated, 101 were classified as NBL, U/PD-H, including 6 cases of undifferentiated subtype (NA-MYCN, n 2; A-MYCN, n 4) and 95 cases of poorly differentiated subtype (NA-MYCN, n 31; A-MYCN, n 64). In accordance with the International Neuroblastoma Pathology Classification, all cases of NBL, U/PD-H, were placed in the UH group, regardless of the patient s age at diagnosis. Clinical Information For the 101 patients with NBL, U/PD-H, clinical characteristics, including age, clinical stage, 15 and protocol assignment, were compared between the A-MYCN group (n 68) and the NA-MYCN group (n 33). Event-free survival (EFS) and overall survival (OS) from the time of study entry were analyzed using the Kaplan Meier method. 16 The log-rank test was used to compare EFS and OS between the A-MYCN group and the NA-MYCN group; these survival comparisons were performed for all 101 patients with NBL, U/PD-H, and
176 CANCER January 1, 2005 / Volume 103 / Number 1 TABLE I System for Assessment of Histologic/Cytologic Features of Neuroblastoma, Undifferentiated/Poorly Differentiated Subtype with High MKI Morphologic feature Cellularity a High Intermediate Low Amount of neuropil Amount of cytoplasm Cellular pleomorphism Nucleolar appearance Definition 2000 cells per hpf 1000 2000 cells per hpf 1000 cells per hpf Not detectable Barely detectable Easily detectable (occupying 50% of tumor tissue area) Abundant (occupying 50% of tumor tissue area) Not detectable Barely detectable Easily detectable Ample cytoplasm (diameter 2 times larger than nuclear diameter) Absent Focally present (25% of tumor tissue area) Present (25 50% of tumor tissue area) Diffusely present (50% of tumor tissue area) Barely detectable Easily detectable but not prominent Small but prominent Large and prominent MKI: mitosis-karyorrhexis index; hpf: high-power field. a Assessed using a BH-2 microscope (Olympus, Melville, NY) equipped with a superwide head (objective, 40; eyepiece, 10). also for the subset of 93 patients with NBL, U/PD-H, who were enrolled in the CCG-3891 (high-risk) study. Histologic/Cytologic Review Pathology slides from 90 of the 101 tumors examined had been filed at the Pathology Reference Laboratory and were available for review in the current study (range, 1 21 slides per tumor; mean, 4.8 slides per tumor). These 90 tumors included 62 from the A- MYCN group and 28 from the NA-MYCN group. The histologic and cytologic features investigated in the current study (by C.K., H.L.M.-M., and H.S.) included cellularity, amount of neuropil, amount of cytoplasm, degree of cellular pleomorphism, and nucleolar appearance (Table 1). Image Analysis for the Evaluation of Nucleolar Area Morphometric analyses of the nucleoli of neuroblastic cells in tumors from the A-MYCN group and the NA- MYCN group were performed. First, 16 tumors from the NA-MYCN group were selected for analysis (patient age range, 1.09 4.71 years; Stage III, n 4; Stage IV, n 12), and then 18 age- and stage-matched tumors (patient age range, 1.10 4.78 years; Stage III, n 3; Stage IV, n 15) were selected from the A-MYCN group. Representative high-resolution digital photomicrographs of each tumor were obtained using a DP-11 digital camera mounted on a BH-2 microscope (Olympus, Melville, NY) with a 100 oil immersion lens. Images were corrected for canvas size, color contrast, and brightness using Photoshop 6.0 software (Adobe, San Jose, CA), and corrected images subsequently were uploaded onto the BIOQUANT Image Analysis System (Nova Prime 6.50.10 MST; BIO- QUANT, Nashville, TN). Calibration was performed using an objective micrometer (Olympus) and the same 100 oil immersion objective lens that was used for photomicrography. For each tumor sample, morphometric analysis was performed using topography and area arrays, with nucleolar perimeter being manually delineated in 50 cells containing visible nucleoli. When multiple nucleoli were present in a single nucleus, an additive mode function was used to ensure that the reported value represented the nucleolar area per cell. The results of all array measurements were downloaded onto an Excel spreadsheet (Microsoft, Redmond, WA); average and median nucleolar areas, along with standard deviations, were calculated using the built-in formula functions. After these calculations were performed, the t test was used to compare the NA-MYCN group and the A-MYCN group with respect to average nucleolar area. RESULTS Clinical Characteristics The median age at diagnosis of NBL, U/PD-H, was 1.78 years (range, 0.0 10.79 years). The distribution of ages at diagnosis peaked between 1 and 1.5 years for patients in the A-MYCN group, with most patients in this group (61 of 68 [89.7%]) having been diagnosed between ages 0.5 and 3.5 years (Fig. 1). In contrast, patients in the NA-MYCN group were evenly distributed with respect to age at diagnosis, with patients as old as age 10.79 years being found in this group. All patients in the A-MYCN group had advanced-stage disease (Stage III, n 12; Stage IV, n 56). The vast majority of patients in the NA-MYCN group (30 of 33 [90.9%]) also had advanced-stage disease (Stage III, n 9; Stage IV, n 21), although 3 patients in this group had Stage II tumors. Sixty-seven patients in the A-MYCN group (age 1 year with Stage III disease, n 11; age 1 year with Stage IV disease, n 10; age 1 year with Stage IV disease, n 46) and 26 patients in the NA-MYCN group (age 1 year, n 26; Stage III disease, n 7; Stage IV disease, n 19) received the CCG-3891 (high-
Large Nucleoli in MYCN-Amplified NB/Kobayashi et al. 177 FIGURE 1. Distribution of ages at diagnosis for patients with neuroblastoma (Schwannian stroma poor), undifferentiated/poorly differentiated subtype with high mitosis-karyorrhexis index. MYCN-amplified cases, n 68; MYCNnonamplified cases, n 33. risk) protocol, whereas 1 patient in the A-MYCN group (age 1 year with Stage III disease) and 7 patients in the NA-MYCN group (age 1 year, n 7; Stage II disease, n 3; Stage III disease, n 2; Stage IV disease, n 2) received the CCG-3881 (low-/intermediate-risk) protocol. The expected 10-year EFS (15.7%) and OS rates (22.2%) for patients in the A-MYCN group were significantly lower than the corresponding rates (56.1% and 69.3%, respectively) for patients in the NA-MYCN group (P 0.0003 and P 0.0001, respectively). When only patients who received the CCG- 3891 protocol were considered, significant differences between the A-MYCN group and the NA-MYCN group in terms of EFS (14.2% vs. 52.9%; P 0.0006) and OS (20.8% vs. 61.3%; P 0.0011) remained (Fig. 2). Histologic/Cytologic Features Table 2 summarizes the histologic features of the 62 tumors in the A-MYCN group and the 28 tumors in the NA-MYCN group. There were no statistical differences between the A-MYCN group and the NA-MYCN group in terms of cellularity, amount of neuropil, amount of cytoplasm, or extent of cellular pleomorphism. Nucleoli were prominent (large or small) in the majority of tumors in both groups (NA-MYCN, 71.4%; A-MYCN, 88.7%); however, 1 or more large, prominent nucleoli were detected in more than half of all tumors in the A-MYCN group (33 of 62 [53.2%]), compared with only 14.3% of all tumors in the NA-MYCN group (4 of 28; P 0.0005) (Fig. 3). It was noted that nucleolar enlargement, if present in a given tumor, was evident in every microscopic field corresponding to that tumor. Nonetheless, the proportion of cells containing prominent nucleoli varied from tumor to tumor; in some cases, almost all FIGURE 2. Prognosis for high-risk patients with neuroblastoma (Schwannian stroma poor), undifferentiated/poorly differentiated subtype with high mitosiskaryorrhexis index, who received the Children s Cancer Group 3891 treatment protocol. (A) Event-free survival (P 0.006). (B) Overall survival (P 0.0011). NA-MYCN: nonamplified MYCN (n 26); A-MYCN: amplified MYCN (n 67). tumor cells contained prominent nucleoli, whereas other tumors were composed of a combination of tumor cells with and without prominent nucleoli. This variability may be attributable in part to poor sample preservation and/or fixation. Morphometric analysis (Fig. 4) revealed that nucleolar area was significantly larger in the A-MYCN group than in the NA-MYCN group. The mean nucleolar area for tumors in the A-MYCN group was 7.63 m 2, compared with 5.53 m 2 in the NA-MYCN group (P 0.004). DISCUSSION pnts represent one of the best models for investigating correlations between genotypic (i.e., molecular) properties and phenotypic (i.e., morphologic) manifestations. In the current study, we analyzed a group of neuroblastomas that had unique histologic features namely, poor or no differentiation and a high MKI. All tumors examined had UH according to the International Neuroblastoma Pathology Classification, and more than two-thirds of these tumors exhibited MYCN amplification. Thus, most of the patients included in the current study received the high-risk protocol in our previous clinical trial (CCG-3891). Our findings indicate that MYCN status (amplified or nonamplified) was significantly associated with age at diagnosis and clinical outcome. In addition, histologic/cytologic examination revealed that in tumors exhibiting MYCN amplification, the nuclear appearance of neuroblastic cells was characterized by the presence of one or more large, prominent nucleoli. NBL, U/PD-H, exhibiting MYCN amplification ac-
178 CANCER January 1, 2005 / Volume 103 / Number 1 TABLE II Morphologic Characteristics of Neuroblastoma, Undifferentiated/Poorly Differentiated Subtype with High MKI Characteristic A-MYCN No. of cases (%) NA-MYCN Cellularity High 54 (87.1) 22 (78.6) Intermediate 7 (11.3) 6 (21.4) Low 1 (1.6) 0 (0) Amount of neuropil 4 (6.5) 2 (7.1) 6 (9.7) 1 (3.6) 49 (79.0) 22 (78.6) 3 (4.8) 3 (10.7) 0 (0) 0 (0) Amount of cytoplasm 8 (12.9) 3 (10.7) 35 (56.5) 13 (46.4) 19 (30.6) 12 (42.9) 0 (0) 0 (0) Pleomorphism 46 (74.2) 20 (71.4) 10 (16.1) 4 (14.3) 3 (4.8) 3 (10.7) 3 (4.8) 1 (3.6) Nucleolar appearance 0 (0) 1 (3.6) 7 (11.3) 7 (25.0) 22 (35.5) 16 (57.1) 33 (53.2) 4 (14.3) Total 62 (100) 28 (100) MKI: mitosis-karyorrhexis index; A-MYCN: amplified MYCN; NA-MYCN: nonamplified MYCN. counted for 10.8% of all pnts in the CCG-3881 and CCG-3891 studies (68 of 628) and appeared to develop in patients with a relatively uniform set of clinical characteristics. Such tumors tended to be diagnosed in advanced clinical stages, with the distribution of patient ages at diagnosis showing a single peak between 0.5 and 1.5 years. Despite the use of an intensive treatment strategy, NBL, U/PD-H, with MYCN amplification was found to carry a poor prognosis. NBL, U/PD-H, without MYCN amplification was relatively rare, accounting for 5.3% of all pnts in the CCG-3881 and CCG-3891 studies (33 of 628). These tumors also were typically diagnosed in advanced clinical stages; however, unlike their counterparts in the A-MYCN group, tumors in the NA-MYCN group were diagnosed with comparable frequency across a range of ages, from infancy to later in childhood. Furthermore, among patients receiving intensive treatment, these tumors were associated with significantly higher survival rates compared with tumors that exhibited MYCN amplification. In the current report, we have confirmed the presence of a significant association between nucleolar enlargement and MYCN amplification in NBL, U/PD-H. Two other reports have documented an association between prominent nucleoli and poor clinical outcome in patients with neuroblastoma. 17,18 Nonetheless, to our knowledge, the current report is the first to clearly demonstrate a significant correlation between MYCN amplification and nucleolar enlargement in undifferentiated neuroblasts. Similar nucleolar enlargement has been observed in differentiating neuroblasts and ganglion cells in tumors that typically do not exhibit MYCN amplification. It should be noted that either single or multiple enlarged nucleoli were found in undifferentiated neuroblasts from tumors exhibiting MYCN amplification and that such nucleolar enlargement was never accompanied by cytoplasmic differentiation. In contrast, the prominent nucleoli found in differentiating neuroblasts and ganglion cells typically are single and situated in eccentrically located vesicular nuclei, and nucleolar enlargement normally is accompanied by cytoplasmic enlargement (cytoplasmic diameter 2 times the nuclear diameter) in this setting. In 1991, Matsumoto et al. 19 reported the presence of large, prominent nucleoli in proliferating cells from seven small cell lung carcinoma cell lines, with these cells exhibiting rapid growth, elevated S-phase fraction, and elevated c-myc or N-myc oncogene expression. In addition, using AgNOR staining (i.e., silver staining of proteins associated with nuclear organizing regions), both Pession et al. 20 (in seven neuroblastoma cell lines) and Derenzini et al. 21 (in xenograft models of seven colon carcinoma cell lines and three neuroblastoma cell lines) found that increased nucleolar area was significantly correlated with shorter tumor cell doubling time. Pession and colleagues also reported that the mean AgNOR-stained area in MYCNamplified neuroblastomas (n 9) was not significantly different from the corresponding area in MYCN-nonamplified neuroblastomas (n 39). Nonetheless, the current study, in which morphometric analysis was used to directly measure nucleolar area in hematoxylin and eosin stained sections, clearly demonstrated that the mean nucleolar area in neuroblastic cells was significantly larger in the A-MYCN group than in the NA-MYCN group. As has been reported in tissue culture cells 22,23 and mouse Sertoli cells, 23 the presence of large, prominent nucleoli in tumors in the A-MYCN group may be attributable to the nuclear localization and/or accumulation of the MYC transcript. Recent advances in clinical, translational, and basic research clearly suggest that the identification of molecular targets will be critical in the development of
Large Nucleoli in MYCN-Amplified NB/Kobayashi et al. 179 FIGURE 3. Typical nuclear features in neuroblastoma (Schwannian stroma poor), undifferentiated/poorly differentiated subtype with high mitosis-karyorrhexis index. (A) Large, prominent nucleoli (arrows) in neuroblastic cells from an MYCN-amplified tumor. (B) Prominent but small nucleoli (arrows) in neuroblastic cells from an MYCN-nonamplified tumor. Original magnification 1000. therapeutic strategies for the management of patients with pnts. In the current study, tumors histologically classified as NBL, U/PD-H, according to the International Neuroblastoma Pathology Classification were found to vary in terms of molecular background and clinical behavior. NBL, U/PD-H, exhibiting MYCN amplification appeared to constitute a uniform group of tumors that carried a poor prognosis, and these tumors were characterized by the frequent presence of one or more large, prominent nucleoli in undifferentiated neuroblastic cells. In contrast, the molecular properties of tumors in the NA-MYCN group have not yet been investigated. In conclusion, we believe that the data presented in the current genotypic, phenotypic, and clinical analysis of neuroblastomas will facilitate further collaboration among those investigating this highly complex disease. FIGURE 4. Mean nucleolar area as determined by image analysis in neuroblastoma (Schwannian stroma poor), undifferentiated/poorly differentiated subtype with high mitosis-karyorrhexis index. Each circle represents the average of 50 nucleolar areas from a given tumor. Horizontal bars represent group averages (P 0.004). A-MYCN: amplified MYCN (n 18); NA-MYCN: nonamplified MYCN (n 16). REFERENCES 1. Brodeur GM, Maris JM. Neuroblastoma. In: Pizzo PA, Poplack DG, editors. Principles and practice of pediatric oncology (4th edition). Philadelphia: Lippincott Williams & Wilkins, 2002:865 937. 2. Shimada H, Chatten J, Newton WA, et al. Histopathologic prognostic factors in neuroblastic tumors: definition of subtypes of ganglioneuroblastoma and an age-linked classification of neuroblastomas. J Natl Cancer Inst. 1984;73:405 413. 3. Shimada H, Ambros IM, Dehner LP, Hata J, Joshi VV, Roald B. Terminology and morphologic criteria of neuroblastic tumors. Recommendations by the International Neuroblastoma Pathology Committee. Cancer. 1999;86:349 363. 4. Shimada H, Umehara S, Monobe Y, et al. International neuroblastoma pathology classification for prognostic evaluation of patients with peripheral neuroblastic tumors. A report from the Children s Cancer Group. Cancer. 2001;92: 2451 2461. 5. Shimada H, Ambros IM, Dehner LP, et al. The International Neuroblastoma Pathology Classification (the Shimada system). Cancer. 1999;86:364 372. 6. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of N-myc in untreated human neuroblastoma correlates with advanced disease stage. Science. 1984;224: 1121 1124. 7. Seeger RC, Brodeur GM, Sather H, et al. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med. 1985;313:1111 1116.
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