- Email: [email protected]
N-myc amplification was rarely detected by fluorescence in situ hybridization in retinoblastoma
Human Pathology (2008) 39, 1172–1175
www.elsevier.com/locate/humpath
Original contribution
N-myc amplification was rarely detected by fluorescence in situ hybridization in retinoblastoma☆ Jeong Hun Kim MD, PhD a,1 , Jin Mi Choi BS b,1 , Young Suk Yu MD a,⁎, Dong Hun Kim MD, PhD c , Jin Hyoung Kim PhD a , Kyu-Won Kim PhD d a
Department of Ophthalmology, College of Medicine, Seoul National University, 110-744 Seoul, South Korea Department of Biochemistry, Queen's University, Kingston, K3L 3N6 Ontario, Canada c Department of Radiology, College of Medicine, Chosun University, 501-717 Gwangju, South Korea d NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 151-742 Seoul, South Korea b
Received 24 August 2007; revised 11 December 2007; accepted 12 December 2007
Keywords: Retinoblastoma; N-myc amplification; Fluorescence in situ hybridization (FISH)
Summary In retinoblastoma, genetic alteration of N-myc amplification different from the alteration of the RB1 gene on chromosome 13q14 has been described. This study is to determine the frequency of N-myc amplification by fluorescence in situ hybridization method in retinoblastoma. This study was prospectively derived from 26 patients who were diagnosed as having unilateral retinoblastoma (highly progressive large retinoblastoma, group 5 in Reese-Ellsworth classification) and underwent enucleation. We performed locus-specific fluorescence in situ hybridization probes for N-myc gene. Our results demonstrated that in only one of 26 patients was N-myc amplification found in retinoblastoma tissue. N-myc amplification has been regarded as one characteristic of retinoblastoma cell line and an adverse prognostic factor. However, our study indicates that N-myc amplification is not frequently found in retinoblastoma. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Retinoblastoma is the most common primary intraocular malignancy in children and is caused by homozygosity for a retinoblastoma gene mutation [1]. The retinoblastoma gene, located on 13q14, is a tumor suppressor in which identified target genes may lead to malignancy. One possible target ☆
This study was supported by a grant from the National R and D Program for Cancer Control, Ministry of Health and Welfare, Seoul, Republic of Korea (05-20240-1). ⁎ Corresponding author. Department of Ophthalmology, College of Medicine, Seoul National University, Seoul 110-744, South Korea. E-mail address: [email protected] (Y. S. Yu). 1 These authors contributed evenly to this work. 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2007.12.008
gene suggested by other studies is N-myc, which is considered to be an essential prognostic factor and is associated with poor prognosis in many neuroblastic tumors, including neuroblastoma [2]. The N-myc gene is a protooncogene that is located on 2p23-24 and encodes for a transcription factor in the family of bHLH-ZIP [3]. The transcription factor has an important regulatory function in early development for multiple differentiation pathways [4]. During neuroectodermal development and differentiation, N-myc gene has determining effects on neuronal/nonneuronal choice of neuronal crest cells [5]. However, N-myc gene expression level is found to be limited in adult organisms because its down-regulation is essential to complete its differentiation toward the specific phenotype [6,7]. It has been reported that down-regulation of
N-myc amplification in retinoblastoma N-myc gene in neuroblastoma cells results in decreased proliferation rate; thus, it has been concluded that N-myc gene regulates proliferation rate in neuroblastoma [7]. In addition to neuroblastoma, homogeneously staining regions involving the N-myc gene amplicons were detected by many studies involving the Y79 retinoblastoma cell line [2,8]. Despite N-myc amplification being a well-known characteristic of the Y79 cell line, the same overexpression could not be detected in retinoblastoma tissues with polymerase chain reaction (PCR) or Southern blot methods [9-11]. Fluorescence in situ hybridization (FISH) is a wellestablished technique for identification of aberrations in interphase nuclei and metaphase chromosomes. FISH is capable of analyzing gene overexpressions to a greater extent than PCR or Southern blot methods [4]. Numerous advantages of FISH over other conventional methods have been reported including its sensitivity, speed, technical simplicity, accuracy, resolution, efficiency, and reproducibility [12-16]. Particularly, it is possible with FISH to discern intercellular heterogeneity in gene amplification and to localize the source of the amplified N-myc signal [17,18]. Retinoblastoma tissues derived from 26 patients were subjected to FISH for detection of N-myc overexpression. Determination of the frequency of N-myc amplification raised the question of both the validity of the Y79 cell line and the identity of N-myc as a cytogenetic characteristic of retinoblastoma.
2. Materials and methods 2.1. Patients This study included 26 patients who were diagnosed as having unilateral retinoblastoma (highly progressive large retinoblastoma, group 5 in Reese-Ellsworth classification) in Seoul National University Children's Hospital and underwent enucleation without any prior treatments of chemotherapy or focal therapies.
2.2. Tissue preparation All human retinoblastoma tissue samples were obtained with informed consent and institutional review board approval and in accordance with the tenets of the Declaration of Helsinki. Primarily enucleated eyeballs from patients diagnosed with retinoblastoma were obtained from the Seoul National University Children's Hospital. After nucleation, the eyeballs were dissected. One part of the dissected eyeball was fixed by immersion in Carnoy solution for histologic examination and the other in 4% formalin for FISH study. Each specimen was then dehydrated through a series of graded ethanol solutions and embedded in paraffin using standard techniques. Paraffin-embedded eyes were sectioned into 4-μm sections. For histologic examination, sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin according to normal histologic procedures.
1173
2.3. Fish on retinoblastoma tissues Four-micrometer–thick paraffin sections were deparaffinized in xylene and dehydrated through gradient ethanol. Dried slides were incubated in 2× SSC solution at 75°C for 10 minutes followed by treatment with proteinase K solution (2 mg/mL) at 37°C for 15 minutes. The slides were rinsed in 2× SSC solution at room temperature. They were then dipped in a 75°C denaturant bath (50% formamide/2× SSC) for 5 minutes and dehydrated in gradient ethanol. Dry slides were placed on a 45°C to 50°C slide warmer. Probe mixture of green-labeled N-myc DNA probe (2p24 N-myc)/red-labeled chromosome 2– specific probe was denatured at 75°C. The probe mix was applied to the slides, which were hybridized in a prewarmed humidified chamber overnight (12-16 hours) at 37°C. Posthybridization washes were done in 0.4× SSC/0.3% NP-40 at 55°C followed by 2× SSC/0.1% NP-40 at room temperature. In series of negative control slides, FISH was performed with deleting the N-myc probe in the mixture. Counterstaining was accomplished with 4′-6-Diamidino-2phenylindole (DAPI). The signals were detected by using fluorescent microscope. We screened a large number of nuclei per sample so as not to miss any N-myc amplified cells. In each case, clear, distinct FISH signals were evaluated by counting 150 nonoverlapping nuclei.
3. Results Clinical findings and histologic prognostic factors of lamina cribrosa or choroidal invasion are summarized in Table 1. In pathological examination, the retinoblastomas, composed of small, round cells with scanty cytoplasm and hyperchromatic nuclei, occupied more than half of the vitreous cavity with or without some intravitreal seedings in all cases. In hematoxylin/eosin staining, all tumors were characterized by the extensive infiltration of small, round cells with high nuclear-to-cytoplasmic ratios, necrosis, and calcification, which were dispersed all over the tumors. Although the rosettes such as Flexner-Wintersteiner rosettes (n = 16, 62%) and Homer-Wright rosettes (n = 7, 27%) were encountered in the tumors, the frequency of fluerettes was rare. In 3 cases, invasion to lamina cribrosa was detected but without any invasion to the choroid.
Table 1 Clinical findings and pathological prognostic factors of patients Clinical and pathological findings of patients Age (mo) Sex (male-female) Infiltration to lamina cribrosa Infiltration to choroidal layer
17.8 (11-35) 15:11 3/26 0/26
1174
Fig. 1 FISH for N-myc amplification. A, Dual-color FISH shows one or two copies of chromosome 2 (red-green dots) in tumor cells of almost all study patients (25 of 26 patients). B, In only one of 26 patients were multiple N-myc signals (red dots) demonstrated. (Magnification ×400.)
The positivity of N-myc amplification by FISH was found in 3.8% (1/26) of patients. As shown in Fig. 1B, N-myc amplification was clearly seen with multiple N-myc signals of red dots. The tumor of positive N-myc amplification did not show the invasion to lamina cribrosa.
4. Discussion This study was undertaken to identify N-myc amplification and its validity as a cytogenetic marker in retinoblastoma tissue. The N-myc gene is generally accepted as a prognostic factor for neuroblastoma, a neuroectodermal cancer similar to retinoblastoma. Based on the similarities between retinoblastoma and neuroblastoma, several studies have attempted to show N-myc amplification in the Y79 retinoblastoma cell line or in tissue samples using various methods including Southern blot, in situ hybridization, and
J. H. Kim et al. reverse transcriptase (RT) PCR [2,8,19]. In these, N-myc amplification in retinoblastoma was demonstrated particularly in analyses using the Y79 cell lines. We previously found, in attempting to find proliferative indices, a relationship between N-myc amplification and proliferative indices, which was demonstrated to be associated with advanced tumor stage and poor clinical outcome [20]. However, even in tumor cells with N-myc gene amplified, a relationship between N-myc amplification and poor outcome could not be found [11,21]. Furthermore, many studies using Southern blot and RT-PCR were not able to find N-myc overexpression in retinoblastoma tumor tissues or cell lines [9-11]. Correspondingly, FISH performed in this study indicates no sign of N-myc amplification in all highly progressive retinoblastoma tested with the exception of one, showing good correlation with Southern blot and RT-PCR analyses. As previously mentioned, FISH is well capable of detecting single gene amplification even when the percentage of tumor in the samples is low [18]. Among currently available techniques, FISH is one of the most reliable methods to identify any chromosomal aberrations including DNA amplification. Thus, the results obtained using FISH in this study clearly demonstrates the absence of N-myc amplification in highly progressive retinoblastoma tissues. The N-myc gene is an essential cytogenetic marker for neuroblastoma and is used as a prognostic indicator; however; the overexpression of the gene is not always observed. It has been reported that the heterogeneity of N-myc amplification depends on degrees of differentiation of the tumor cells [4]. The relationship between N-myc amplification and the degree of tumor cell differentiation can also be elucidated in retinoblastoma tumors. Retinoblastoma tumors form in the eyes of young children, and the cells of origin of retinoblastoma tumors are suspected to be embryonic cells [9]. Retinoblastoma tumors induced by mutations in both RB-1 alleles prevent normal differentiation. The N-myc gene regulates cell cycle in early development but is no longer expressed as cells are terminally differentiated. Consequently, normal fetal retinas manifest normal expression levels of the N-myc gene, whereas adult retina does not have any expression. Therefore, expression level of N-myc will be sustained at the embryonic level if normal fetal retinal normal cell differentiation was prevented because of the loss of heterozygosity in retinoblastoma alleles. Thus, as a previous report, the expression of the N-myc gene in tumor cells may not necessarily be due to misregulation of RB-1, but may be due to the embryonic origin of the tumor [9]. From the results obtained in this investigation, N-myc amplification may not be essential in retinoblastoma malignancy. Therefore, N-myc amplification may be a characteristic of particular cell lines such as Y79, not a general characteristic of retinoblastoma. Further studies are needed to establish representative cell lines as well as cytogenetic markers for retinoblastoma.
N-myc amplification in retinoblastoma
References [1] Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci 1971;68:820-3. [2] Lee WH, Murphree AL, Benedict WF. Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 1984;309:458-60. [3] Galderisi U, Jori FP, Giordano A. Cell cycle regulation and neural differentiation. Oncogene 2003;22:5208-19. [4] Sartelet H, Grossi L, Pasquier D, et al. Detection of N-myc amplification by FISH in immature areas of fixed neuroblastomas: more efficient than southern blot/PCR. J Pathol 2002;198:83-91. [5] Wakamatsu Y, Watanabe Y, Nakamura H, et al. Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neural differentiation. Development 1997;124:1953-62. [6] Queva C, Hurlin PJ, Foley KP, et al. Sequential expression of the MAD family of transcriptional repressors during differentiation and development. Oncogene 1998;16:967-77. [7] Galderisi U, Di Bernardo G, Cipollaro M, et al. Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product. J Cell Biochem 1999;73:97-105. [8] Bartova E, Kozubek S, Gajova H, et al. Cytogenetics and cytology of retinoblastomas. J Cancer Res Clin Oncol 2003;129:89-99. [9] Squire J, Goddard AD, Canton M, et al. Tumour induction by the retinoblastoma mutation is independent of N-myc expression. Nature 1986;322:555-7. [10] Choi SW, Lee TW, Yang SW, et al. Loss of retinoblastoma gene and amplification of N-myc gene in retinoblastoma. J Korean Med Sci 1993;8:73-7. [11] Lillington DM, Goff LK, Kingston JE, et al. High level amplification of N-myc is not associated with adverse histology or outcome in primary retinoblastoma tumours. Br J Cancer 2002;87:779-82.
1175 [12] Tajiri T, Shono K, Fujii Y, et al. Highly sensitive analysis for N-myc amplification in neuroblastoma based on fluorescence in situ hybridization. J Pediatr Surg 1999;34:1615-9. [13] Hachitanda Y, Saito M, Mori T, et al. Application of fluorescence in situ hybridization to detect N-myc gene amplification on paraffinembedded tissue sections of neuroblastoma. Med Pediatr Oncol 1997; 29:135-8. [14] Eckschlager T, McClain K. Comparison of fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR) for detection of residual neuroblastoma cells. Neoplasma 1996;43:301-3. [15] Aldosari N, Bigner SH, Burger PC, et al. MYCC and MYCN oncogene amplification in medulloblastoma. A fluorescence in situ hybridization study on paraffin sections from the Children's Oncology Group. Arch Pathol Lab Med 2002;126:540-4. [16] Visakorpi T, Hyytinen E, Kallioniemi A, et al. Sensitive detection of chromosome copy number aberrations in prostate cancer by fluorescence in situ hybridization. Am J Pathol 1994;145:624-30. [17] Kucheria K, Jobanputra V, Talwar R, et al. Human molecular cytogenetics: diagnosis, prognosis, and disease management. Teratog Carcinog Mutagen 2003;S1:225-33. [18] Shapiro DN, Valentine MB, Rowe ST, et al. Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma. Am J Pathol 1993;142:1339-46. [19] Sakai K, Kanda N, Shiloh Y, et al. Molecular and cytologic analysis of DNA amplification in retinoblastoma. Cancer Genet Cytogenet 1985; 17:95-112. [20] Kim CJ, Chi JG, Choi HS, et al. Proliferation not apoptosis as a prognostic indicator in retinoblastoma. Virchows Arch 1999;434: 301-5. [21] Mairal A, Pinglier E, Gilbert E, et al. Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses. Genes Chromosome Cancer 2000;28:370-9.
www.elsevier.com/locate/humpath
Original contribution
N-myc amplification was rarely detected by fluorescence in situ hybridization in retinoblastoma☆ Jeong Hun Kim MD, PhD a,1 , Jin Mi Choi BS b,1 , Young Suk Yu MD a,⁎, Dong Hun Kim MD, PhD c , Jin Hyoung Kim PhD a , Kyu-Won Kim PhD d a
Department of Ophthalmology, College of Medicine, Seoul National University, 110-744 Seoul, South Korea Department of Biochemistry, Queen's University, Kingston, K3L 3N6 Ontario, Canada c Department of Radiology, College of Medicine, Chosun University, 501-717 Gwangju, South Korea d NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, 151-742 Seoul, South Korea b
Received 24 August 2007; revised 11 December 2007; accepted 12 December 2007
Keywords: Retinoblastoma; N-myc amplification; Fluorescence in situ hybridization (FISH)
Summary In retinoblastoma, genetic alteration of N-myc amplification different from the alteration of the RB1 gene on chromosome 13q14 has been described. This study is to determine the frequency of N-myc amplification by fluorescence in situ hybridization method in retinoblastoma. This study was prospectively derived from 26 patients who were diagnosed as having unilateral retinoblastoma (highly progressive large retinoblastoma, group 5 in Reese-Ellsworth classification) and underwent enucleation. We performed locus-specific fluorescence in situ hybridization probes for N-myc gene. Our results demonstrated that in only one of 26 patients was N-myc amplification found in retinoblastoma tissue. N-myc amplification has been regarded as one characteristic of retinoblastoma cell line and an adverse prognostic factor. However, our study indicates that N-myc amplification is not frequently found in retinoblastoma. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Retinoblastoma is the most common primary intraocular malignancy in children and is caused by homozygosity for a retinoblastoma gene mutation [1]. The retinoblastoma gene, located on 13q14, is a tumor suppressor in which identified target genes may lead to malignancy. One possible target ☆
This study was supported by a grant from the National R and D Program for Cancer Control, Ministry of Health and Welfare, Seoul, Republic of Korea (05-20240-1). ⁎ Corresponding author. Department of Ophthalmology, College of Medicine, Seoul National University, Seoul 110-744, South Korea. E-mail address: [email protected] (Y. S. Yu). 1 These authors contributed evenly to this work. 0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2007.12.008
gene suggested by other studies is N-myc, which is considered to be an essential prognostic factor and is associated with poor prognosis in many neuroblastic tumors, including neuroblastoma [2]. The N-myc gene is a protooncogene that is located on 2p23-24 and encodes for a transcription factor in the family of bHLH-ZIP [3]. The transcription factor has an important regulatory function in early development for multiple differentiation pathways [4]. During neuroectodermal development and differentiation, N-myc gene has determining effects on neuronal/nonneuronal choice of neuronal crest cells [5]. However, N-myc gene expression level is found to be limited in adult organisms because its down-regulation is essential to complete its differentiation toward the specific phenotype [6,7]. It has been reported that down-regulation of
N-myc amplification in retinoblastoma N-myc gene in neuroblastoma cells results in decreased proliferation rate; thus, it has been concluded that N-myc gene regulates proliferation rate in neuroblastoma [7]. In addition to neuroblastoma, homogeneously staining regions involving the N-myc gene amplicons were detected by many studies involving the Y79 retinoblastoma cell line [2,8]. Despite N-myc amplification being a well-known characteristic of the Y79 cell line, the same overexpression could not be detected in retinoblastoma tissues with polymerase chain reaction (PCR) or Southern blot methods [9-11]. Fluorescence in situ hybridization (FISH) is a wellestablished technique for identification of aberrations in interphase nuclei and metaphase chromosomes. FISH is capable of analyzing gene overexpressions to a greater extent than PCR or Southern blot methods [4]. Numerous advantages of FISH over other conventional methods have been reported including its sensitivity, speed, technical simplicity, accuracy, resolution, efficiency, and reproducibility [12-16]. Particularly, it is possible with FISH to discern intercellular heterogeneity in gene amplification and to localize the source of the amplified N-myc signal [17,18]. Retinoblastoma tissues derived from 26 patients were subjected to FISH for detection of N-myc overexpression. Determination of the frequency of N-myc amplification raised the question of both the validity of the Y79 cell line and the identity of N-myc as a cytogenetic characteristic of retinoblastoma.
2. Materials and methods 2.1. Patients This study included 26 patients who were diagnosed as having unilateral retinoblastoma (highly progressive large retinoblastoma, group 5 in Reese-Ellsworth classification) in Seoul National University Children's Hospital and underwent enucleation without any prior treatments of chemotherapy or focal therapies.
2.2. Tissue preparation All human retinoblastoma tissue samples were obtained with informed consent and institutional review board approval and in accordance with the tenets of the Declaration of Helsinki. Primarily enucleated eyeballs from patients diagnosed with retinoblastoma were obtained from the Seoul National University Children's Hospital. After nucleation, the eyeballs were dissected. One part of the dissected eyeball was fixed by immersion in Carnoy solution for histologic examination and the other in 4% formalin for FISH study. Each specimen was then dehydrated through a series of graded ethanol solutions and embedded in paraffin using standard techniques. Paraffin-embedded eyes were sectioned into 4-μm sections. For histologic examination, sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin according to normal histologic procedures.
1173
2.3. Fish on retinoblastoma tissues Four-micrometer–thick paraffin sections were deparaffinized in xylene and dehydrated through gradient ethanol. Dried slides were incubated in 2× SSC solution at 75°C for 10 minutes followed by treatment with proteinase K solution (2 mg/mL) at 37°C for 15 minutes. The slides were rinsed in 2× SSC solution at room temperature. They were then dipped in a 75°C denaturant bath (50% formamide/2× SSC) for 5 minutes and dehydrated in gradient ethanol. Dry slides were placed on a 45°C to 50°C slide warmer. Probe mixture of green-labeled N-myc DNA probe (2p24 N-myc)/red-labeled chromosome 2– specific probe was denatured at 75°C. The probe mix was applied to the slides, which were hybridized in a prewarmed humidified chamber overnight (12-16 hours) at 37°C. Posthybridization washes were done in 0.4× SSC/0.3% NP-40 at 55°C followed by 2× SSC/0.1% NP-40 at room temperature. In series of negative control slides, FISH was performed with deleting the N-myc probe in the mixture. Counterstaining was accomplished with 4′-6-Diamidino-2phenylindole (DAPI). The signals were detected by using fluorescent microscope. We screened a large number of nuclei per sample so as not to miss any N-myc amplified cells. In each case, clear, distinct FISH signals were evaluated by counting 150 nonoverlapping nuclei.
3. Results Clinical findings and histologic prognostic factors of lamina cribrosa or choroidal invasion are summarized in Table 1. In pathological examination, the retinoblastomas, composed of small, round cells with scanty cytoplasm and hyperchromatic nuclei, occupied more than half of the vitreous cavity with or without some intravitreal seedings in all cases. In hematoxylin/eosin staining, all tumors were characterized by the extensive infiltration of small, round cells with high nuclear-to-cytoplasmic ratios, necrosis, and calcification, which were dispersed all over the tumors. Although the rosettes such as Flexner-Wintersteiner rosettes (n = 16, 62%) and Homer-Wright rosettes (n = 7, 27%) were encountered in the tumors, the frequency of fluerettes was rare. In 3 cases, invasion to lamina cribrosa was detected but without any invasion to the choroid.
Table 1 Clinical findings and pathological prognostic factors of patients Clinical and pathological findings of patients Age (mo) Sex (male-female) Infiltration to lamina cribrosa Infiltration to choroidal layer
17.8 (11-35) 15:11 3/26 0/26
1174
Fig. 1 FISH for N-myc amplification. A, Dual-color FISH shows one or two copies of chromosome 2 (red-green dots) in tumor cells of almost all study patients (25 of 26 patients). B, In only one of 26 patients were multiple N-myc signals (red dots) demonstrated. (Magnification ×400.)
The positivity of N-myc amplification by FISH was found in 3.8% (1/26) of patients. As shown in Fig. 1B, N-myc amplification was clearly seen with multiple N-myc signals of red dots. The tumor of positive N-myc amplification did not show the invasion to lamina cribrosa.
4. Discussion This study was undertaken to identify N-myc amplification and its validity as a cytogenetic marker in retinoblastoma tissue. The N-myc gene is generally accepted as a prognostic factor for neuroblastoma, a neuroectodermal cancer similar to retinoblastoma. Based on the similarities between retinoblastoma and neuroblastoma, several studies have attempted to show N-myc amplification in the Y79 retinoblastoma cell line or in tissue samples using various methods including Southern blot, in situ hybridization, and
J. H. Kim et al. reverse transcriptase (RT) PCR [2,8,19]. In these, N-myc amplification in retinoblastoma was demonstrated particularly in analyses using the Y79 cell lines. We previously found, in attempting to find proliferative indices, a relationship between N-myc amplification and proliferative indices, which was demonstrated to be associated with advanced tumor stage and poor clinical outcome [20]. However, even in tumor cells with N-myc gene amplified, a relationship between N-myc amplification and poor outcome could not be found [11,21]. Furthermore, many studies using Southern blot and RT-PCR were not able to find N-myc overexpression in retinoblastoma tumor tissues or cell lines [9-11]. Correspondingly, FISH performed in this study indicates no sign of N-myc amplification in all highly progressive retinoblastoma tested with the exception of one, showing good correlation with Southern blot and RT-PCR analyses. As previously mentioned, FISH is well capable of detecting single gene amplification even when the percentage of tumor in the samples is low [18]. Among currently available techniques, FISH is one of the most reliable methods to identify any chromosomal aberrations including DNA amplification. Thus, the results obtained using FISH in this study clearly demonstrates the absence of N-myc amplification in highly progressive retinoblastoma tissues. The N-myc gene is an essential cytogenetic marker for neuroblastoma and is used as a prognostic indicator; however; the overexpression of the gene is not always observed. It has been reported that the heterogeneity of N-myc amplification depends on degrees of differentiation of the tumor cells [4]. The relationship between N-myc amplification and the degree of tumor cell differentiation can also be elucidated in retinoblastoma tumors. Retinoblastoma tumors form in the eyes of young children, and the cells of origin of retinoblastoma tumors are suspected to be embryonic cells [9]. Retinoblastoma tumors induced by mutations in both RB-1 alleles prevent normal differentiation. The N-myc gene regulates cell cycle in early development but is no longer expressed as cells are terminally differentiated. Consequently, normal fetal retinas manifest normal expression levels of the N-myc gene, whereas adult retina does not have any expression. Therefore, expression level of N-myc will be sustained at the embryonic level if normal fetal retinal normal cell differentiation was prevented because of the loss of heterozygosity in retinoblastoma alleles. Thus, as a previous report, the expression of the N-myc gene in tumor cells may not necessarily be due to misregulation of RB-1, but may be due to the embryonic origin of the tumor [9]. From the results obtained in this investigation, N-myc amplification may not be essential in retinoblastoma malignancy. Therefore, N-myc amplification may be a characteristic of particular cell lines such as Y79, not a general characteristic of retinoblastoma. Further studies are needed to establish representative cell lines as well as cytogenetic markers for retinoblastoma.
N-myc amplification in retinoblastoma
References [1] Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci 1971;68:820-3. [2] Lee WH, Murphree AL, Benedict WF. Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 1984;309:458-60. [3] Galderisi U, Jori FP, Giordano A. Cell cycle regulation and neural differentiation. Oncogene 2003;22:5208-19. [4] Sartelet H, Grossi L, Pasquier D, et al. Detection of N-myc amplification by FISH in immature areas of fixed neuroblastomas: more efficient than southern blot/PCR. J Pathol 2002;198:83-91. [5] Wakamatsu Y, Watanabe Y, Nakamura H, et al. Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neural differentiation. Development 1997;124:1953-62. [6] Queva C, Hurlin PJ, Foley KP, et al. Sequential expression of the MAD family of transcriptional repressors during differentiation and development. Oncogene 1998;16:967-77. [7] Galderisi U, Di Bernardo G, Cipollaro M, et al. Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product. J Cell Biochem 1999;73:97-105. [8] Bartova E, Kozubek S, Gajova H, et al. Cytogenetics and cytology of retinoblastomas. J Cancer Res Clin Oncol 2003;129:89-99. [9] Squire J, Goddard AD, Canton M, et al. Tumour induction by the retinoblastoma mutation is independent of N-myc expression. Nature 1986;322:555-7. [10] Choi SW, Lee TW, Yang SW, et al. Loss of retinoblastoma gene and amplification of N-myc gene in retinoblastoma. J Korean Med Sci 1993;8:73-7. [11] Lillington DM, Goff LK, Kingston JE, et al. High level amplification of N-myc is not associated with adverse histology or outcome in primary retinoblastoma tumours. Br J Cancer 2002;87:779-82.
1175 [12] Tajiri T, Shono K, Fujii Y, et al. Highly sensitive analysis for N-myc amplification in neuroblastoma based on fluorescence in situ hybridization. J Pediatr Surg 1999;34:1615-9. [13] Hachitanda Y, Saito M, Mori T, et al. Application of fluorescence in situ hybridization to detect N-myc gene amplification on paraffinembedded tissue sections of neuroblastoma. Med Pediatr Oncol 1997; 29:135-8. [14] Eckschlager T, McClain K. Comparison of fluorescent in situ hybridization (FISH) and the polymerase chain reaction (PCR) for detection of residual neuroblastoma cells. Neoplasma 1996;43:301-3. [15] Aldosari N, Bigner SH, Burger PC, et al. MYCC and MYCN oncogene amplification in medulloblastoma. A fluorescence in situ hybridization study on paraffin sections from the Children's Oncology Group. Arch Pathol Lab Med 2002;126:540-4. [16] Visakorpi T, Hyytinen E, Kallioniemi A, et al. Sensitive detection of chromosome copy number aberrations in prostate cancer by fluorescence in situ hybridization. Am J Pathol 1994;145:624-30. [17] Kucheria K, Jobanputra V, Talwar R, et al. Human molecular cytogenetics: diagnosis, prognosis, and disease management. Teratog Carcinog Mutagen 2003;S1:225-33. [18] Shapiro DN, Valentine MB, Rowe ST, et al. Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma. Am J Pathol 1993;142:1339-46. [19] Sakai K, Kanda N, Shiloh Y, et al. Molecular and cytologic analysis of DNA amplification in retinoblastoma. Cancer Genet Cytogenet 1985; 17:95-112. [20] Kim CJ, Chi JG, Choi HS, et al. Proliferation not apoptosis as a prognostic indicator in retinoblastoma. Virchows Arch 1999;434: 301-5. [21] Mairal A, Pinglier E, Gilbert E, et al. Detection of chromosome imbalances in retinoblastoma by parallel karyotype and CGH analyses. Genes Chromosome Cancer 2000;28:370-9.