- Email: [email protected]
Detection of MYCN amplification in three neuroblastoma cell lines by non-radioactive chromosomal in situ hybridization
Detection of MYCN Amplification in Three Neuroblastoma Cell Lines by Non-Radioactive Chromosomal In Situ Hybridization Tracey L. McRobert, Christina Rudduck, Ursula R. Kees, and O. Margaret Garson
ABSTRACT: A non-radioactive chromosomal in situ hybridization technique utilizing a bintin-streptavidin-polyalkaline-phosphatase complex was successfully applied to three ne,urablastoma cell lines for detection of MYCN amplification. These cell lines, designated PER-106, PER-107, and PER-108, were derived from consecutive bone marrow samples taken from a patient with stage IV neuroblastoma. The cell line derived at diagnosis (PER-106) exhibited MYCN amplification in the form of variable numbers of double-minute chromosomes, small fragments, and rings of varying sizes. This observed variability of MYCN amplification may explain the reported heterogeneity of both MYCN mRNA and protein expression among individual cells of some neuroblastomas. The cell lines derived from subsequent samples (PER-107 and PER-108) contained amplified MYCN as two consistent homogeneously staining regions in every cell. These, were located on the shor~ arms of chromosomes 6 and 14. Thus, amplified MYCN was identified in each cell line and demonstrated the, concurrent evolution of amplification with cytogenetic abnormalities.
INTRODUCTION Neuroblastoma is one of the more c o m m o n solid tumors of early c h i l d h o o d [1]. It is an aggressive tumor with a highly variable clinical course where the majority of (:ases (70%) have undergone metastatic spread at the time of diagnosis [2]. Prognosis of children with neuroblastoma is greatly influenced by the stage of disease at diagnosis, where Evans stages IlI and IV have the worst prognosis 13]. Other prognostic factors include age at diagnosis, site of the primary tumor, and urinary catecholamine levels. In 1983, the MYCN oncogene was first identified in human neuroblastoma as an amplified gene having a limited sequence homology to the c-myc oncogene. It was isolated from homogeneously staining region (hsr) and doubleminute (dmin)-bearing neuroblastoma cells by several research groups [4-6] and was found to map to region 2p24 in normal cells [71. The MYCN oncogene may be amplified in both neuroblastoma tumors and cell lines and correlates strongly with rapid disease progression and poor progno-
From the Department ofCytagenetics (T. L. M., C. R., O. M. G.), St. Vincent's ltaspital. Fitzroy, Victoria. Australia~ and Leukaemia llesearch Laboratory {U. R. K.I, Clinical Immunolog)" Research Unit. Princess Margaret Hospital, Perth. Western Australia. Address reprint requests to: Associate ProJessor O. M. (;arsan. Department of Cytogenetics, St. Vincent's tlospital. Fitzroy. Victaria, Australia, 3065. Received April 10, 1991; accepted September 26, 1991. 128 Cancer Genet Cytogene.l59:128-134 11992) 0165-4608,'92~$05,00
sis [8]. Therefore, it is an i n d e p e n d e n t prognostic indicator that may be clinically useful as a guide for treating even the favorable stages II and IV-S of neuroblastoma [9, 10]. Detection of MYCN amplification is usually achieved by Southern analysis, a technique which allows an estimate of MYCN copy number for the overall cell population. Most studies have shown MYCN amplification to be consistent in the i n d i v i d u a l tumor examined at different times in vivo [11, 12]. If it is not present at diagnosis, MYCN amplification does not seem to appear with disease progression. Conversely, progressive disease is very likely to occ:ur in patients with MYCN amplification at diagnosis [13]. However, heterogeneity of amplification between individual cells, if present, will not be detected using Southern analysis. Another approach is to detect MYCN amplification by (:hromosomal in situ hybridization, which is traditionally performed using radioactively labeled probes [141. Recently, nonradioactive systems have been developed using biotinylated probes detected by a variety of immunogenic, fluorescent, and enzymatic labels. These techniques are quick, yielding a result within 24 hours, they have high resolution, and although they are not quite as sensitive as radioactive techniques, unique sequences as small as 3 kb have been detected [15]. We have therefore used a biotin-streptavidin-polyalkaline-phosphatase system to investigate the MYCN amplification in three neuroblastoma cell lines. By studying (:ell lines derived from a patient with neuroblastoma at three different stages of the disease, it was possible to determine O 1992 Elsevier Science Publishing Co.. lilt:. 655 ..\vmumof the Americas. New York. NY lo()10
M Y C N Amplification in Neuroblastoma
how gene amplification and cytogenetic abnormalities evolved with disease progression.
MATERIALS AND METHODS Case Report A 17-month-old Caucasian boy presented in October, 1984 with disseminated neuroblastoma including gross bone marrow involvement (Stage IV). He responded to chemotherapy and the residual primary tumor was subsequently exc:ised, but the disease recurred 13 months later, again showing gross marrow involvement together with skull and orbital secondaries. Death occurred 15 months after diagnosis, at which time the marrow was almost c:ompletely replaced by tumor cell clumps. Three cell lines were derived from consecutive bone marrow samples taken at diagnosis, and 13 and 15 months after diagnosis. These were designated PER-106, PER-107, and PER-108 respectively, with PER-106 consisting mainly of cells growing in suspension and forming large aggregates of small round cells [16]. PER 107 and 108, derived after disease recurrence, grew substrate adherent. Chromosome Preparation The neuroblastoma cells were cultured in RPMI 1640 medium with 20% Fetal Calf Serum (FCS)/Penic:illin-Streptomycin (50 IU/mL), L-Glutamine (2 mM), 2-mercaptoethanol (10 5 M), pyruvate (1 mM), and non-essential amino acids, in tissue culture flasks in a humidified 37°C incubator containing 5% CO2 in air. The fluorodeoxyuridine synchronization technique of Webber and Garson [17] was used to improve the quantity and quality of metaphase spreads. Harvesting commenc:ed after an 8-hour release time. Colcemid was added to give a final c:onc:entration of 0.8 p.g/mL and cultures were then incubated for either 1.5 hours or overnight, at 37°C. Adherent cells were detached with Trypsin : EDTA (Flow Laboratories "16-89149) at 37°C for 5 minutes. Chromosome preparations were made by standard cytogenetic techniques and were trypsin (;-banded [18J and C-banded [19]. Karyotypes designated according to the classification of the International System for Human Cytogenetic: Nomenc:lature [20] were constructed from each cell line. Metaphases were also stained with 10% Giemsa for 10 minutes, to ascertain the presence and number of dmins. In Situ Hybridization The 1.0-kb EcoRI-BamHl Human M Y C N probe used (Amersham code RPN.1316X) is equivalent to the pNB-1 probe used by Schwab et al. [6]. The probe was labeled with Biotin-16-dUTP (Boehringer catalogue no. 1093 070) using a Nick Translation kit (Boehringer catalogue no. 976 776). The in situ hybridization was performed according to our modification [21] of the protocol of Harper et al. [14] modified by Pinkel et al. [22]. Probe Detection A combination of the techniques described by Pinkel et al. [22] and Lewis et al. [23] was used to detect the probe.
129
Amplification of the signal was achieved by sequential application of Avidin DN (Vector catalogue no. A-3100) and Biotinylated Anti-Avidin DN (Vector catalogue no. BA-0300), at 10/~g/mL and 5/~g/mL, respectively. Thereafter, a Streptavidin-Biotin-Complex Alkaline-Phosphatase kit was used following the recommendations in the kit (Dakopatts Code No. K391) as previously described [21].
RESULTS Cytogenetics Giemsa staining of PER-106 revealed tumor cells with variable numbers of dmin (0-150), fragments (0-10), and ring chromosomes (0-12). Ring chromosomes were found in six of 22 metaphases analyzed, whereas all cells contained dmins. Eight G-banded metaphases identified the karyotype as 46,XY,÷psu dic(1)(qter--~cen::p13--~qter),+11, t(11;17)(q13;q11),-19,+f,-r,+dmin (Fig. 1). C-banding confirmed the dicentric nature of the abnormality of c:hromosome 1. G-banding of PER 107 revealed the presence of two hsr, one at 6p25 and the other at 14p13 in all cells, dmin, fragments, and rings were not detected, but the other numerical and structural cytogenetic abnormalities present at diagnosis were observed, with in addition, a deletion of chromosome 8 at p21 and a deletion of one chromosome 11 at q21. The abnormalities were found in all nine cells fully karyotyped; the same two hsr were observed in an additional 30 cells. Analysis of PER-I08 showed the same two hsr but further karyotypic evolution, namely, deletion of chromosome 4 at q25 in all 17 cells karyotyped and a deletion at 18q21 in 9 of 17 cells analyzed (Fig. 2). In addition, the length of the hsr relative to the total lengths of chromosomes 6 and 14, respectively, were measured and no differences were found between the two cell lines. In Situ Hybridization In situ hybridization applied to cell line PER-106 clearly showed that M Y C N was amplified in all of the dmin, flagments, and ring chromosomes within each cell (Fig. 3). The hsr of the cell lines PER-107 and PER-108 both displayed M Y C N amplification. DISCUSSION Previous analysis of patient survival in neuroblastoma by stage, age, and MYCN copy number has indicated that MYCN amplification is highly correlated with rapid tumor progression and a poor prognosis [8, 91. In this study, MYCN amplification has been found to be involved with the development of an aggressive stage IV neuroblastoma, from which three cell lines were derived. These cell lines, derived from bone marrow infiltrated with neuroblastoma and taken first at diagnosis, when the disease recurred. and just prior to death from the one patient, have been studied cytogenetically and by non-radioactive chromosomal in situ hybridization using an MYCN probe. Cytogenetic indicators of a poor prognosis were observed in the cell line at diagnosis, i.e., the near-diploid
2
3
20
19
15
t)¢
7
O Lib
~ q~
~ m
"~
''"'""
8
'~
~
9
16
On
. 10
21
4
17
;a, 22
11
m
Figure 1 G-banded karyotype of the PER106 cell line: 46,XY.psu dic(1){qter-,cen::p13-,qter},+11,t{11;17) (q13;q11),- 19,+ f,+r,- dmin.
14
Iit
6
H 11 Jr .
131
N
X
I
1
I( It 11
~'
18
12
L,~ ,t
II
o
131
BI
---4'
t~
q
l
l
r
~
4 4
0
{l}
~J
+.-~
x.,~-
•- -
--i
Q
t~
If}
X
I/}
r-
132
T.L. McRobert et al.
a O
Figure 3
In situ hybridization of the PER106 cell line showing the amplific:ation of MYCN on the rings and dmin.
count, dmins, and structural abnormalities, particularly involving the short arm of chromosome 1 [24]. Observation of such changes helps to demonstrate the clonal nature of most neoplasms and the importance, in tumor progression, of sequential genetic changes within the neoplastic: clone. In our case, primary karyotypic changes involving lp and the t(11;17) may have been involved in the initiation of the tumor. Trisomy of 17q is a non-random abnormality observed in neuroblastoma. The amplification of MYCN, together with the secondary changes involving 4[q25), 8(p21), and 18(q21), may have contributed to tumor progression. Amplification of MYCN was successfully demonstrated in all three cell lines. However, the chromosomal manifestation of the amplification differed between the lines showing evolution of dmin, fragments, and rings in the line established at diagnosis to hsrs in the subsequent relapse cell lines. Heterogeneity of MYCN amplification was also demonstrated in the diagnostic cell line (PER-106) as the number of dmin per cell was shown to vary markedly in this cell line. This is not unexpected since dmin chromosomes randomly segregate during cell division as they lack centromeres, and can therefore be unevenly distributed among daughter cells. This observed heterogeneity of MYCN DNA amplification between cells may explain in part the reported heterogeneity of MYCN mRNA [25, 26] and MYCN protein expression [27, 28[ among individual neuroblastoma cells in the one tumor. It is also possible that some heterogeneity of mRNA expression is the result of a subpopulation of cells displaying increased transcriptional efficiency of MYCN, resulting from a regulatory
change. This idea is supported by observations that enhanced MYCN expression may not always correlate with MYCN gene amplification [29-31]. The evolution of MYCN amplification from dmins to hsrs has previously been reported [32] and suggests that hsrs provide selective advantage, or have a greater phenotypic stability, relative to that of cells containing drain. In this study no cells were observed with both dmins and hsrs, however, the PER-106 cell line contained M Y C N amplified small fragments and rings of various sizes, in addition to dmin, it is not known whether these rings developed over time during the culture of this cell line or were present at diagnosis, since the original specimen had not been studied cytogenetically. To our knowledge, the amplification of MYCN in the form of rings has not previously been described, but allows speculation as to the mechanisms of gene amplification. Extending the predicted model of amplification [33], the ring chromosomes may represent an intermediate step between drains and hsrs. It is possible that the dmin replicate and increase in size up to the stage where they may appear as large ring chromosomes carrying tandem repeats of the amplicon. The ring could then be integrated into the genome of a dmin-bearing cell as the precursor of an hsrbearing sub-population of cells. Heterogeneity of MYCN amplification between individual neuroblastoma cells needs to be investigated further. The ability to detect a minority of MYCN-amplified cells within a non-amplified population using this technique may prove to be significant and denote poor prognosis, given that based on Southern analysis, the same sample
MYCN A m p l i f i c a t i o n in N e u r o b l a s t o m a
m a y be classified i n a p p r o p r i a t e l y into a m o r e favorable p r o g n o s t i c group. We suggest that the n o n - i s o t o p i c in situ h y b r i d i z a t i o n t e c h n i q u e u t i l i z e d in this study c o u l d be r o u t i n e l y a p p l i e d to b o n e m a r r o w s a m p l e s and t u m o r s in order to i d e n t i f y a m p l i f i e d genes in i n t e r p h a s e and metaphase cells. It c o u l d also be u t i l i z e d to d e t e r m i n e if gene a m p l i f i c a t i o n occurs m o r e f r e q u e n t l y than is cytologic:ally obvious. T h i s c o u l d i n v o l v e identification of a m p l i f i c a t i o n in ring form or the i d e n t i f i c a t i o n of u n r e c o g n i z e d hsrs, as has been the case w i t h a t u m o r s p e c i m e n from a patient w h e r e a marker c h r o m o s o m e was identified by this techn i q u e as an hsr(17q) [21]. This work was supported in part by the Anti-Cancer Council of Victoria (T. L. M., C. R., O. M. G.), the National Health and Medical Researc:h Council (C. R., O. M. G.], the Children's l,eukaemia Research Fund of the Princess Margaret Children's Medical Research Foundation (U. R. K.), anct the Cancer Foundation of Western Australia (U. R. K.).
REFERENCES 1. Young JL, Miller RW (1974): Incidence of malignant tumors in U.S. children. J Pediatr 86:254-258. 2. Evans AE (1980): Staging and treatment of neuroblastoma. Cancer 45:1799-1802. 3. Evans AE, D'Angio GJ, Randolph J (1971]: A proposed staging for children with neuroblastoma. Cancer 27:374-378. 4. Kanda N, Schreck R, Air F, Bruns (;, Baltimore D, Latt S (1983]: Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: Facilitation by fluorescence-activated flow sorting of metaphase chromosomes. Proc Natl Ac:ad Sci USA 80:4069-4073. 5. Kohl NE, Kanda N, Schrec:k RR, Bruns G, Latt SA, Gilbert F, Alt FW (1983): Transposition and amplification of oncogene related sequences in human neuroblastomas. Cell 35:359367. 6. Schwab M, Alitalo K, Klempnauer K, Varmus HE, Bishop JM, Gilbert F, Brodeur G, Goldstein M, Trent J (1983): Amplified DNA with limited homology to myc cellular onc:ogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature 305:245-248. 7. Schwab M, Varmus HE, Bishop JM, Grzesc:hik K. Naylor SL, Sakaguchi AY, Brodeur (;, Trent J (1984): Chromosome, localization in normal human cells and neuroblastomas of a gene related to c-myc. Nature 308:288-291. 8. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM (1984): Amplification of N-myc: in untreated human neuroblastnmas (:nrrelates with advanced disease stage. Science 224:1121-1124. 9. Seeger RC, Brodeur GM, Sather H. Dalton A, Siegel SE, Wong KY, Hammond D (1985): Association of multiple copies of the N-myc onc:ogene with rapid progression of neuroblastoma. N Engl J Med 313:1111-1116. 10. Nakagawara A, Ikeda K, Tsuda T, Higashi K, Okabe T (1987): Amplification of N-myc oncogene in stage 11 and IVS neuroblastomas may be a prognostic indicator. ] Pediatr Surg 22:415-418. 11. Brodeur GM, Hayes FA, Green AA, Casper JT, Wasson J, Wallath S, Seeger RC (1987): Consistent N-myc copy number in simultaneous or consecutive neuroblastoma samples from sixty individual patients. Cancer Res 47:4248-4253. 12. Tsuda H, Shimosato Y, Uptcm MP, Yokata J, Terade M, Ohira M, Sugimura T, Hirohashi S (1988): Retrospective study on amplification of N-myc and c-myc genes in pediatric solid
133
13.
14.
15.
16.
17.
18.
19.
21).
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
tumors and its association with prognosis and tumor differentiation. Lab Invest 59:321-327. Brodeur GM, Fong C, Morita M, Griffith R, Hayes FA, Seeger RC (1988): Molecular analysis and clinical significance of Nmyc amplification and chromosome lp monosomy in human neuroblastomas. Prog Clin Biol Res 271:3-15. Harper ME, Ulrich A, Saunders GR (1981): l,ocalization of the human insulin gene to the distal end of the short arm of chromosome 11. Proc: Natl Acad Sci USA 78:4458-4460. Pinkel D, l,andegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988): Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Ac:ad Sci USA 85:9138-9142. Kees UR, Ford J, Dawson VM, Ranford PR, Armstrong JA (1992): Three neuroblastoma cell lines established from consecutive bone marrow samples of one patient show distinct morphological features, amplification MYCN, and surface marker expression. Canc:er (;enet Cytogenet 59:119-127. Webber I,M, Garson OM (1983): Fluorodeoxyuridine synchronization of bone marrow cultures. Cancer Genet Cytogenet 8:123-132. Seabright M (1972): The use of proteolytic enzyme for the mapping of structural rearrangements in the chromosomes of man. Chromosoma 36:204-210. Salamanca F, Armendares S (1974): C-bands in human metaphase chromosomes treated by barium hydroxide. Ann Genet 17:135-136. ISCN (19851: An International System for Human Cytogenetic Nomenclature. Harnden DG, Klinger DP, eds; Published in collaboration with Cytogenet Cell Genet, Karger, Basel. Rudduck C, Lukeis RE, McRobert TL, (;how CW, Garson Olvl (1992): Chromosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe. Cancer Genet Cytogenet (submitted). Pinkel I), Straume T, Gray JW (19861: Cytogenetic analysis using quantitative, high-sensitivity fluorescenc:e hybridization. Proc Natl Acad Sc:i USA 83:2934-2938. Lewis FA, Griffiths S, Dunnicliff R, Wells M, Dudding N, Bird CC (1987): Sensitive in situ hybridization technique using biotin-streptavidin-polyalkaline-phosphatase complex. J Clin Pathol 40:163-166. Brodeur GM, Fong C-Y (1989): Molecular biology and genetics of human neuroblastoma. Cancer Genet Cytogenet 41:153-174. Grady-Leopardi EF, Schwab M, Ablin AR, Rosenau W (1986): Detection of N-myc onc:ogene expression in human neuroblastoma by in situ hybridization and blot analysis: relationship to clinical outcome. Cancer Res 46:3196-3199. Cohen PS, Seeger RC, Triche T], Israel MA {1988}: Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization. Am J Clin Pathol 131:391-397. Ikegaki N, Polakova K, Prince I,, Kennett RH (1988): Expression and localization of the N-myc protein in human neuroblastoma cells: analysis of the effect of gamma interferon treatment and distribution of the N-myc protein in the nucleus. Prog Clin Biol Res 271:133-144. Seeger RC, Wada R, Brodeur GM, Moss TJ, Bjork RL, Sousa L, Slamon DJ (1988): Expression of N-myc by neuroblastomas with one or multiple copies of the oncogene. Prog Clin Biol Res 271:41-49. Rosen N, Reynolds CP, Thiele CJ, Biedler JL, Israel MA (1986): Increased N-myc expression following progressive growth of human neuroblastoma. Cancer Res 46:4139-4142. Bartram CR, Berthold F (1987): Amplification and expression of the N-myc" gene in neuroblastoma. Eur J Pediatr 146:162165.
134
31. Nisen PD, Waber PG, Rich MA, Pierce S, Garvin JR, Gilbert F, Lanzkowsky P (1988): Nomyc oncogene RNA expression in neuroblastoma. J Natl Cancer Inst 80:1633-1637. 32. Biedler JL, Ross RA, Shanske S, Spengler BA (1980): Human neuroblastoma cytogenetics: Search for significance of homo-
T . L . M c R o b e r t et al.
geneously staining regions and double minute chromosomes. In: Advances in Neuroblastoma Research, Evans AE, ed. New York, Raven Press, pp. 81-96. 33. Roberts JM, Buck LB, Axel R (1983): A structure for amplified DNA. Cell 33:53-63.
ABSTRACT: A non-radioactive chromosomal in situ hybridization technique utilizing a bintin-streptavidin-polyalkaline-phosphatase complex was successfully applied to three ne,urablastoma cell lines for detection of MYCN amplification. These cell lines, designated PER-106, PER-107, and PER-108, were derived from consecutive bone marrow samples taken from a patient with stage IV neuroblastoma. The cell line derived at diagnosis (PER-106) exhibited MYCN amplification in the form of variable numbers of double-minute chromosomes, small fragments, and rings of varying sizes. This observed variability of MYCN amplification may explain the reported heterogeneity of both MYCN mRNA and protein expression among individual cells of some neuroblastomas. The cell lines derived from subsequent samples (PER-107 and PER-108) contained amplified MYCN as two consistent homogeneously staining regions in every cell. These, were located on the shor~ arms of chromosomes 6 and 14. Thus, amplified MYCN was identified in each cell line and demonstrated the, concurrent evolution of amplification with cytogenetic abnormalities.
INTRODUCTION Neuroblastoma is one of the more c o m m o n solid tumors of early c h i l d h o o d [1]. It is an aggressive tumor with a highly variable clinical course where the majority of (:ases (70%) have undergone metastatic spread at the time of diagnosis [2]. Prognosis of children with neuroblastoma is greatly influenced by the stage of disease at diagnosis, where Evans stages IlI and IV have the worst prognosis 13]. Other prognostic factors include age at diagnosis, site of the primary tumor, and urinary catecholamine levels. In 1983, the MYCN oncogene was first identified in human neuroblastoma as an amplified gene having a limited sequence homology to the c-myc oncogene. It was isolated from homogeneously staining region (hsr) and doubleminute (dmin)-bearing neuroblastoma cells by several research groups [4-6] and was found to map to region 2p24 in normal cells [71. The MYCN oncogene may be amplified in both neuroblastoma tumors and cell lines and correlates strongly with rapid disease progression and poor progno-
From the Department ofCytagenetics (T. L. M., C. R., O. M. G.), St. Vincent's ltaspital. Fitzroy, Victoria. Australia~ and Leukaemia llesearch Laboratory {U. R. K.I, Clinical Immunolog)" Research Unit. Princess Margaret Hospital, Perth. Western Australia. Address reprint requests to: Associate ProJessor O. M. (;arsan. Department of Cytogenetics, St. Vincent's tlospital. Fitzroy. Victaria, Australia, 3065. Received April 10, 1991; accepted September 26, 1991. 128 Cancer Genet Cytogene.l59:128-134 11992) 0165-4608,'92~$05,00
sis [8]. Therefore, it is an i n d e p e n d e n t prognostic indicator that may be clinically useful as a guide for treating even the favorable stages II and IV-S of neuroblastoma [9, 10]. Detection of MYCN amplification is usually achieved by Southern analysis, a technique which allows an estimate of MYCN copy number for the overall cell population. Most studies have shown MYCN amplification to be consistent in the i n d i v i d u a l tumor examined at different times in vivo [11, 12]. If it is not present at diagnosis, MYCN amplification does not seem to appear with disease progression. Conversely, progressive disease is very likely to occ:ur in patients with MYCN amplification at diagnosis [13]. However, heterogeneity of amplification between individual cells, if present, will not be detected using Southern analysis. Another approach is to detect MYCN amplification by (:hromosomal in situ hybridization, which is traditionally performed using radioactively labeled probes [141. Recently, nonradioactive systems have been developed using biotinylated probes detected by a variety of immunogenic, fluorescent, and enzymatic labels. These techniques are quick, yielding a result within 24 hours, they have high resolution, and although they are not quite as sensitive as radioactive techniques, unique sequences as small as 3 kb have been detected [15]. We have therefore used a biotin-streptavidin-polyalkaline-phosphatase system to investigate the MYCN amplification in three neuroblastoma cell lines. By studying (:ell lines derived from a patient with neuroblastoma at three different stages of the disease, it was possible to determine O 1992 Elsevier Science Publishing Co.. lilt:. 655 ..\vmumof the Americas. New York. NY lo()10
M Y C N Amplification in Neuroblastoma
how gene amplification and cytogenetic abnormalities evolved with disease progression.
MATERIALS AND METHODS Case Report A 17-month-old Caucasian boy presented in October, 1984 with disseminated neuroblastoma including gross bone marrow involvement (Stage IV). He responded to chemotherapy and the residual primary tumor was subsequently exc:ised, but the disease recurred 13 months later, again showing gross marrow involvement together with skull and orbital secondaries. Death occurred 15 months after diagnosis, at which time the marrow was almost c:ompletely replaced by tumor cell clumps. Three cell lines were derived from consecutive bone marrow samples taken at diagnosis, and 13 and 15 months after diagnosis. These were designated PER-106, PER-107, and PER-108 respectively, with PER-106 consisting mainly of cells growing in suspension and forming large aggregates of small round cells [16]. PER 107 and 108, derived after disease recurrence, grew substrate adherent. Chromosome Preparation The neuroblastoma cells were cultured in RPMI 1640 medium with 20% Fetal Calf Serum (FCS)/Penic:illin-Streptomycin (50 IU/mL), L-Glutamine (2 mM), 2-mercaptoethanol (10 5 M), pyruvate (1 mM), and non-essential amino acids, in tissue culture flasks in a humidified 37°C incubator containing 5% CO2 in air. The fluorodeoxyuridine synchronization technique of Webber and Garson [17] was used to improve the quantity and quality of metaphase spreads. Harvesting commenc:ed after an 8-hour release time. Colcemid was added to give a final c:onc:entration of 0.8 p.g/mL and cultures were then incubated for either 1.5 hours or overnight, at 37°C. Adherent cells were detached with Trypsin : EDTA (Flow Laboratories "16-89149) at 37°C for 5 minutes. Chromosome preparations were made by standard cytogenetic techniques and were trypsin (;-banded [18J and C-banded [19]. Karyotypes designated according to the classification of the International System for Human Cytogenetic: Nomenc:lature [20] were constructed from each cell line. Metaphases were also stained with 10% Giemsa for 10 minutes, to ascertain the presence and number of dmins. In Situ Hybridization The 1.0-kb EcoRI-BamHl Human M Y C N probe used (Amersham code RPN.1316X) is equivalent to the pNB-1 probe used by Schwab et al. [6]. The probe was labeled with Biotin-16-dUTP (Boehringer catalogue no. 1093 070) using a Nick Translation kit (Boehringer catalogue no. 976 776). The in situ hybridization was performed according to our modification [21] of the protocol of Harper et al. [14] modified by Pinkel et al. [22]. Probe Detection A combination of the techniques described by Pinkel et al. [22] and Lewis et al. [23] was used to detect the probe.
129
Amplification of the signal was achieved by sequential application of Avidin DN (Vector catalogue no. A-3100) and Biotinylated Anti-Avidin DN (Vector catalogue no. BA-0300), at 10/~g/mL and 5/~g/mL, respectively. Thereafter, a Streptavidin-Biotin-Complex Alkaline-Phosphatase kit was used following the recommendations in the kit (Dakopatts Code No. K391) as previously described [21].
RESULTS Cytogenetics Giemsa staining of PER-106 revealed tumor cells with variable numbers of dmin (0-150), fragments (0-10), and ring chromosomes (0-12). Ring chromosomes were found in six of 22 metaphases analyzed, whereas all cells contained dmins. Eight G-banded metaphases identified the karyotype as 46,XY,÷psu dic(1)(qter--~cen::p13--~qter),+11, t(11;17)(q13;q11),-19,+f,-r,+dmin (Fig. 1). C-banding confirmed the dicentric nature of the abnormality of c:hromosome 1. G-banding of PER 107 revealed the presence of two hsr, one at 6p25 and the other at 14p13 in all cells, dmin, fragments, and rings were not detected, but the other numerical and structural cytogenetic abnormalities present at diagnosis were observed, with in addition, a deletion of chromosome 8 at p21 and a deletion of one chromosome 11 at q21. The abnormalities were found in all nine cells fully karyotyped; the same two hsr were observed in an additional 30 cells. Analysis of PER-I08 showed the same two hsr but further karyotypic evolution, namely, deletion of chromosome 4 at q25 in all 17 cells karyotyped and a deletion at 18q21 in 9 of 17 cells analyzed (Fig. 2). In addition, the length of the hsr relative to the total lengths of chromosomes 6 and 14, respectively, were measured and no differences were found between the two cell lines. In Situ Hybridization In situ hybridization applied to cell line PER-106 clearly showed that M Y C N was amplified in all of the dmin, flagments, and ring chromosomes within each cell (Fig. 3). The hsr of the cell lines PER-107 and PER-108 both displayed M Y C N amplification. DISCUSSION Previous analysis of patient survival in neuroblastoma by stage, age, and MYCN copy number has indicated that MYCN amplification is highly correlated with rapid tumor progression and a poor prognosis [8, 91. In this study, MYCN amplification has been found to be involved with the development of an aggressive stage IV neuroblastoma, from which three cell lines were derived. These cell lines, derived from bone marrow infiltrated with neuroblastoma and taken first at diagnosis, when the disease recurred. and just prior to death from the one patient, have been studied cytogenetically and by non-radioactive chromosomal in situ hybridization using an MYCN probe. Cytogenetic indicators of a poor prognosis were observed in the cell line at diagnosis, i.e., the near-diploid
2
3
20
19
15
t)¢
7
O Lib
~ q~
~ m
"~
''"'""
8
'~
~
9
16
On
. 10
21
4
17
;a, 22
11
m
Figure 1 G-banded karyotype of the PER106 cell line: 46,XY.psu dic(1){qter-,cen::p13-,qter},+11,t{11;17) (q13;q11),- 19,+ f,+r,- dmin.
14
Iit
6
H 11 Jr .
131
N
X
I
1
I( It 11
~'
18
12
L,~ ,t
II
o
131
BI
---4'
t~
q
l
l
r
~
4 4
0
{l}
~J
+.-~
x.,~-
•- -
--i
Q
t~
If}
X
I/}
r-
132
T.L. McRobert et al.
a O
Figure 3
In situ hybridization of the PER106 cell line showing the amplific:ation of MYCN on the rings and dmin.
count, dmins, and structural abnormalities, particularly involving the short arm of chromosome 1 [24]. Observation of such changes helps to demonstrate the clonal nature of most neoplasms and the importance, in tumor progression, of sequential genetic changes within the neoplastic: clone. In our case, primary karyotypic changes involving lp and the t(11;17) may have been involved in the initiation of the tumor. Trisomy of 17q is a non-random abnormality observed in neuroblastoma. The amplification of MYCN, together with the secondary changes involving 4[q25), 8(p21), and 18(q21), may have contributed to tumor progression. Amplification of MYCN was successfully demonstrated in all three cell lines. However, the chromosomal manifestation of the amplification differed between the lines showing evolution of dmin, fragments, and rings in the line established at diagnosis to hsrs in the subsequent relapse cell lines. Heterogeneity of MYCN amplification was also demonstrated in the diagnostic cell line (PER-106) as the number of dmin per cell was shown to vary markedly in this cell line. This is not unexpected since dmin chromosomes randomly segregate during cell division as they lack centromeres, and can therefore be unevenly distributed among daughter cells. This observed heterogeneity of MYCN DNA amplification between cells may explain in part the reported heterogeneity of MYCN mRNA [25, 26] and MYCN protein expression [27, 28[ among individual neuroblastoma cells in the one tumor. It is also possible that some heterogeneity of mRNA expression is the result of a subpopulation of cells displaying increased transcriptional efficiency of MYCN, resulting from a regulatory
change. This idea is supported by observations that enhanced MYCN expression may not always correlate with MYCN gene amplification [29-31]. The evolution of MYCN amplification from dmins to hsrs has previously been reported [32] and suggests that hsrs provide selective advantage, or have a greater phenotypic stability, relative to that of cells containing drain. In this study no cells were observed with both dmins and hsrs, however, the PER-106 cell line contained M Y C N amplified small fragments and rings of various sizes, in addition to dmin, it is not known whether these rings developed over time during the culture of this cell line or were present at diagnosis, since the original specimen had not been studied cytogenetically. To our knowledge, the amplification of MYCN in the form of rings has not previously been described, but allows speculation as to the mechanisms of gene amplification. Extending the predicted model of amplification [33], the ring chromosomes may represent an intermediate step between drains and hsrs. It is possible that the dmin replicate and increase in size up to the stage where they may appear as large ring chromosomes carrying tandem repeats of the amplicon. The ring could then be integrated into the genome of a dmin-bearing cell as the precursor of an hsrbearing sub-population of cells. Heterogeneity of MYCN amplification between individual neuroblastoma cells needs to be investigated further. The ability to detect a minority of MYCN-amplified cells within a non-amplified population using this technique may prove to be significant and denote poor prognosis, given that based on Southern analysis, the same sample
MYCN A m p l i f i c a t i o n in N e u r o b l a s t o m a
m a y be classified i n a p p r o p r i a t e l y into a m o r e favorable p r o g n o s t i c group. We suggest that the n o n - i s o t o p i c in situ h y b r i d i z a t i o n t e c h n i q u e u t i l i z e d in this study c o u l d be r o u t i n e l y a p p l i e d to b o n e m a r r o w s a m p l e s and t u m o r s in order to i d e n t i f y a m p l i f i e d genes in i n t e r p h a s e and metaphase cells. It c o u l d also be u t i l i z e d to d e t e r m i n e if gene a m p l i f i c a t i o n occurs m o r e f r e q u e n t l y than is cytologic:ally obvious. T h i s c o u l d i n v o l v e identification of a m p l i f i c a t i o n in ring form or the i d e n t i f i c a t i o n of u n r e c o g n i z e d hsrs, as has been the case w i t h a t u m o r s p e c i m e n from a patient w h e r e a marker c h r o m o s o m e was identified by this techn i q u e as an hsr(17q) [21]. This work was supported in part by the Anti-Cancer Council of Victoria (T. L. M., C. R., O. M. G.), the National Health and Medical Researc:h Council (C. R., O. M. G.], the Children's l,eukaemia Research Fund of the Princess Margaret Children's Medical Research Foundation (U. R. K.), anct the Cancer Foundation of Western Australia (U. R. K.).
REFERENCES 1. Young JL, Miller RW (1974): Incidence of malignant tumors in U.S. children. J Pediatr 86:254-258. 2. Evans AE (1980): Staging and treatment of neuroblastoma. Cancer 45:1799-1802. 3. Evans AE, D'Angio GJ, Randolph J (1971]: A proposed staging for children with neuroblastoma. Cancer 27:374-378. 4. Kanda N, Schreck R, Air F, Bruns (;, Baltimore D, Latt S (1983]: Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: Facilitation by fluorescence-activated flow sorting of metaphase chromosomes. Proc Natl Ac:ad Sci USA 80:4069-4073. 5. Kohl NE, Kanda N, Schrec:k RR, Bruns G, Latt SA, Gilbert F, Alt FW (1983): Transposition and amplification of oncogene related sequences in human neuroblastomas. Cell 35:359367. 6. Schwab M, Alitalo K, Klempnauer K, Varmus HE, Bishop JM, Gilbert F, Brodeur G, Goldstein M, Trent J (1983): Amplified DNA with limited homology to myc cellular onc:ogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour. Nature 305:245-248. 7. Schwab M, Varmus HE, Bishop JM, Grzesc:hik K. Naylor SL, Sakaguchi AY, Brodeur (;, Trent J (1984): Chromosome, localization in normal human cells and neuroblastomas of a gene related to c-myc. Nature 308:288-291. 8. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM (1984): Amplification of N-myc: in untreated human neuroblastnmas (:nrrelates with advanced disease stage. Science 224:1121-1124. 9. Seeger RC, Brodeur GM, Sather H. Dalton A, Siegel SE, Wong KY, Hammond D (1985): Association of multiple copies of the N-myc onc:ogene with rapid progression of neuroblastoma. N Engl J Med 313:1111-1116. 10. Nakagawara A, Ikeda K, Tsuda T, Higashi K, Okabe T (1987): Amplification of N-myc oncogene in stage 11 and IVS neuroblastomas may be a prognostic indicator. ] Pediatr Surg 22:415-418. 11. Brodeur GM, Hayes FA, Green AA, Casper JT, Wasson J, Wallath S, Seeger RC (1987): Consistent N-myc copy number in simultaneous or consecutive neuroblastoma samples from sixty individual patients. Cancer Res 47:4248-4253. 12. Tsuda H, Shimosato Y, Uptcm MP, Yokata J, Terade M, Ohira M, Sugimura T, Hirohashi S (1988): Retrospective study on amplification of N-myc and c-myc genes in pediatric solid
133
13.
14.
15.
16.
17.
18.
19.
21).
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
tumors and its association with prognosis and tumor differentiation. Lab Invest 59:321-327. Brodeur GM, Fong C, Morita M, Griffith R, Hayes FA, Seeger RC (1988): Molecular analysis and clinical significance of Nmyc amplification and chromosome lp monosomy in human neuroblastomas. Prog Clin Biol Res 271:3-15. Harper ME, Ulrich A, Saunders GR (1981): l,ocalization of the human insulin gene to the distal end of the short arm of chromosome 11. Proc: Natl Acad Sci USA 78:4458-4460. Pinkel D, l,andegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988): Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc Natl Ac:ad Sci USA 85:9138-9142. Kees UR, Ford J, Dawson VM, Ranford PR, Armstrong JA (1992): Three neuroblastoma cell lines established from consecutive bone marrow samples of one patient show distinct morphological features, amplification MYCN, and surface marker expression. Canc:er (;enet Cytogenet 59:119-127. Webber I,M, Garson OM (1983): Fluorodeoxyuridine synchronization of bone marrow cultures. Cancer Genet Cytogenet 8:123-132. Seabright M (1972): The use of proteolytic enzyme for the mapping of structural rearrangements in the chromosomes of man. Chromosoma 36:204-210. Salamanca F, Armendares S (1974): C-bands in human metaphase chromosomes treated by barium hydroxide. Ann Genet 17:135-136. ISCN (19851: An International System for Human Cytogenetic Nomenclature. Harnden DG, Klinger DP, eds; Published in collaboration with Cytogenet Cell Genet, Karger, Basel. Rudduck C, Lukeis RE, McRobert TL, (;how CW, Garson Olvl (1992): Chromosomal localization of amplified N-myc in neuroblastoma cells using a biotinylated probe. Cancer Genet Cytogenet (submitted). Pinkel I), Straume T, Gray JW (19861: Cytogenetic analysis using quantitative, high-sensitivity fluorescenc:e hybridization. Proc Natl Acad Sc:i USA 83:2934-2938. Lewis FA, Griffiths S, Dunnicliff R, Wells M, Dudding N, Bird CC (1987): Sensitive in situ hybridization technique using biotin-streptavidin-polyalkaline-phosphatase complex. J Clin Pathol 40:163-166. Brodeur GM, Fong C-Y (1989): Molecular biology and genetics of human neuroblastoma. Cancer Genet Cytogenet 41:153-174. Grady-Leopardi EF, Schwab M, Ablin AR, Rosenau W (1986): Detection of N-myc onc:ogene expression in human neuroblastoma by in situ hybridization and blot analysis: relationship to clinical outcome. Cancer Res 46:3196-3199. Cohen PS, Seeger RC, Triche T], Israel MA {1988}: Detection of N-myc gene expression in neuroblastoma tumors by in situ hybridization. Am J Clin Pathol 131:391-397. Ikegaki N, Polakova K, Prince I,, Kennett RH (1988): Expression and localization of the N-myc protein in human neuroblastoma cells: analysis of the effect of gamma interferon treatment and distribution of the N-myc protein in the nucleus. Prog Clin Biol Res 271:133-144. Seeger RC, Wada R, Brodeur GM, Moss TJ, Bjork RL, Sousa L, Slamon DJ (1988): Expression of N-myc by neuroblastomas with one or multiple copies of the oncogene. Prog Clin Biol Res 271:41-49. Rosen N, Reynolds CP, Thiele CJ, Biedler JL, Israel MA (1986): Increased N-myc expression following progressive growth of human neuroblastoma. Cancer Res 46:4139-4142. Bartram CR, Berthold F (1987): Amplification and expression of the N-myc" gene in neuroblastoma. Eur J Pediatr 146:162165.
134
31. Nisen PD, Waber PG, Rich MA, Pierce S, Garvin JR, Gilbert F, Lanzkowsky P (1988): Nomyc oncogene RNA expression in neuroblastoma. J Natl Cancer Inst 80:1633-1637. 32. Biedler JL, Ross RA, Shanske S, Spengler BA (1980): Human neuroblastoma cytogenetics: Search for significance of homo-
T . L . M c R o b e r t et al.
geneously staining regions and double minute chromosomes. In: Advances in Neuroblastoma Research, Evans AE, ed. New York, Raven Press, pp. 81-96. 33. Roberts JM, Buck LB, Axel R (1983): A structure for amplified DNA. Cell 33:53-63.