Key words: chip technology, arrays, neuroblastoma, breast cancer, prognosis, chromosome

Abstract
Regulatory or structural alterations of cellular oncogenes have been implicated in the causation of cancers. Amplification represents one of the major molecular pathways by which gene expression is constitutively enhanced above the level of physiologically normal variation. Consequently, the significance of oncogene amplification in tumorigenesis originally had emerged from expression profiling of tumor cells by oncogene arrays. Amplified oncogenes have been found associated with more aggressive tumor variants and in selected settings are clinical markers to determine patient prognosis.

Introduction
Since its discovery in drug-resistant eukaryotic cells, somatic amplification of specific genes has been implicated in an increasing variety of adaptive responses of cells to environmental stresses [reviewed in 1,2]. Increase of the gene dosage by DNA amplification is a common genetic mechanism for upregulating gene expression. Scheduled amplification can be part of the developmental program, e.g., the amplification of chorion genes in ovaries of the fruitfly [3] or of actin genes during myogenesis in the chicken [4]. Unscheduled DNA amplification has been seen in response to exposure to cytotoxic drugs [reviews ed in 5-7] or during tumorigenesis [reviewed in 8]. In both scenarios, enhanced expression has been found consequent to gene amplification. Amplification values usually are above 5 copies, often can reach 500 or more gene copies. Reports of values lower than 5 should be taken with caution, they could result from ploidy-changes or even from experimental variations.

Cytogenetics of amplified DNA
Cytogenetic analyses have brought to light the frequency of DNA amplifications in tumor cells and have provided a starting point to define the contribution for tumorigenesis that comes from an increase of the dosage of cellular oncogenes by amplification. Cytogenetic manifestations for DNA amplification have been encountered as yet exclusively in tumor cells. They do not seem to be restricted to vertebrate cells but have been found in malignant cells of insects as well [9]. Among solid human tumors, double minutes (DMs) and homogeneously staining regions (HSRs) (Fig.1) can be found in virtually any type at least in some cases, and detection is usually a matter of patience. In experimental tumors larger structures refered to as c-bandless chromosomes (CMs) have been detected [10] The chance to detect DMs or HSRs is increased when tumor cells are established in culture and when cells carrying amplification are selected for. In no instance has amplification been found as a tissue culture artifact. Detection of DMs and HSRs in direct preparations of solid tumors often is difficult. This may be due to either the general difficulty of obtaining good karyotypes from solid tumors, or to the heterogeneity of tumor cells. It is well established that tumor cell populations are heterogeneous for many characteristics, and it is possible that amplification varies among members of the tumor cell populations. For instance, growth in peripheral regions of the tumor could be subjected to selective forces different from those that operate in a more central environment. Failure to detect amplified DNA in a particular type of tumor by analyzing DNA with known gene probes does not exclude the presence of amplified DNA. We should be open to the possibility that the human genome contains genes involved in growth control in addition to the around 50 that have been identified and designated "cellular oncogenes". Identifying and defining amplified DNA in tumor cells has become a strategy for the isolation of additional cellular genes involved in growth control and possibly in tumorigenesis.

Recently developed technologies, in particular "comparative genomic hybridisation" (CGH) have pointed out numerous chromosomal loci as candidates for harbouring amplified genes [11,12]. Many of these measurements should be interpreted with caution as CGH generally do not allow a reliable quantitation of DNA dosage.

Identifying amplified oncogenes
While cytogenetic identification of DMs or HSRs in tumor cells in general allows to predict the presence of an amplified oncogene, at least in cases when drug resistance can be excluded, the identity of the oncogene remains obscure. Given the fact that genes usually are expressed at significantly enhanced level consequent to amplification, the early identifications of amplified oncogenes employed expression profiling using an oncogene array (Figure 2). This approach for the first time had allowed to identify a cellular oncogene (RasK) that mapped both to DMs and to an HSR [13]. Subsequent studies by numerous laboratories have unveiled a plethora of amplified oncogenes in virtually every type of cancer [reviewed in 1]. In the majority of cases a single oncogene appears amplified, the independent non-syntenic amplification of different oncogenes has been documented occasionally [14].

The amplified DNA in tumor cells is usually much larger than the transcription unit of a particular proto-oncogene. Simple continuous amplified DNA regions in the range of one to several hundred kilobases prevail in neuroblastomas, while complex, and sometimes discontinuous regions of up to 20 Mb are seen in breast cancers [15,16]. Smaller amplified regions usually encompass only a single gene, such as MYCN in neuroblastomas [17]. By contrast, larger amplified domains often contain several syntenic co-amplified genes, such as the 11q13 encompassing two FGF genes INT2 (FGF3) and HST (FGF4), CYCD1, as well as the noncoding region BCL1 [18] in various carcinomas, or the 12q13-14 amplification involving MDM2, CDK4, and GLI (and presumably many more genes) in sarcomas [19] and occasionally neuroblastomas [14]. The amplification of non-syntenic genes, such as of MYCN (2p24) and MDM2/CDK4/GLI (12q13), which is occasionally seen in neuroblastomas, or of multiple genomic regions in many breast cancers [15] indicates that certain cells are genetically susceptible to amplification. However, the molecular identity of the gene(s) that might be related to instability remains enigmatic.

Amplified oncognes as predictor of clinical outcome
Amplified oncogenes have been found sporadically in a broad spectrum of tumor types, recurrent amplification of a particular gene or genomic regions appears limited to few tumor types. The picture of the prevalence of amplification in tumor cells may be incomplete, however, as new technologies, such as CGH, point to an ever-increasing number of mostly undefined genomic regions in which amplification might have occurred. Certain types of human tumors harbour an amplified oncogene at frequencies of 20-30%, raising the question of a possible association with clinical parameters. Two types of tumors have emerged where the determination of oncogene copy number has turned out to be useful for assessing patient prognosis.

MHCN (2p24) in neuroblastomas
Amplified MYCN in neuroblastomas is prototypic for the significance of proto-oncogene amplification in tumorigenesis [20,21]. A lower frequency of MYCN amplifications has been identified in other types of neuronal tumors, such as retinoblastomas, small cell lung cancers, glioblastomas, and astrocytomas. HSRs are generally located on different chromosomes, not at the resident site 2p24 of MYCN [42]. Amplified copies can be present either extrachromosomally as DMs or intrachromosomally as HSRs. Amplification values in neuroblastomas may range between 5 and more than 500-fold, usually values of around 50- to 100-fold are seen in tumors. Neuroblastoma cells lacking amplification are not necessarily single copy for MYCN. Instead, MYCN can be duplicated at 2p24, as shown by fluorescence in situ hybridization (FISH) [22]. It is unclear whether duplication represents a prelude to amplification or an alternative pathway for activating the oncogenic potential of MYCN.

The complexity of amplified DNA encompassing MYCN can range from 100 kb to more than 1 Mb. A core 100- to 200-kb domain encompassing MYCN has been found consistently without rearrangements. In most HSRs, several hundred kilobase DNA segments of unit size length without noticeable rearrangement as compared to the normal genomic organization are present in an ordered direct repeat head-to-tail tandem arrangement [23]. The size of the DNA surrounding MYCN raises the possibility that additional genes may be co-amplified. From several technological strategies to identify co-amplified genes MYCN has emerged as the only consistently amplified gene, although in about 50% of the MYCN-positive cases has the DDX1 gene, which maps within 400 kb 5' of MYCN [24] been found co-amplified, both in retinoblastomas [25] and in neuroblastomas [24]. However, in no case has DDX1 been found as the only amplified gene, suggesting that MYCN is functionally responsible for the maintenance of the 2q24-amplified DNA.

Most of the in vitro established neuroblastoma cell lines have amplified MYCN. The proportion of tumors with amplified MYCN is approximately 20%, although there are variations among different studies [26-29]. Amplified MYCN has been found only in more aggressive variants of neuroblastoma, where it connotes a dire prognosis. In low stage neuroblastomas it has emerged as a powerful independent marker to predict poor patient outcome [30-32], in addition to histopathological parameters, and therapeutic approaches are designed in part on MYCN status.

ERBB2 (17q11-12) in breast cancer
Among the various proto-oncogenes amplified in primary breast cancer, ERBB2, also known as HER-2 or NEU located in 17q11-12, is the best studied. Ovarian cancer has amplified ERBB2 as well, but in other tumors the amplification of ERBB2 is poorly documented.

Amplified ERBB2 was originally encountered in the breast cancer cell line MAC117 [33]. In an extensive survey, approximately 20-25% of primary breast cancers showed amplified ERB2 [34] with amplification values in most cases up to 20. A similar amplification frequency was found in ovarian cancers [35]. An extensive DNA sequence survey did not reveal mutations in the ERBB2 coding region, which is consistent with the concept that increased dosage of the wild-type gene has a role in tumorigenesis. Enhanced expression was uniformly associated with amplification in both tumor types. Few attempts to understand the molecular topography of the co-amplified DNA have been made. The only gene found co-amplified is the closely linked ERBA [36]. As overexpression of ERBA was not detected, the significance of the co-amplification remains unclear. Both the genetic complexity and the full informative content of the 17q11-12 amplified DNA are largely unknown.

Clinical parameters for the prognosis of breast cancer include size of the primary tumor, tumor stage at diagnosis, hormonal receptor status, and number of axillary lymph nodes with metastatic disease. Among these, the number of nodes involved is by far the best prognostic factor. An extensive study revealed that there was essentially no correlation between gene amplification and estrogen or progesterone receptor status, size of tumors, or age at diagnosis. A good correlation was detected, however, between amplification and number of positive nodes. Amplification was found to be a significant predictor or both overall survival and time to relapse [35] and appears to be superior to all other prognostic parameters except for positive lymph nodes. Generally similar observations were made in subsequent studies [37,38]. In ovarian cancer, there is a statistically significant correlation between gene amplification and survival [35]. It remains to be seen how this correlation can be used in the design of therapeutic regimens.

Amplification mechanisms
Malignant tumors characteristically arise from a multiplicity of events within the emerging cancer cell [39]. Prominent among these events are various forms of genetic damage, which may contribute to both the initiation and the continued progression of tumorigenesis. Although an amplified oncogene may be present in a major proportion of cells of an individual tumor [40], it is currently impossible to assign oncogene amplification a particular role in the multistep evolution of cancer cells.

Are there common molecular pathways through which different oncogenes are amplified in tumor cells? In the absence of any experimental condition by which the amplification of oncogenes could be triggered in tumor cells, it is impossible to directly inspect the evolution of amplified DNA. We can only compare structural elements of the end stage of the amplification process, which obviously result from a combination of molecular events during the initiation of amplification and the further, possibly cell type specific, processing of the amplified DNA. If we look at the end product, we can broadly recognize two different structural arrangements of amplified oncogenes. First, the amplified DNA can be intra-chromosomal, residing at the chromosomal site of the single copy genes involved. This arrangement is exemplified by the 11q13-amplified DNA [41]. Second, the amplified DNA can be extra-chromosomal and/or integrated in a chromosome different from the one harbouring the single copy gene. This topography applies for MYCN in neuroblastomas [42].

The molecular pathways by which MYCN is amplified have remained enigmatic. The end-point of the amplification appears to be tandem arrangement of unit size amplicons in an HSR on a chromosomal position different from 2p24 with retention of the single copy MYCN at 2p24. Among various models this arrangement would best agree with a replication-excision model (Fig. 2A). The DNA would undergo locally an additional round of replication, and the extrareplicated DNA could be excised from the replication structure and persist as extrachromosomal element, which could be visualized as a DM. At some point this extrachromosomal DNA could integrate into any chromosomal site and undergo several cycles of in situ amplification. This would explain the regular arrangement of amplification units in HSRs. The extrachromosomal generation of large amplification units that would then integrate is unlikely, as such structures have not been observed. Also, independent integration of extrachromosomal elements in the same chromosome would be unlikely to result in regular tandem arrangements. A BFB-like mechanism starting at fragile sites [43-45] appears unlikely for MYCN amplification. Although duplication of DNA, the immediate consequence of a BFB cycle, has been observed occasionally at the MYCN locus [22], the arrangement is in tandem, and not inverted. Similarly, the arrangement of amplicons in the HSR is in tandem. And finally, the HSR itself resides always in a position which is distant from 2p24, while DNA amplified by BFB cycles would be expected to remain on chromosome 2.

Conclusions
The amplification of oncogenes in human tumor cells has now attracted attention over many years for several reasons. First, it is one of the major molecular pathways by which the oncogenic potential of cellular oncogenes is activated. Second, like several other types of genetic alteration it reflects the genetic instability of the tumor cell. Finally, amplification of oncogenes frequently characterizes particularly aggressive subsets of tumors. Prototoypic is the amplified MYCN in neuroblastomas, which represented the clinical debut of onogenes and which is now widely used in evaluating the prognosis and designing therapeutic regimens for patients with neuroblastoma. Identification of amplified MYCN has been achieved by early expression profiling using oncogene arrays. It can be anticipated that the search for amplified DNA, now by modern approaches such as CGH, will remain a productive experimental strategy for exploring further the role of amplification in tumorigenesis.

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Figure 1
Chromosomal manisfestations of amplified oncogenes in cancer cells (a) G-banded metaphase of a human cancer cell with two homogeneously staining regions (HSRs) on derivative chromosome X (arrows); (b) fluorescence in situ hybridization (FISH) with a MYC probe shows one single copy signal (at right) plus highly amplified signal on HSRs (left); (c, d) Amplified MYC in double minutes (DMs): DAPI-stained metaphase with DMs (c) and FISH reveals amplified MYC in DMs (d); (e, f) amplified MYC in more complex chromosome structures: metaphase with unusually large DMs plus conspicuous ring chromosome (e) FISH reveals amplified MYC in large DMs, in the ring chromosome and in an HSR, all co-existing in the same cell.

Figure 2
Expression profiling of oncogenes in murine tumor cell line Y1 to detect oncogene amplification. Oncogene specific DNA was spotted on a nitrocellular filter, which subsequently was probed with radioactively labelled cDNA generated by reverse transcription of total polyadenylated RNA extracted from the tumor cells. A strong signal was seen for K-ras, which upon DNA analysis, turned out to 30-60fold amplified (from 20). Reprinted by permission from Nature (303, 497-501, 1983) copyright (1983) Macmillan Magazines Ltd. (the word Nature must be hyperlinked to the Nature homepage at www.nature.com)

Figure 3
Possible models illustrating amplification of the gene MYCN in neuroblastomas. Amplification could start if DNA undergoes unscheduled replication during cell division (A), or if part of the DNA is excised following loop formation (B). In normal cells the MYCN gene is localized as a single copy on chromosome 2, whereas tumor cells with amplification have up to several hundred copies, generally located in an HSR on another chromosome (in this case chromosome 11). The original copy of MYCN at 2p24 is retained, which would be in line with model A (from 46; with permission by Springer-Verlag).

Figure 4
Amplification through BFB cycles starting at fragile sites. Double chromatid breaks, such as at a fragile site (insert), and subsequent repair could result in chromatid fusion telomeric to an oncogene. At anaphase the fused chromatids form a bridge, where two copies of an oncogene would be arrranged invertedly in head-to head-position. If this structure breaks asymmetrically, the daughter cells receive either a chromatid with duplicated oncogene or a chroatid, from which the gene is deleted. Perpetuating this BFB cycle would eventually result in amplified inverted copies of the oncogene in extension of the original position of the single copy gene (from 46; with permission by Springer-Verlag).

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