Gene amplification as double minutes or homogeneously staining regions in solid tumors: origin and structure

Abstract

Double minutes (dmin) and homogeneously staining regions (hsr) are the cytogenetic hallmarks of genomic amplification in cancer. Different mechanisms have been proposed to explain their genesis. Recently, our group showed that the MYC-containing dmin in leukemia cases arise by excision and amplification (episome model). In the present paper we investigated 10 cell lines from solid tumors showing MYCN amplification as dmin or hsr. Particularly revealing results were provided by the two subclones of the neuroblastoma cell line STA-NB-10, one showing dmin-only and the second hsr-only amplification. Both subclones showed a deletion, at 2p24.3, whose extension matched the amplicon extension. Additionally, the amplicon structure of the dmin and hsr forms was identical. This strongly argues that the episome model, already demonstrated in leukemias, applies to solid tumors as well, and that dmin and hsr are two faces of the same coin. The organization of the duplicated segments varied from very simple (no apparent changes from the normal sequence) to very complex. MYCN was always overexpressed (significantly overexpressed in three cases). The fusion junctions, always mediated by nonhomologous end joining, occasionally juxtaposed truncated genes in the same transcriptional orientation. Fusion transcripts involving NBAS (also known as NAG), FAM49A, BC035112 (also known as NCRNA00276), and SMC6 genes were indeed detected, although their role in the context of the tumor is not clear.

Figure 1.

Partial metaphases showing cohybridization FISH experiments using WCP specific for chromosome 2 (green) and RP11-480N14 (chr2:15,999,497–16,004,580), containing the MYCN gene (red). GLC8, STA-NB-4, STA-NB-8, STA-NB-10/dmin, and STA-NB-13 showed MYCN amplification in form of dmin (example provided at right). The remaining cell lines displayed hsr amplification in a single location (SK-N-BE, STA-NB-10/hsr) or in multiple locations (GLC14, STA-NB-3, STA-NB-15). Note the absence of the RP11-480N14 signal on one of the two chromosomes 2 in STA-NB-10 clones (arrows).

Figure 2.

Details of array CGH data (chr2:14,500,000–19,500,000) related to both STA-NB-10/dmin (triangles) and STA-NB-10/hsr (circles) subclones. The pattern of amplified/nonamplified regions perfectly overlaps. Note also that the nonamplified regions, internal to the amplicon, are present in single copy.

Figure 3.

Definition of the boundaries of the deletion on chromosome 2 present in the cell line STA-NB-10/dmin using FISH. (a,b) Partial metaphases showing examples of FISH experiments using fosmid probes spanning the distal (a) and proximal (b) breakpoints of the deleted region, cohybridized with the BAC containing the MYCN gene. (c,d) The position on the human sequence of fosmid clones in a and b, respectively. (Vertical red lines) Amplicon boundaries that were defined by sequencing the amplicon junction (see text and Supplemental Fig. 4). The definition of the deletion breakpoints was obtained as follows. Distal breakpoint. Fosmid G248P87145E2 (chr2:15,461,044–15,505,473; 44.4 kb) gave a signal on del(2) that, by eye, consistently appeared to be half the intensity of the signal on normal chromosome 2 (10 analyzed metaphases; its black and white original signal is separately reported in a). The middle part of the fosmid was therefore assumed to harbor the telomeric break of the deletion at ∼chr2:15,483 kb. G248P87145E2 is present also on the amplicon (a, inset), but the signal intensity is biased by the amplification. The normal intensity on del(2) of fosmid G248P800648F12 (chr2:15,428,940–15,471,782), and the absence of any signal on the dmin, was consistent with the assumption in a. Proximal breakpoint. Fosmid G248P87325H8 (chr2:18,656,707–18,695,100; 38.4 kb) consistently gave a signal of half intensity on del(2) (10 analyzed metaphases; its black and white original signal is separately reported in b). The centromeric break of the deletion was therefore assumed to lay at ∼chr2:18,575 kb. Note that sequences of G248P87145E2 are present on the amplicon (b, inset), but, again, the signal intensity is biased by the amplification. The intensity on del(2) of signals yielded by fosmids G248P89303A7, G248P85351E4, and G248P86949C3 was consistent with this hypothesis. The deletion, therefore, spanned approximately the interval chr2:15,483–18,575 kb. Because of growth difficulty of the STA-NB-10/hsr cell line, not all FISH experiments performed on STA-NB-10/dmin were also performed in hsr subclone (see Supplemental Table 1). (e,f) Evidence for the deletion on one of the two copies of chromosome 2. Two fosmid clones flanking the deleted region (red and green) were hybridized with a probe (RP11-723P4, violet) mapping within the deletion but absent in the amplicon (see text). The BAC was chosen in order to avoid the high background due to the amplification. The distance between the red and green probes on normal chromosome 2 (red arrow) is substantially reduced on the del(2) (white arrow), as expected. (f) Rationale of the FISH experiment in e.

Figure 4.

(Lower part of each section) Schematic representation of the structure of the amplicon. Different blocks composing the amplicons are represented by arrows of different color. (Upper parts) Arrangement of the amplified blocks in the reference sequence (hg18). (Gray line segments) Nonamplified regions; (vertical figures) boundaries of the breaks, at single-base resolution if sequenced (Supplemental Fig. 4), or as a small interval (maximum interval size: 2.5 kb); (?) uncloned fusion regions. In STA-NB-10 the deleted regions (del) internal to the amplified region are indicated. The lengths of the arrows are only approximately proportional the DNA segment length.

Figure 5.

Sequence of Junctions I, III, and VI of STA-NB-10/dmin and STA-NB-10/hsr (see Fig. 4), found identical at the single base-pair level. Note the AT microhomology at the junction.

Figure 6.

Cohybridization FISH experiments used to characterize hsr insertion sites. MYCN was used in all experiments to mark the hsr, and it was cohybridized with contiguous probes flanking the insertion (a,f) or with clones yielding split signals (b–e). (a) In STA-NB-3, the insertion is within the short arm of chromosome 13, between the centromere (alphoid probe, red) and the NOR region (RP5-1174A5). (c) In STA-NB-15, the BAC RP11-449J21 (red) displayed signals distally, proximally, and within the hsr (arrowheads), likely due to a duplication event occurring simultaneously to the hsr insertion process. (e,f) Cell line GLC14 showed two insertion sites. Map positions of all the clones are reported in Supplemental Table 1.