Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene

Abstract

The evolution of somatic genetic aberrations in breast cancer has remained poorly understood. The most common chromosomal abnormality is hyperdiploidy, which is thought to arise via a transient hypodiploid state. However, hypodiploidy persists in 1 to 2% of breast tumors, which are characterized by a poor prognosis. We studied the genetic aberrations in 15 flow cytometrically hypodiploid breast cancers by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). Surprisingly, numerous copy number gains were detected in addition to the copy number losses. The number of gains per tumor was 4.3 +/- 3.2 and that of losses was 4.5 +/- 3.3 (mean +/- SD), which is similar to that previously observed in hyperdiploid breast cancers. Gains at chromosomes or chromosomal regions at 11q13, 1q, 19, and 16p and losses of 2q, 4, 6q, 9p, 13, and 18 were most commonly observed. Compared with unselected breast carcinomas, hypodiploid tumors showed certain differences. Loss of chromosome 4 (53%) and gain of 11q13 (60%) were significantly more common in hypodiploid tumors. The gain at 11q13 was found by FISH to harbor amplification of the Cyclin D1 oncogene, which is therefore three to four times more common in hypodiploid than in unselected breast cancers (15 to 20%). Structural chromosomal aberrations (such as Cyclin D1 amplification) were present both in diploid and hypodiploid tumor cell populations, as assessed by FISH and CGH after flow cytometric sorting. Together these results indicate that hypodiploid tumors form a distinct genetic entity of invasive breast cancer, although they probably share a common genetic evolution pathway where structural chromosomal aberrations precede gross DNA ploidy changes.

Figure 1.

Flow cytometric DNA histogram demonstrating hypodiploidy in a breast cancer sample. A small DNA diploid G0/G1 stemline with a DNA index of 1.00 and a larger DNA hypodiploid stemline with a prominent G0/G1 peak with a DNA index of 0.93 (corresponding to 85% of all nuclei analyzed). The S-phase fraction (S) was 4.4%, indicating a low tumor proliferation rate. G2 refers to G2/M phase of the cell cycle for the hypodiploid stemline. C and T stand for chicken and trout red blood cells, which were used as internal controls.

Figure 2.

Summary of the gains and losses in 15 hypodiploid breast cancers by CGH. Lines on the right side of the chromosome indicate copy number gains, and those on the left side indicate copy number losses. Thick lines indicate high-level amplification.

Figure 3.

The comparison of the frequencies of the most common gains (A) and losses (B) in 15 hypodiploid (black columns) to 55 unselected (gray columns) breast cancers (Tirkkonen et al¹⁵). Only aberrations common (>10%) for either type of tumor population are shown. Hypodiploid breast tumors show distinct pattern of genetic aberrations compared with unselected tumors. The frequency of loss of chromosome 4 and amplification of 11q13 (11q) are statistically significant.

Figure 4.

Examples of two-color FISH in hypodiploid tumors. Hybridized signals are visualized either in green or red fluorescence, and nuclei are counterstained with DAPI (blue). A tumor sample (I) shows loss of one copy of chromosomes 2 (green) and 18 (red) and a tumor (J) shows loss of one copy of chromosome 4 (green), whereas chromosome X (red) has retained both copies. DAPI (blue) was used for counterstaining of the nuclei. The corresponding CGH copy number profiles of chromosomes 2, 4, 18, and X are shown in below the FISH images.

Figure 5.

High-level amplification of Cyclin D1 oncogene at 11q13 in a hypodiploid breast tumor. A: CGH green-to-red fluorescence profile showing a copy number increase at 11q13. B: FISH of the same tumor demonstrating amplification of the Cyclin D1 gene (numberous green signals). A chromosome 11 centromere probe was as a reference red signals. DAPI blue was used as a counterstain.

Figure 6.

Comparison of averaged CGH profiles from flow cytometrically sorted diploid and hypodiploid subpopulations of tumor B. Green-to-red fluorescence ratio profiles from chromosomes 8, 11, and 16 demonstrate that gain of 11q13 and 16p are present both in diploid and hypodiploid populations and that the latter shows an additional loss of the entire chromosome 8.

Figure 7.

Evidence by FISH that subregional genetic aberrations are present in both diploid and hypodiploid tumor cell populations. A: Tumor C showing nuclei with a single copy of chromosome 8 (green) and a duplication of MYC oncogene (red) and cells with two copies of chromosome 8 and four copies of MYC. b: Tumor E showing amplification of Cyclin D1 gene (numberous green signals) in cells with a single copy and those with two copies of chromosome 11 (red signals).