Chromosomal breakage-fusion-bridge events cause genetic intratumor heterogeneity

Abstract

It has long been known that rearrangements of chromosomes through breakage-fusion-bridge (BFB) cycles may cause variability of phenotypic and genetic traits within a cell population. Because intercellular heterogeneity is often found in neoplastic tissues, we investigated the occurrence of BFB events in human solid tumors. Evidence of frequent BFB events was found in malignancies that showed unspecific chromosome aberrations, including ring chromosomes, dicentric chromosomes, and telomeric associations, as well as extensive intratumor heterogeneity in the pattern of structural changes but not in tumors with tumor-specific aberrations and low variability. Fluorescence in situ hybridization analysis demonstrated that chromosomes participating in anaphase bridge formation were involved in a significantly higher number of structural aberrations than other chromosomes. Tumors with BFB events showed a decreased elimination rate of unstable chromosome aberrations after irradiation compared with normal cells and other tumor cells. This result suggests that a combination of mitotically unstable chromosomes and an elevated tolerance to chromosomal damage leads to constant genomic reorganization in many malignancies, thereby providing a flexible genetic system for clonal evolution and progression.

Figure 1

BFB cycles. (A) Ring chromosomes that have undergone a sister chromatid exchange or a torsion may form bridges at anaphase that subsequently break and rejoin into new rings. As bridges may break at any point between the centromeres, the rings in the daughter cells can be different from each other and from the ring in the mother cell. (B) Dicentric chromosomes and chromosomes involved in telomeric associations can also form bridges at anaphase. The broken ends may rejoin subsequently or rearrange with other broken chromosomes, forming new variants of dicentric chromosomes.

Figure 2

Chromosome banding, FISH, and Giemsa stain analyses. (A) Ring chromosome and telomeric associations in a low-grade MFH. (B) Anaphase bridge in the same case. (C) A dicentric chromosome (arrow) visualized by FISH with a probe for all human centromeres (red) in the pancreatic carcinoma cell line LPC5. (D) Chromatin bridges between interphase nuclei in an atypical lipomatous tumor with ring chromosomes containing amplified sequences from 12q; the bridges are positive with the 12q probes 751a4 (red) and 2 g11 (green). (E) Combinations (n = 9) of structural rearrangements involving chromosome 9 material (white classification color), found by spectral karyotyping of MFH1. (F) Dicentric chromosomes containing material from chromosome 3 (dark blue) shown by combined binary and ratio labeling FISH (Upper) and inverted 4′,6-diamidino-2-phenylindole staining (Lower) in MFH2. (G) Combinations (n = 13) of structural rearrangements involving material from chromosome 1 detected by whole-chromosome painting (red) in MM. (H) Complete (at the top and right) and broken (on the left) internuclear chromatin bridges positive for whole-chromosome-9 paint (green) in MFH1.

Figure 3

The relationship between anaphase bridging and mitotically unstable chromosomes. ABC frequency is proportional to RDT frequency in borderline/low and highly malignant tumors. The correlation coefficients are 0.88 and 0.72, respectively (Spearman rank order correlation).

Figure 4

Elimination of chromosomal instability after irradiation. (A) In fibroblasts (GM498B and GM3349B), the RDT and ABC frequencies reached normal levels three passages after irradiation, whereas the frequency of cells containing stable chromosome aberrations (S), i.e., translocations, additions, deletions, and inversions increased slightly during 10 passages; S ≤ 6% before irradiation. (B) In MLS, the RDT and ABC frequencies reached normal levels at passages 3 and 2. The OSA and MM cell lines have elevated RDT and ABC levels inherently. Irradiated OSA cells did not return to their base level until passage 8. MM reached the ABC and RDT base levels at passages 10 and 12 (not shown), respectively. The RDT values were corroborated by FISH with a probe for all human centromeres in the fibroblasts and OSA (data not shown).

Figure 5

Formation (white) and elimination rates (gray) of cells carrying mitotically unstable chromosomes in tumor and fibroblast cultures, estimated by the model ΔRDT = F × (1 − RDT) − E × RDT, where ΔRDT is the difference in RDT frequency between two generations, RDT is the RDT frequency at the lower generation number, F is the newly formed fraction/generation, and E is the eliminated fraction/generation. ΔRDT was assumed to be equal to 0 at the average baseline RDT value. E and F values were calculated for each interval between passages during the elimination phase. Median values are indicated by column height, and ranges by vertical bars. The elimination rates were significantly lower in OSA and MM compared with MLS and the fibroblast lines GM498B and GM3349B (P ≤ 0.002; two-tailed Mann–Whitney U test), whereas the formation rates did not differ significantly.