Unraveling the chromosome 17 patterns of FISH in interphase nuclei: an in-depth analysis of the HER2 amplicon and chromosome 17 centromere by karyotyping, FISH and M-FISH in breast cancer cells

Abstract

Background: In diagnostic pathology, HER2 status is determined in interphase nuclei by fluorescence in situ hybridization (FISH) with probes for the HER2 gene and for the chromosome 17 centromere (CEP17). The latter probe is used as a surrogate for chromosome 17 copies, however chromosome 17 (Chr17) is frequently rearranged. The frequency and type of specific structural Chr17 alterations in breast cancer have been studied by using comparative genomic hybridization and spectral karyotyping, but not fully detailed. Actually, balanced chromosome rearrangements (e.g. translocations or inversions) and low frequency mosaicisms are assessable on metaphases using G-banding karyotype and multicolor FISH (M-FISH) only.

Methods: We sought to elucidate the CEP17 and HER2 FISH patterns of interphase nuclei by evaluating Chr17 rearrangements in metaphases of 9 breast cancer cell lines and a primary culture from a triple negative breast carcinoma by using G-banding, FISH and M-FISH.

Results: Thirty-nine rearranged chromosomes containing a portion of Chr17 were observed. Chromosomes 8 and 11 were the most frequent partners of Chr17 translocations. The lowest frequency of Chr17 abnormalities was observed in the HER2-negative cell lines, while the highest was observed in the HER2-positive SKBR3 cells. The MDA-MB231 triple negative cell line was the sole to show only non-altered copies of Chr17, while the SKBR3, MDA-MB361 and JIMT-1 HER2-positive cells carried no normal Chr17 copies. True polysomy was observed in MDA-MB231 as the only Chr17 alteration. In BT474 cells polysomy was associated to Chr17 structural alterations. By comparing M-FISH and FISH data, in 8 out of 39 rearranged chromosomes only CEP17 signals were detectable, whereas in 14 rearranged chromosomes HER2 and STARD3 genes were present without CEP17 signals. HER2 and STARD3 always co-localized on the same chromosomes and were always co-amplified, whereas TOP2A also mapped to different derivatives and was co-amplified with HER2 and STARD3 on SKBR3 cells only.

Conclusion: The high frequency of complex Chr17 abnormalities suggests that the interpretation of FISH results on interphase nuclei using a dual probe assay to assess gene amplification should be performed "with caution", given that CEP17 signals are not always indicative of normal unaltered or rearranged copies of Chr17.

Figure 1

Analysis of Chr17 using G-Banding, dual-color FISH ( HER2 /CEP17 , STARD3 /CEP17 and TOP2A /CEP17) and M-FISH in the MCF7, T47D, ZR-75-1 and MDA-MB231 not HER2 amplified breast cancer cell lines. Rearranged chromosomes containing a portion of Chr17 are visualized by G-Banding technique on the left and by M-FISH on the right. For M-FISH the classified color of Chr17 is shown in pink, the translocation partners are numbered on the right hand side of the chromosomes and the frequency at which each abnormality was observed is indicated in brackets at the end of each abnormality. CEP17, HER2, STARD3 and TOP2A are shown in the middle by dual-color FISH (HER2/CEP17, STARD3/CEP17, TOP2A/CEP17, respectively) whenever mapped to the corresponding derivatives (CEP17 is green-labeled; HER2, STARD3 and TOP2A genes are red-labeled).

Figure 2

Analysis of Chr17 using G-Banding, dual-color FISH ( HER2 /CEP17 , STARD3 /CEP17 and TOP2A /CEP17) and M-FISH in KPL4 HER2 amplified breast cancer cell line showing four translocated Chr17 in addition to the normal-appearing copies of Chr17 and in one triple negative breast cancer case (TNBC) showing five rearranged copies of Chr17. Rearranged chromosomes containing a portion of Chr17 are visualized by G-Banding technique on the left and by M-FISH on the right. For M-FISH the classified color of Chr17 is shown in pink, the translocation partners are numbered on the right hand side of the chromosomes and the frequency at which each abnormality was observed is indicated in brackets at the end of each abnormality. CEP17, HER2, STARD3 and TOP2A are shown in the middle by dual-color FISH (HER2/CEP17, STARD3/CEP17, TOP2A/CEP17, respectively) whenever mapped to the corresponding derivatives (CEP17 is green-labeled; HER2, STARD3 and TOP2A genes are red-labeled). In the TNBC cells the chromosome in which we identified Chr17 material only is a der(17)del(17)(p11.2)del(17)(q11.2) with a deletion on both short and long arm involving 17q12-q21.

Figure 3

Analysis of Chr17 using G-Banding, dual-color FISH ( HER2 /CEP17 , STARD3 /CEP17 and TOP2A /CEP17) and M-FISH in BT474 and MDA-MB361 HER2 amplified breast cancer cell lines showing six translocated copies of Chr17. Rearranged chromosomes containing a portion of Chr17 are visualized by G-Banding technique on the left and by M-FISH on the right. For M-FISH the classified color of Chr17 is shown in pink, the translocation partners are numbered on the right hand side of the chromosomes and the frequency at which each abnormality was observed is indicated in brackets at the end of each abnormality. CEP17, HER2, STARD3 and TOP2A are shown in the middle by dual-color FISH (HER2/CEP17, STARD3/CEP17, TOP2A/CEP17, respectively) whenever mapped to the corresponding derivatives (CEP17 is green-labeled; HER2, STARD3 and TOP2A genes are red-labeled).

Figure 4

Analysis of Chr17 using G-Banding, dual-color FISH ( HER2 /CEP17 , STARD3 /CEP17 and TOP2A /CEP17) and M-FISH in SKBR3 and JIMT-1 HER2 amplified breast cancer cell lines showing four or six translocated copies of Chr17. Rearranged chromosomes containing a portion of Chr17 are visualized by G-Banding technique on the left and by M-FISH on the right. For M-FISH the classified color of Chr17 is shown in pink, the translocation partners are numbered on the right hand side of the chromosomes and the frequency at which each abnormality was observed is indicated in brackets at the end of each abnormality. CEP17, HER2, STARD3 and TOP2A are shown in the middle by dual-color FISH (HER2/CEP17, STARD3/CEP17, TOP2A/CEP17, respectively) whenever mapped to the corresponding derivatives (CEP17 is green-labeled; HER2, STARD3 and TOP2A genes are red-labeled).

Figure 5

Representative FISH images of the MDA-MB231, T47D and ZR-75-1 breast cancer cells and one TNBC case using HER2 /CEP17, STARD3 /CEP17 and TOP2A /CEP17 dual-color probes. Metaphase spreads are shown and boxes indicate representative interphase nuclei for each case. None of these cell lines showed amplification of the HER2, STARD3 or TOP2A genes. Gene signals are red-labeled, CEP17 signals are green-labeled.

Figure 6

Representative FISH images of the MCF7, BT474, MDA-MB361 and SKBR3 breast cancer cell lines using HER2 /CEP17, STARD3 /CEP17 and TOP2A /CEP17 dual-color probes. Metaphase spreads are shown and boxes indicate representative interphase nuclei for each case. FISH images for the BT474, MDA-MB361 and SKBR3 cells demonstrated HER2 and STARD3 gene amplification, while TOP2A gene amplification was observed in the SKBR3 cells only. Gene signals are red-labeled, CEP17 signals are green-labeled.

Figure 7

Representative FISH images of the JIMT-1 and KPL4 breast cancer cell lines using HER2 /CEP17, STARD3 /CEP17 and TOP2A /CEP17 dual-color probes. Metaphase spreads are shown and boxes indicate representative interphase nuclei for each case. FISH images for the JIMT-1 and KPL4 cells demonstrate HER2 and STARD3 gene amplification, while TOP2A gene amplification was not observed in these cells. Gene signals are red-labeled, CEP17 signals are green-labeled.