Evaluation of breast cancer polyclonality by combined chromosome banding and comparative genomic hybridization analysis

Abstract

Cytogenetically unrelated clones have been detected by chromosome banding analysis in many breast carcinomas. Because these karyotypic studies were performed on short-term cultured samples, it may be argued that in vitro selection occurred or that small clones may have arisen during culturing. To address this issue, we analyzed 37 breast carcinomas by G-banding and comparative genomic hybridization (CGH), a fluorescent in situ hybridization--based screening technique that does not require culturing or tumor metaphases. All but two of the 37 karyotypically abnormal cases presented copy number changes by CGH. The picture of genomic alterations revealed by the two techniques overlapped only partly. Sometimes the CGH analysis revealed genomic imbalances that belonged to cell populations not picked up by the cytogenetic analysis and in other cases, especially when the karyotypes had many markers and chromosomes with additional material of unknown origin, CGH gave a more reliable overall picture of the copy number gains and losses. However, besides sometimes revealing cell populations with balanced chromosome aberrations or unbalanced changes that nevertheless remained undetected by CGH, G-banding analysis was essential to understand how the genomic imbalances arose in the many cases in which both techniques detected the same clonal abnormalities. Furthermore, because CGH pictures only imbalances present in a significant proportion of the test sample, the very detection by this technique of imbalances belonging to apparently small, cytogenetically unrelated clones of cells proves that these clones must have been present in vivo. This constitutes compelling evidence that the cytogenetic polyclonality observed after short-term culturing of breast carcinomas is not an artifact.

Figure 1

Graphic comparison of the genomic gains (A) and losses (B) detected by G-banding (green) and CGH (red) from 1p36 to Xq28 (all cases pooled). For every case, the presence or absence of imbalance in every chromosome band was computed in a spreadsheet. The total number of imbalances detected by each technique in every band was then used to prepare the graphic comparison. Imbalances in some areas of the genome are equally often detected by both techniques (e.g., gain of 1q and losses of 3p, 6q, 8p, 11q, and 16q). However, CGH seems to detect more often gains of 3q, 6q, 8q, 11, 16p, 17q and 20q, whereas G-banding more often detects losses of 1p, 2q, 4, 9q, 15q, and 17q. These differences might be explained by the preferential detection of disparate clones by each technique or by the frequent presence of the said chromosomal segments in marker chromosomes or in chromosomes that by G-banding are seen to have addition of unknown material.

Figure 2

G-banding karyogram (A) and CGH profile (B) showing the same clonal cell population in a breast carcinoma (case 80/93) with complex genomic changes. See Table 1 for a full description of the acquired clonal aberrations. The changes present in this metaphase that are not indicated in the karyotype are nonclonal.

Figure 3

Partial karyograms and CGH profiles showing the increased understanding of the acquired genetic changes obtained by the combined analysis. (A) The genomic imbalances detected by CGH (rev ish enh(1q,16p)) in case 46/93 are due to one (left) and three (right) supernumerary der(1q;16p) present in two related subclones. The loss of one chromosome 16 occurred only in one subclone, which explains why the ratio profile did not reach 0.75 at 16q. (B) The presence of a der(1q;16p) instead of a normal chromosome 16 explains the gain of 1q and loss of 16q seen by CGH in case 467/93. The imbalances detected by CGH in chromosome 11 showed that the ring chromosome contained multiple copies of 11p14p15 and 11q12q14 but no 11q22qter material. The ring was unstable and was larger in some cells (right). (C) Chromosome banding analysis showed that the rev ish enh(8q,10q21q22) and rev ish dim(8p) detected by CGH in case 503/92 derived from the presence of multiple copies of an abnormal chromosome 8 having an insertion of a segment of 10q in its long arm as well as loss of 8p material. In this case, the gain of 1q and loss of 16q are independent events, because the gain of 1q resulted from an i(1q) and not from a der(1q;16p). The inv(9) is not detectable by CGH.

Figure 4

Imbalances brought about by two cytogenetically unrelated clones (A and B) were simultaneously detected by CGH (C) in case 136/93, indicating that both clones were large in vivo and, by inference, were part of the tumor parenchyma. See Table 1 for a full description of the acquired aberrations. The changes not indicated in the karyotype are nonclonal.

Figure 5

Amplifications (ratio >2.0) were mostly seen in cases with complex abnormalities detected by either G-banding or CGH. Top row: amp(1q25q31), amp(6p23p25), amp(7p21p22), and amp(7p11p14,7q11q21). Middle row: amp(8q23), amp(8q24), amp(8q), and amp(10p12p14). Bottom row: amp(11q13), amp(17q11q21), amp(17q22q24), and amp(20q12q13).