Genomic differences between estrogen receptor (ER)-positive and ER-negative human breast carcinoma identified by single nucleotide polymorphism array comparative genome hybridization analysis

Abstract

Background: Estrogen receptor (ER) remains one of the most important biomarkers for breast cancer subtyping and prognosis, and comparative genome hybridization has greatly contributed to the understanding of global genetic imbalance. The authors used single-nucleotide polymorphism (SNP) arrays to compare overall copy number aberrations (CNAs) as well as loss of heterozygosity (LOH) of the entire human genome in ER-positive and ER-negative breast carcinomas.

Methods: DNA was extracted from frozen tumor sections of 21 breast carcinoma specimens and analyzed with a proprietary 50K XbaI SNP array. Copy number and LOH probability values were derived for each sample. Data were analyzed using bioinformatics and computational software, and permutation tests were used to estimate the significance of these values.

Results: There was a global increase in CNAs and LOH in ER-negative relative to ER-positive cancers. Gain of the long arm of chromosome 1 (1q) and 8q were the most obvious changes common in both subtypes: An increase in the chromosome 1 short arm (1p)/1q ratio was observed in ER-negative samples, and an increased 16p/16q ratio was observed in ER-positive samples. Significant CNAs (adjusted P<.05) in ER-negative relative to ER-positive tumors included 5q deletion, loss of 15q, and gain of 2p and 21q. Copy-neutral LOH (cnLOH) common to both ER-positive and ER-negative samples included 9p21, the p16 tumor suppressor locus, and 4q13, the RCHY1 (ring finger and CHY zinc finger domain-containing 1) oncogene locus. Of particular interest was an enrichment of 17q LOH among the ER-negative tumors, potentially suggesting breast cancer 1 gene (BRCA1) mutations.

Conclusions: SNP array detected both genetic imbalances and cnLOH and was capable of discriminating ER-negative breast cancer from ER-positive breast cancer.

Figure 1

The genetic heterogeneity of breast cancer specimens is illustrated. The distribution of the log2 ratio of each sample is shown. Each point represents a probe; data were down-sampled to 10,000 markers per sample.

Figure 2

The copy number state of all samples in the study is illustrated. Each panel represents a chromosome, with chromosomes 1 through 12 shown on the left (from bottom to top), chromosomes 13 through 22 shown on the right, and chromosome X shown at the top. Samples within a panel include estrogen receptor (ER)-positive samples (below the dashed blue line) and ER-negative samples (above the dashed line). Colors indicate copy number status (purple, deletion; red, duplication); greater color saturation indicates greater copy number aberration.

Figure 3

Statistical test results are illustrated for differential genomic copy number aberrations in estrogen receptor (ER)-negative relative to ER-positive breast carcinomas. The red bars present the genomic identification of significant targets in cancer (GISTIC) analysis of gained segments, and blue bars indicate the GISTIC analysis of deleted segments. Light and dark shades of gray present the z-statistic results of gained and deleted segments, respectively. The red dashed line marks multiple-comparison, adjusted P values (1 – pAdj) of .05. (A) This is an overview of the adjusted P values for all genomic segments from the GISTIC and z-statistic analyses. The x-axis depicts the genomic coordinates arranged in chromosome number order with the short arm (p) to the left and the long arm (q) to the right within each chromosome. The y-axis represents the results from testing the difference of log2 ratios between ER-positive cancers and ER-negative cancers, with the values representing (1 – pAdj) times the test result’s sign; the sign represents the direction of the 2-tailed statistical test result, in which a positive sign (upper half) indicates relative gain in ER-positive samples compared with ER-negative samples (ie, a relative loss in ER-negative samples), and a negative sign (lower half) indicates relative loss in ER-positive samples (ie, relative gain in ER-negative samples). Values near ±1 are statistically highly significant, whereas values near 0 are not. (B) This is a detailed view of the adjusted P values of chromosome 1 genomic segments. (C) This is a detailed view of the adjusted P values of chromosome 16 genomic segments.

Figure 4

Copy-neutral loss of heterozygosity (cnLOH) of chromosome 17 is more frequent in estrogen receptor (ER)-negative (ER−) breast cancer (right of vertical blue dashed line) than in ER-positive (ER+) breast cancer (left of vertical blue dashed line). LOH without reduction in copy number in the 21 sample dataset is indicated in red; and overall LOH, including that associated with deletions, is indicated in pink. Missing values are indicated in white (4.3% genome-wide). Smaller genome coordinates representing the short arm are shown on the bottom.