Relationship of extreme chromosomal instability with long-term survival in a retrospective analysis of primary breast cancer

Abstract

Background: Chromosomal instability (CIN) is thought to be associated with poor prognosis in solid tumors; however, evidence from preclinical and mouse tumor models suggest that CIN may paradoxically enhance or impair cancer cell fitness. Breast cancer prognostic expression signature sets, which reflect tumor CIN status, efficiently delineate outcome in estrogen receptor ER-positive breast cancer in contrast to ER-negative breast cancer, suggesting that the relationship of CIN with prognosis differs in these two breast cancer subtypes.

Methods: Direct assessment of CIN requires single-cell analysis methods, such as centromeric FISH, aimed at determining the variation around the modal number of two or more chromosomes within individual tumor nuclei. Here, we document the frequency of tumor CIN by dual centromeric FISH analysis in a retrospective primary breast cancer cohort of 246 patients with survival outcome.

Results: There was increased CIN and clonal heterogeneity in ER-negative compared with ER-positive breast cancer. Consistent with a negative impact of CIN on cellular fitness, extreme CIN in ER-negative breast cancer was an independent variable associated with improved long-term survival in multivariate analysis. In contrast, a linear relationship of increasing CIN with poorer prognosis in ER-positive breast cancer was observed, using three independent measures of CIN.

Conclusions: The paradoxical relationship between extreme CIN and cancer outcome in the ER-negative cohorts may explain why prognostic expression signatures, reflecting tumor CIN status, fail to predict outcome in this subgroup.

Impact: Assessment of tumor CIN status may support risk stratification in ER-negative breast cancer and requires prospective validation.

Figure 1. Distribution of average Modal Centromeric Deviation across the Leeds primary breast cancer cohort

Figure 1A: Flowchart of experimental procedures and breast cancer cohort CIN analysis. Figure 1B: Histogram distribution of the percentage of nuclei with deviation from the modal centromere signals and allocation of MCD cohorts 1-4. The y-axis shows the frequency of the MCD score. The MCD scores were grouped into four cohorts with similar range. Figure 1C: Heatmap of centromeric distribution for chromosome 2 and 15. Each column represents one tumour ranked according to MCD scores, with corresponding ER status: ER-negative (red), ER-positive (black) and HER2 status: HER2 negative (red), HER2 positive (black). The rows of the heatmap indicate the centromere number of each chromosome per nucleus and colours the percentage of nuclei having the particular centromere number (blue = 0%, red = 100%). For diagrammatic purposes the figure only portrays centromere counts between one and five.

Figure 2. Relationship of CIN scores to Shannon Diversity Index

Correlation of modal centromere deviation with Shannon Diversity Index for chromosome 2 (Figure 2A) and chromosome 15 (Figure 2B). The MCD4 extreme CIN cohort has the highest mean (Chromosome 15 and 2) Shannon Diversity Index (Figure 2C) Representative images of MCD4 cohort tumours (Figure 2D). Centromeric probes of chromosome 2 and chromosome 15 were labelled in red and green respectively.

Figure 3. Relationship of histopathological parameters to MCD cohort

Figure 3A: Distribution of Estrogen and HER2 receptor status across each MCD cohort. The frequency of ER− (including HER2+/− tumours), ER+ (including HER2+/− tumours) and HER2+ (including ER+/− tumours) breast cancers within each MCD cohort. Figure 3B: Relationship of histopathological parameters to MCD cohort in ER-negative breast cancer.

Figure 4. Association of Modal Centromeric Deviation Cohort with Breast cancer death

Kaplan-Meier survival curves for the four MCD cohorts across all ER-negative breast cancer patients. The y-axis shows breast cancer specific survival probabilities, the x-axis shows survival time in months.

Figure 5. Association of Chromosomal Instability with clinical outcome. CIN status is determined by quartile distribution using CIN70, aCGH structural chromosome complexity score and MCD (CEP2 and 15)

Figure 5A: Hazard ratios for ER-negative breast cancer across CIN quartiles determined by CIN70, combined structural chromosomal complexity (aCGH) score and MCD. Figure 5B: Hazard ratios for ER-negative breast cancer associated with quartiles of MammaPrint® and GGI (Genomic Grade Index) expression signatures. Figure 5C: Hazard ratios for ER-positive breast cancer with CIN scored by CIN70, combined structural instability (aCGH) and MCD 5D: Hazard ratios for ER-positive breast cancer associated with quartiles of MammaPrint® and GGI (Genomic Grade Index) expression signatures. Hazard ratios represent risk of death for centromeric analysis and risk of recurrence or development of metastasis for gene expression/CGH analyses.