P4-09-30: Relationship between DNA Ploidy, Biomarker Expression and Molecular Subtypes of Invasive Breast Cancer.
Authors: Sarode VR , Rahardja D , Morrison DH , Peng Y , Rao R , Euhus D 
Published: June 2012

Background: DNA ploidy has been shown to have prognostic significance in patients with breast cancer. Studies in the past have mainly utilized flow cytometry (FCM) for measuring DNA ploidy. However FCM has several disadvantages, the instrument cannot distinguish benign from malignant cells and it cannot be performed on small tumor samples. The relationship between DNA ploidy and biomarker expression in breast cancer has not been well studied. Recently, gene expression analysis has demonstrated distinct subtypes of breast cancer. Expression of ER, PR and Her2 by IHC has been used as a surrogate tool for the molecular classification of breast cancer. Aim: To determine the relationship between DNA ploidy, biomarkers (ER, PR, HER2, Ki67 and p53) expression and molecular subtypes of invasive breast cancer (IBCA) using image analysis.

Design: DNA analysis was performed on Feulgen stained sections from the same tumor block used for biomarker analysis. DNA indices and ploidy were analyzed using the Autocyte Pathology Workstation (Tripath, Burlington, NC). Briefly, a total of 200–300 nuclei were collected and mean DNA index reported. DNA index was obtained by measuring the optical density of tumor cells in comparison with those of the non-neoplastic stromal cells in the sample using the latter as the diploid reference (value of 1.0). Tumors were classified as diploid (DNA indices of 0.90 to 1.1), aneuploid (DNA indices of &lt;0.89 and &gt; 1.1) and multiploid tumors; multiple indices. Patient's age, tumor size, histologic grade, stage, biomarker status and breast cancer subtypes were correlated with ploidy status.

Results: Of the 248 cases of IBCA, 176 had aneuploid DNA and 72 diploid. Aneuploid tumors were predominantly grade 3, 72.1% versus 27.9% of diploid (p&lt;0.0001). Tumor ploidy had no significant association with age (&lt;40 years) versus older age (&gt;50 years), p=0.118. The mean tumor size in aneuploid and diploid tumors was 4.2 cm and 3.2 cm respectively (p=0.009). Aneuploid tumors were frequently ER and PR negative compared to diploid (p&lt;0.0001) with a mean ER level of 31.7% compared to 72.2% in diploid tumors (p&lt;0.0001). The mean PR level was 17.2% in aneuploid versus 34.0% in diploid tumors (p=0.0009). The ki67 index was 52.4% in aneuploid compared to 29.1% in diploid (p&lt;0.0001). The p53 expression was 30.3% in aneuploid versus 14.4% in diploid tumors. Aneuploid tumors were frequently of advanced T stage compared to diploid tumors (p=0.0048). There was no significant association with N stage (p=0.734). By multivariate analysis after adjusting for age, grade, T and N stage, ER (p=0.0021), PR (p=0.0003), HER2 over-expression (p= 0.0028), Ki67 (p=0.0383), T stage (p= 0.0172 and grade (&lt;0.0001) were significantly associated with aneuploidy. Of breast cancer subtypes, Her2 (p&lt;0.0001), triple negative (p&lt;0.0001), luminal B subtype (p=0.006) were frequently aneuploid and luminal A was frequently diploid (p=0.051).

Conclusion: DNA ploidy measured by image analysis has significant association with biomarker expression. Increased DNA content is associated with poor prognostic features and aggressive subtypes of breast cancer.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-30.