Our data from a large group of women in a defined population indicate that screening mammography may miss tumors of lobular or mucinous histology and some rapidly proliferating, high-grade tumors.

In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

Analysis of allelic loss in archival tumor specimens is constrained by quality and quantity of tissue and by technical limitations on the number of chromosomal sites that can be efficiently evaluated in conventional analyses using polymorphic microsatellite markers. Newly developed array-based assays have the potential to yield genome-wide data from small amounts of tissue but have not been validated for use with routinely processed specimens. We used the Affymetrix HuSNP assay, composed of 1494 single nucleotide polymorphism sites, to compare allelic loss results obtained from both formalin-fixed and frozen breast tissue samples. Tumor cells were separated from normal epithelia and nonepithelial cells by dissection and bivariate cytokeratin/DNA flow sorting; normal breast cells from the same patient served as constitutive normal. Allele results from the HuSNP array averaged 96% reproducibility between duplicates and were concordant between the fixed and frozen normal samples. We also analyzed DNA from the same samples after whole-genome amplification (primer extension preamplification). Although overall signal intensities were lower, the genotype data from the primer extension preamplification material was concordant with genomic DNA data from the same samples. Results from genomic normal tissue DNA averaged informative single nucleotide polymorphism at 379 (25%) loci genome-wide. Although data points were clustered and some segments of chromosomes were not informative, our data indicated that the Affymetrix HuSNP assay could provide an efficient and valid genome-wide analysis of allelic imbalance in routinely processed and whole genome-amplified pathology specimens.

The MAPK/ERK pathway is a cascade of serine/threonine protein kinases activated by various extracellular factors to control cell growth, differentiation, migration and death. A number of somatic mutations occur that lead to constitutive activation of this pathway in multiple human cancers. BRAF mutations were detected in over 60% of melanomas and may account for a significant proportion of colon cancer patients whose tumors are resistant to anti-EGFR therapy. ARQ 736 is a novel inhibitor of BRAF and is selectively potent in killing cancer cell lines harboring at least one mutated BRAF allele. The objective of this study is to identify potential protein biomarkers for use in connection with ARQ 736 treatment. A number of proteins released by BRAF (V600E) mutant A375 human melanoma cells into conditioned media detected by antibody arrays (R&D human angiogenesis array ARY007 and human cytokine array ARY005) are reduced following exposure to ARQ 736. These effects were not seen in the ARQ 736-resistant cell line DLD1, a k-ras mutant colon carcinoma line. Potential protein biomarkers identified include GM-CSF, Serpin E1, VEGF, amphiregulin, IGFBP-2, IGFBP-3, pentraxin-3 and IL-8. To investigate whether changes in secreted proteins observed in vitro in response to ARQ 736 treatment could be recapitulated in vivo, a similar analysis of plasma was performed at the termination of an A375 human tumor xenograft study. In this study, ARQ 736 administered intraperitoneally at 300 mg/kg on a Q1Dx13 schedule resulted in a 54% inhibition of growth of A375 human melanoma tumor xenografts. Constant administration of ARQ 736 by Alzet osmotic mini-pumps resulted in a 77% inhibition of tumor growth. Pharmacodynamic evidence of BRAF inhibition by ARQ 736 was demonstrated by a reduction in p-ERK staining of tumors via immunohistochemistry. Plasma from the ARQ 736 treated mice showed a reduction in human angiogenesis and cytokine related proteins relative to the control group. These include GM-SCF, Serpin E1, sICAM, and IL-8. The markedly reduced plasma IL-8 levels in response to ARQ 736 treatment detected by the antibody array were subsequently confirmed with a quantitative ELISA for human IL-8 (6-8 fold reduction). These results provide a basis for future clinical biomarker studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2517.

Supplementary Table 2 from Array Comparative Genomic Hybridization Analysis of Genomic Alterations in Breast Cancer Subtypes