Abstract 5267: Relationship between gene and tumor marker expression in primary breast carcinoma and lymph node metastases of the same patient
Authors: Andres Sarah A. , Wittliff James L. 
Published: June 2012

Abstract
Purpose: Our goal is to develop genomics-based tests of primary breast carcinoma for assessing risk of recurrence by comparing gene and tumor marker expression of primary lesions with those of lymph node and distant metastases. Using microarray analyses from our studies and other reports, 32 candidate genes were selected from carcinoma and stromal cells, whose expression appears related to clinical outcome of breast cancer (Andres et al., Cancer Res 69(Suppl): 403s-404s, 2009). Expression of candidate genes was evaluated with estrogen (ER) and progestin receptor (PR) protein levels to ascertain their relationship during lymph node invasion.
Procedures: ER and PR levels were determined by either ligand binding or enzyme immunoassay. RNA was isolated from tissue sections of de-identified frozen biopsies of invasive ductal carcinoma using the RNeasy® Mini kit (Qiagen) and analyzed for quality and quantity (Agilent Bioanalyzer). cDNA for qPCR measurements was prepared in Tris-HCl buffer with KCl, MgCl2, DTT (Invitrogen), dNTPs (Invitrogen), RNasin® (Promega) and Superscript® RT III (Invitrogen). qPCR reactions were performed using Power Sybr® Green PCR Master Mix (Applied Biosystems), forward/reverse primers and cDNA obtained from the reverse transcription reaction. Relative gene expression was calculated by the ddCt method, using β-actin as a reference and Universal Human Reference RNA (Stratagene) as a calibrator.
Results: Although ER and PR protein levels varied between the primary and lymph node metastasis, no patient with an ER- or PR- primary exhibited a positive ER/PR result in the metastasis. Only 3 patients with an ER+ or PR+ primary exhibited a loss of receptor expression in the lymph node metastasis. Expression of 15 of 32 genes (EVL, GABRP, TRIM29, IL6ST, RABEP1, SLC39A6, FUT8, PFKP, XBP1, MCM6, YBX1, LRBA, GATA3, MAPRE2, CKS2) was discordant in most primary lesion-lymph node metastasis pairs. However, expression of NAT1, ESR1, ST8SIA1, TBC1D9, CENPA, PLK1, ATAD2, PTP4A2, CX3CL1 GMPS and SLC43A3 exhibited concordance in the majority of primary-metastasis pairs. Only 3 patients exhibited differences in expression levels of SCUBE2, TPBG, TCEAL1, DSC2, MELK and BUB1 in primary-nodal metastasis pairs.
Conclusions: To ascertain genomic changes in primary breast carcinomas and lymph node metastases, we compared levels of 32 candidate genes whose expression appears related to clinical outcome of breast cancer. Seldom was gene expression in the metastasis greater than that of the primary. Gene expression and biological activities of their protein products are used to classify subsets of primary breast cancers according to metastatic spread. These results serve as the foundation for developing a genomics-based test to predict risk of breast cancer recurrence. Supported in part by grants from Phi Beta Psi Charity Trust and a CTSP Grant from the Commonwealth of Kentucky.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5267. doi:10.1158/1538-7445.AM2011-5267