The aim of this study is to systematically evaluate the prognostic effect of the immune system in breast cancer.

The hypothesis of this study is that the immune system can have a prognostic role in tumors with high expression of proliferation-associated genes.

The population-based cohort study consists of 200 consecutive lymph node-negative breast cancer patients treated at the Department of Obstetrics and Gynecology of the Johannes Gutenberg University Mainz between 1988 and 1998. Patients were all treated with surgery and did not receive any systemic therapy in the adjuvant setting. The established prognostic factors (histologic grade, tumor size, age at diagnosis, and steroid receptor status) were collected from the original pathology reports of the gynecologic pathology division within our department. Patients were treated with either modified radical mastectomy (n = 75) or breast conserving surgery followed by irradiation (n = 125) and were without evidence of regional lymph node and distant metastasis at the time of surgery. The median age of the patients at surgery was 60 y (range, 34– 89 y). The median time of follow up was 92 months.

The study was approved by the ethical review board of the medical association of Rhineland-Palatinate [no. 837.139.05 (4797)].

For all tumors, samples were snap frozen and stored at
80 °C. Tumor cell content exceeded 40% in all samples. Approximately 50 mg of frozen breast tumor tissue were crushed in liquid nitrogen. RLT buffer was added and the homogenate was centrifuged through a QIAshredder column (Qiagen). From the eluate, total RNA was isolated with the RNeasy Kit (Qiagen) according to the manufacturer’s instructions. RNA yield was determined by UV absorbance, and RNA quality was assessed by analysis of rRNA band integrity on an Agilent 2100 Bioanalyzer RNA 6000 LabChip kit (Agilent Technologies).

The Affymetrix HG-U133A array and GeneChip System was used to quantify the relative transcript abundance in the breast cancer tissues. Starting from 5-Ag total RNA, labeled cRNA was prepared using the Roche Microarray cDNA Synthesis, Microarray RNA Target Synthesis (T7) and Microarray Target Purification Kit according to the manufacturer’s instructions. Raw.cel file data were processed by MAS 5.0 software. In the analysis settings, the global scaling procedure was chosen, which multiplied the output signal intensities of each array to a mean target intensity (TGT) of 500. Samples with suboptimal average signal intensities (i.e., scaling factors >25) or glyceraldehyde-3-phosphate dehydrogenase 3'/5' ratios >5 were relabeled and rehybridized on new arrays. Raw.cel file, MAS 5.0 processed and patient data have been deposited in National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and are accessible through GEO Series accession no. GSE11121.