The criteria for the sporadic patients (n = 97) were: primary invasive breast carcinoma less than 5 cm (T1 or T2), no axillary metastases (N0), age at diagnosis less than 55 years, calendar year of diagnosis 1983–1996, no previous malignancies; all patients were treated by modified radical mastectomy (n = 35) or breast-conserving treatment (n = 62), including axillary lymph node dissection followed by radiotherapy. Five patients of the metastases group received adjuvant systemic therapy consisting of chemotherapy (n = 3) or hormonal therapy (n = 2), all other patients did not receive additional treatment. All patients were followed at least annually for a period of at least 5 years. The criteria for hereditary patients (n = 20) were: carriers of a germline mutation in BRCA1 or BRCA2, and primary invasive breast carcinoma; no other selection criterion was applied. This study was approved by the Medical Ethical Committee of the Netherlands Cancer Institute. Tumour material was snap-frozen in liquid nitrogen within 1 h after surgery. A haematoxylin and eosin stained section was prepared before and after cutting slides for RNA isolation for assessment of the percentage of tumour cells. Only samples with greater than 50% tumour cells were selected, mean 67% and median 70% for all groups studied. Formalin-fixed, paraffin-embedded tumour tissue was used to evaluate the following: tumour type (according to the World Health Organisation classification), histological grade (grade 1–3), and the presence of angioinvasive growth and extensive lymphocytic infiltrate. ER expression was determined by immunohistochemical staining (negative when less than 10% of the nuclei showed staining, all others ER positive). We used 30 sections of 30-µm thickness for total RNA isolation. Total RNA was isolated with RNAzolB, and finally dissolved in RNase-free H2O. Twenty-five micrograms of total RNA was treated with DNase using the Qiagen RNase-free DNase kit and RNeasy spin columns. Total RNA treated with DNase was dissolved in RNase-free H2O to a final concentration of 0.2 µg µl-1.
Label
Cy3
Label protocol
cRNA was generated by in vitro transcription using T7 RNA polymerase on 5 µg total RNA and labelled with Cy3 or Cy5 (CyDye, Amersham Pharmacia Biotech). Five micrograms of Cy-labelled cRNA from one breast cancer tumour was mixed with the same amount of reverse colour Cy-labelled product from a pool, which consisted of an equal amount of cRNA from each individual sporadic patient
The criteria for the sporadic patients (n = 97) were: primary invasive breast carcinoma less than 5 cm (T1 or T2), no axillary metastases (N0), age at diagnosis less than 55 years, calendar year of diagnosis 1983–1996, no previous malignancies; all patients were treated by modified radical mastectomy (n = 35) or breast-conserving treatment (n = 62), including axillary lymph node dissection followed by radiotherapy. Five patients of the metastases group received adjuvant systemic therapy consisting of chemotherapy (n = 3) or hormonal therapy (n = 2), all other patients did not receive additional treatment. All patients were followed at least annually for a period of at least 5 years. The criteria for hereditary patients (n = 20) were: carriers of a germline mutation in BRCA1 or BRCA2, and primary invasive breast carcinoma; no other selection criterion was applied. This study was approved by the Medical Ethical Committee of the Netherlands Cancer Institute. Tumour material was snap-frozen in liquid nitrogen within 1 h after surgery. A haematoxylin and eosin stained section was prepared before and after cutting slides for RNA isolation for assessment of the percentage of tumour cells. Only samples with greater than 50% tumour cells were selected, mean 67% and median 70% for all groups studied. Formalin-fixed, paraffin-embedded tumour tissue was used to evaluate the following: tumour type (according to the World Health Organisation classification), histological grade (grade 1–3), and the presence of angioinvasive growth and extensive lymphocytic infiltrate. ER expression was determined by immunohistochemical staining (negative when less than 10% of the nuclei showed staining, all others ER positive). We used 30 sections of 30-µm thickness for total RNA isolation. Total RNA was isolated with RNAzolB, and finally dissolved in RNase-free H2O. Twenty-five micrograms of total RNA was treated with DNase using the Qiagen RNase-free DNase kit and RNeasy spin columns. Total RNA treated with DNase was dissolved in RNase-free H2O to a final concentration of 0.2 µg µl-1.
Label
Cy5
Label protocol
cRNA was generated by in vitro transcription using T7 RNA polymerase on 5 µg total RNA and labelled with Cy3 or Cy5 (CyDye, Amersham Pharmacia Biotech). Five micrograms of Cy-labelled cRNA from one breast cancer tumour was mixed with the same amount of reverse colour Cy-labelled product from a pool, which consisted of an equal amount of cRNA from each individual sporadic patient
Hybridization protocol
standard
Scan protocol
Labelled cRNAs were fragmented to an average size of approximately 50–100 nucleotides by heating at 60 °C in the presence of 10 mM ZnCl2, added to a hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine, 50 mM MES, pH 6.5, and formamide to a final concentration of 30%, final volume 3 ml at 40 °C. Hu25K microarrays represented the 24,479 biological oligonucleotides plus 1,281 control probes. Sequences for microarrays were selected from RefSeq (a collection of non-redundant mRNA sequences; http://www.ncbi.nlm.nih.gov/LocusLink/refseq.html) and from expressed sequence tag (EST) contigs (http://www.phrap.org/est_assembly/human/gene_number_methods.html). Each mRNA or EST contig was represented on the Hu25K microarray by a single 60-polymer oligonucleotide chosen by the oligonucleotide probe design programme. After hybridization, slides were washed and scanned using a confocal laser scanner (Agilent Technologies). Fluorescence intensities on scanned images were quantified, corrected for background noise and normalized.
Description
human breast tumor RNA
Data processing
Normalization between the channels was accomplished by normalizing each channel to the mean intensities of all genes. Batch effect correction using the Kolmogorov-Smirnov weighted mean method was used to reduce variability between patients. Patients were first categorized into batches. Then, for each probe, Kolmogorov-Smirnov statistics were calculated for every batch permutation. Weights were calculated by using the sum normalized sums of the KS distance matrix. Weights were used to calculate the weighted mean expression value of the given probe. Weighted average mean correction was then performed on each batch.
