Cytogenetic and molecular studies have identified imbalanced chromosomal regions leading to the characterization of several candidate genes. Differences in gene expression were examined in the blood by whole genome microarray analysis among individuals with double or single primary malignancies and healthy individuals. Twenty-four individuals with at least two primary malignancies of the breast and/or colon and/or ovary were compared with 32 individuals with single breast, colon or ovarian cancer. The single malignancy group had a median duration of disease of 9 years (range 5-23 years). Validation was obtained by examining each patient separately with quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for the determined genes. Overall a large number of genes were determined to be deregulated. From the classifiers built, a 9-probe signature was determined between second primary and single tumor patients. Four other genes were determined to be repressed (p<1x10(-4)) in individuals with two primary malignancies when compared with individuals with a single malignancy and also when comparing single malignancies and healthy subjects. The levels of gene deregulation were confirmed by validation with quantitative RT-PCR analysis. Functional analysis, suggested that these genes are associated with protein biosynthesis and folding, inhibition of apoptosis and intracellular signalling via GTP cascade. The outcome of the present study was 13 genes had a statistically significant difference in expression between individuals with double primary malignancies compared to individuals with single primary malignancies. Nine of those were confirmed by the classifier analysis.