Replication timing aberrations and aneuploidy in peripheral blood lymphocytes of breast cancer patients

Abstract

Background: Peripheral blood lymphocytes of patients with hematological malignancies or solid tumors, such as renal cell carcinoma or prostate cancer, display epigenetic aberrations (loss of synchronous replication of allelic counterparts) and genetic changes (aneuploidy) characteristic of the cancerous phenotype. This study sought to determine whether such alterations could differentiate breast cancer patients from cancer-free subjects.

Methods: The HER2 locus-an oncogene assigned to chromosome 17 whose amplification is associated with breast cancer (BCA)-and the pericentromeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosome 17. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization technology applied to phytohemagglutinin-stimulated lymphocytes of 20 women with BCA and 10 control subjects.

Results: We showed that both the HER2 and CEN17 loci in the stimulated BCA lymphocytes altered their characteristic pattern of synchronous replication and exhibited asynchronicity. In addition, there was an increase in chromosome 17 aneuploidy. The frequency of cells displaying asynchronous replication in the patients' samples was significantly higher (P < 10(-12) for HER2 and P < 10(-6) for CEN17) than the corresponding values in the control samples. Similarly, aneuploidy in patients' cells was significantly higher (P < 10(-9)) than that in the controls.

Conclusions: The HER2 and CEN17 aberrant replication differentiated clearly between BCA patients and control subjects. Thus, monitoring the replication of these genes offers potential blood markers for the detection and monitoring of breast cancer.

Figure 1

Fluorescent signals in PHA-stimulated lymphocytes at interphase after FISH with the CEN17 probe. (A and B) Cells with two singlets (SS cells), in which neither allele has replicated. (C and D) Cells with two doublets (DD cells), in which both alleles have replicated. (E and F) Cells with one singlet and one doublet (SD cells), which are S-phase cells in which one allele has replicated, whereas its partner still has to do so.

Figure 2

Distribution of blood samples, according to their HER2 versus CEN17 SD values. BCA indicates breast cancer patients; CON, control subjects. The arrows point to samples obtained form BRCA1 mutation carriers. Note the clear separation between the CON and the BCA samples.

Figure 3

Mean values of (A) SD, (B) SS, and (C) DD for HER2 and CEN17. Values within the upper boxes of each frame present the level of significance (P) of the differences between the control group (10 cases) and the breast cancer group (20 cases) for the designated locus.

Figure 4

Distribution of blood samples according to their SD value and aneuploidy frequencies for CEN17. (A) Aneuploidy frequency of chromosome 17 versus SD value for CEN17. (B) Multisomic versus monosomic frequencies of chromosome 17. The aneuploidy value is the sumof the correspondingmonosomic andmultisomic values. The arrows in each frame point to samples obtained form BRCA1 mutation carriers.

Figure 5

Mean and SD values of frequency of cells with aneuploidy of chromosome 17. Values in the upper boxes present the level of significance (P) of the differences between the control group (10 cases) and the breast cancer group (20 cases) for the designated type of chromosomal aberration; the aneuploidy value is the sum of the monosomic and multisomic corresponding values.