Regulatory BC200 RNA in peripheral blood of patients with invasive breast cancer

Abstract

Regulatory brain cytoplasmic 200 RNA (BC200 RNA) is highly expressed in human mammary carcinoma cells. Here, we ask whether BC200 RNA becomes detectable in peripheral blood of patients with invasive breast cancer. Using quantitative reverse-transcription PCR (qRT-PCR) methodology, we observed that BC200 RNA blood levels were significantly elevated, in comparison with healthy subjects, in patients with invasive breast cancer prior to tumorectomy (p=0.001) and in patients with metastatic breast cancer (p=0.003). In patients with invasive breast cancer who had recently undergone tumorectomy, BC200 RNA blood levels were not distinguishable from levels in healthy subjects. However, normality analysis revealed a heterogeneous distribution of patients in this group, including a subgroup of individuals with high residual BC200 RNA blood levels. In blood from patients with invasive breast cancer, BC200 RNA was specifically detected in the mononuclear leukocyte fraction. The qRT-PCR approach is sensitive enough to detect as few as three BC200 RNA-expressing tumor cells. Our work establishes the potential of BC200 RNA detection in blood to serve as a molecular indicator of invasive breast malignancy.

Keywords: Rna; biological markers; blood cell count; cancer; hematologic tests.

Figure 1

BC200 RNA blood levels in patients with breast cancer (A) High levels of BC200 RNA are detected in peripheral blood from a patient with metastatic breast cancer (MBC) Total RNA was isolated from whole blood The RT-PCR product was resolved on a 12% agarose gel (bands at the bottom of the gel represent primers used in the PCR reaction) Medical history: The patient was diagnosed with breast cancer 2 years prior to BC200 RNA analysis She underwent surgery and adjuvant therapy, including six cycles of doxorubicin and cyclophosphamide followed by anastrozole (Arimidex) Two years later, she was diagnosed with bone metastases and was scheduledto undergo a new treatment cycle with paclitaxel (Taxol) and trastuzumab (Herceptin) Blood for BC200 RNA analysis was drawn after diagnosis of recurrent disease but before Taxol/Herceptin treatment It appears that in this case, that is, prior to the initiation of adjuvant therapy, BC200 RNA blood levels are higher than average BC200 RNA blood levels in metastatic disease (B) Bar diagram shows qRTPCR analysis of BC200 RNA expression levels in whole blood from healthy subjects and from three groups of patients with breast cancer (presurgery, postsurgery, metastatic disease) BC200 RNA amplification data were normalized to ARP mRNA Data are shown in the format mean±SEM, with each data point representing three experiments for each blood sample analyzed BC200 RNA blood levels are shown as fold increase in comparison with the control group (healthy subjects), calculated as 2^−ΔΔCt (Materials and methods section) Statistical analysis: non-parametric Kruskal-Wallis analysis (p=0.005) followed by non-parametric Mann-Whitney U-test; **p=0.003, ***p=0.001 (C) BC200 RNA blood levels in former patients with breast cancer with no evidence of recurrent or residual disease are similar to those in healthy subjects Statistical analysis: Kruskal-Wallis (p=0.668).

Figure 2

Sample distribution analysis of Primary Disease I and Primary Disease II patient groups (normal and detrended normal Q-Q plots) Normality analysis of Primary Disease I (primary disease patients prior to tumorectomy) sample distribution shows no significant deviation from normal distribution (green line, A) Conversely, analysis of Primary Disease II group (postsurgery primary disease patients) sample distribution shows a significant departure from normality (green line, B) Kolmogorov-Smirnov, p<0.0005.

Figure 3

ROC analysis of BC200 RNA expression in peripheral blood of patients with invasive breast cancer To ascertain the discriminative diagnostic power of the BC200 RNA blood test, receiver operating characteristic (ROC) analysis was performedby plotting sensitivity against (1−specificity), comparing BC200 RNA levels in blood from healthy subjects with BC200 RNA levels in presurgery patients with invasive breast cancer (group Primary Disease I) The obtained ROC curve shows an area under the curve of 089 (95% CI of 077 to 10), indicating a high discriminative efficacy of the BC200 RNA test.

Figure 4

(A and B) Limiting dilution experiments with MCF-7 breast cancer cell RNA in Baby Hamster Kidney (BHK) cell RNA (A) The panel shows qtr.-PCR amplification plots for BC200 RNA Total RNA isolated from MCF-7 cells, representing the respective number of cells indicated, was diluted with total RNA from 10⁴ BHK cells and subjected to qtr.-PCR amplification The curves show that BC200 RNA amplification allowed detection of as few as three MCF-7 cells The zero MCF-7 cells sample failed to reach amplification threshold after 40 cycles, indicating that no BC200 RNA was detected (B) Control amplification experiments were performed with acidic ribosomal protein (ARP) mRNA In contrast to BC200 amplification, ARP amplification resulted in plots that were grouped together, indicating constant expression levels independent of MCF-7 cell numbers (C and D) Limiting dilution experiments: MCF-7 breast cancer cell RNA in RNA from blood of healthy subjects (C) The panel shows qtr.-PCR amplification plots for BC200 RNA Total RNA isolated from MCF-7 cells (from 3 to 15 cells) was diluted with total RNA (10 ng) from blood of healthy human subjects BC200 RNA qtr.-PCR amplification allowed detection of three MCF-7 cells (D) Control amplification experiments were performed with GAPDH mRNA As in the experiments with ARP mRNA, GAPDH amplification plots were grouped together.

Figure 5

Expression of BC200 RNA in tumor cell lines Expression levels in MCF-10A cells, MCF-7 cells, BHK cells, and HeLa cells were established by qRT-PCR BC200 RNA expression was significantly elevated in MCF-7 cells and HeLa cells, in comparison with MCF-10A cells (the latter normalized to 1) Quantitative analysis: one-way ANOVA, p=0.0003; Dunnett’s post hoc analysis, comparison of MCF-7 cells (p<0.001) and of HeLa cells (p<0.01) with MCF-10A cells BHK cells were usedas a negative control as expression of BC200 RNA is specific to primates.