Distinct and complementary information provided by use of tissue and DNA microarrays in the study of breast tumor markers

Abstract

Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining two-thirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing approximately 600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.

Figure 1.

Expression levels of ERBB2 and MUC1 mRNA levels measured by cDNA array analysis and real-time quantitative PCR amplification. ERBB2 and MUC1 mRNA expression levels measured using cDNA arrays (artificially ×30 for visual effect) (left) and real-time quantitative PCR amplification (artificially ×30 for visual effect) (right). Results for each tumor (from top to bottom) are represented as opposite bars. For ERBB2: ρ_s = 0.78, P < 0.0001; for MUC1: ρ_s = 0.88, P < 0.0001.

Figure 2.

Expression of proteins studied by IHC on TMAs. A: H&E staining of a paraffin block section (25 × 30 mm) from the TMA containing 216 arrayed tumor (3 × 55) and control samples. B: Anti-angiogenin staining. C: Anti-FGFR1 staining. D: Anti-GATA3 staining. E: Anti-PRLR staining. From B to E, the first section is from normal breast tissue, the second and third from tumor tissue (the second illustrates a moderate staining whereas the third illustrates a strong staining). Original magnifications, ×50.

Figure 3.

Transformation of continuous cDNA array data into discontinuous data. mRNA expression levels measured by cDNA array are plotted for each sample in an increasing order. For each gene, classes are determined on visual inspection and are separated by vertical bars on the graphs. Results for ER-α (ESR1), prolactin receptor (PRLR), mucin 1 (MUC1), and ERBB2 are shown.

Figure 4.

Comparison of data obtained by cDNA array and IHC on TMA. Results for each tumor (from top to bottom) are represented as opposite bars, with the value of IHC (quick score) on the left, and the value of the cDNA array analyses (artificially ×30 for visual effect) on the right. Values for ER, GATA3, and ERBB2 show good correlation between the two methods, whereas values for P53, THBS1, and MUC1 do not show such correlation.

Figure 5.

Similar variations in expression levels of two groups of proteins. A: The expression levels of ER, BCL2, and GATA3 as measured by IHC on TMAs correlated, as determined by simple linear regression analysis. B: Similarly, the expression levels of FGFR1, TACC1, and TACC2 correlated.

Figure 6.

Kaplan-Meier plots of patient overall survival. Left: Survival according to MUC1 mRNA and protein expression levels. Right: Survival according to THBS1 mRNA and protein expression levels (labeled high and low). High and low protein levels correspond to strong plus moderate versus weak plus negative (see Table 3▶ ), respectively, and high and low mRNA levels correspond to classes 2 and 3 versus class 1 (see Figure 4▶ ), respectively.

Figure 7.

Expression of MUC1 protein studied by IHC on a tissue-microarray. A: H&E staining of a paraffin block section (25 × 30 mm) from the TMA containing 647 arrayed samples, including 592 tumors and 55 controls. B: MUC1 staining: normal breast tissue (left), apical (middle), and cytoplasmic (right) staining in tumors. C: Kaplan-Meier plot of patient overall survival: survival differs significantly according to MUC1 protein localization. A: Absence of staining or apical localization; B: cytoplasmic or circumferential membrane localization.