Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

Abstract

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months.

Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score.

Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses.

Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Trial registration: ClinicalTrials.gov NCT00004205.

Figures

Figure 1

Comparison of scores computed from intact RNA and partially degraded RNA from FFPE material. Scores were determined for RNA from FF material and RNA from corresponding FFPE tumor material of 82 patients. Scatter plots are shown between scores from FF and FFPE tissues representing ER_8 (A), PGR_5 (B), HER2_2 (C) and PRO_10 (D) for each tumor. Pearson correlations are indicated.

Figure 2

Comparison of scores and immunohistochemical analysis. Correlation of histological grading and PRO_10 score. The 342 tumors were classified according to histological grading. The data are shown as boxplots with median (solid line), interquartile ranges (boxes) and minimum and maximum non-outlier values (whiskers). The PRO_10 scores higher and lower than the median are indicated as red and blue dots, respectively for each grade.

Figure 3

Survival data based on molecular scores. Kaplan-Meier plots for DFS. Patients were stratified into grade I (blue), II (green) and III (red line) (A), into low (blue) and high (red) PRO_10 scores in all samples (B) and in Grade II samples (C). The RISK_25 score is shown for all samples (D) and for tumors of patients with lymph node positive (N+) cancer (E). Median values of the scores were used as cut-offs. The p-values correspond to Log-rank test.

Figure 4

Expected rate of disease-free survival (DFS). The expected rate of events at 60 months (solid line) is shown as a function of PRO_10 (A), PGR_5 (B) and RISK_25 scores (C). The 95% confidence intervals are indicated (dashed lines). Vertical lines represent the median of all scores (solid line) and 25% and 75% quantiles (dashed lines).