Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer

Abstract

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology.

Fig. 1

Scheme of preanaytical steps from tissue removal to archiving of the FFPE tumor material and related experiments that were performed in this study

Fig. 2

EndoPredict test results achieved after different storage times and temperatures before fixation. The solid line represents the reference score which was derived from the mean EP score of the tissue sections that have been stored for 10 min at 20 °C before fixation. Dotted lines represent the reference score ±3× its standard deviation as determined by precision study [24]. Measurements were performed in duplicate (n = 2). Mean values are presented, with standard errors of the means indicated by bars

Fig. 3

EndoPredict test results achieved after different fixation times. The solid line represents the reference score which was derived from the mean EP score of the tissue sections that have been fixed for 20 h in neutral buffered formalin. Dotted lines represent the reference score ±3× its standard deviation as determined by precision study [24]. Measurements were performed in duplicate (n = 2). Mean values are presented, with standard errors of the means indicated by bars

Fig. 4

Correlation between the EP (a) and EPclin (b) scores of paired whole and tumor-enriched, manually microdissected tissue sections from ER+/HER2− breast cancer patients (n = 39). Pearson correlation coefficient of EP scores was 0.97 with a concordance of EndoPredict risk classification of 87 %. Pearson correlation coefficient of EPclin scores was 0.98 with a concordance of EndoPredict risk classification of 95 %. Black filled circles represent tissue samples with TCC ≥30 %, open circles tissue sections with TCC <30 %

Fig. 5

Correlation of EP scores between stored and nonstored FFPE tissue sections. EP scores have been determined at the beginning of the study, after 12 months at +4 °C (a) or after 12 months at +20 °C (b), respectively. Pearson correlation coefficients were 0.99 and >0.99 with a concordance of EndoPredict risk classification of 100 %