Reliable PCR quantitation of estrogen, progesterone and ERBB2 receptor mRNA from formalin-fixed, paraffin-embedded tissue is independent of prior macro-dissection

Abstract

Gene expression analysis on messenger RNA (mRNA) purified from formalin-fixed, paraffin-embedded tissue is increasingly used for research purposes. Tissue heterogeneity may question specificity and interpretation of results from mRNA isolated from a whole slide section, and thresholds for minimal tumor content in the paraffin block or macrodissection are used to avoid contamination from non-neoplastic tissue. The aim was to test if mRNA from tissue surrounding breast cancer affected quantification of estrogen receptor α (ESR1), progesterone receptor (PGR) and human epidermal growth factor receptor 2 (ERBB2), by comparing gene expression from whole slide and tumor-enriched sections, and correlating gene expression from whole slide sections with corresponding immunohistochemistry. Gene expression, based on mRNA extracted from a training set (36 paraffin blocks) and two validation sets (133 + 1,083 blocks), were determined by quantitative reverse transcription polymerase chain reaction for all samples, as well as by microarray for 133 validation samples. In the training set, agreement between high vs. low mRNA expression from whole slide and tumor-enriched sections was absolute for ESR1 and ERBB2, and 83 % for PGR. Overall agreements, when comparing mRNA expression to immunohistochemistry, were 100 % (ERBB2), 89 % (ESR1) and 83 % (PGR), which was confirmed in the validation sets. Percentage of tumor in the sections did not influence the results. In conclusion, reliable quantification of ESR1, PGR and ERBB2 mRNA expression can be obtained from a whole slide section, and correlates well with immunohistochemistry. Prior removal of surrounding tissue was found to be unnecessary even with minimal tumor content in the section.

Robustness of biomarker determination in breast cancer by RT-qPCR: impact of tumor cell content, DCIS and non-neoplastic breast tissue

“…In the respective experiments, we found that changes in the relative gene expression were insignificant down to 20–39% TCC. This is in agreement with data reported by Tramm and coworkers, who showed an excellent agreement between gene expression data for ESR1, PGR and ERBB2 from whole tissue and from macrodissected extracts [ 30 ]. Likewise, macrodissection did not affect the prognostic significance of RNA expression of cancer-associated genes in primary breast tumor samples [ 12 ].…”

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

“…This indicates that the relative gene expression of the analyzed genes is even preserved in samples with a TCC below 30 %. Similar results were obtained by Tramm and colleagues who showed that the surrounding non-neoplastic tissue does not affect the quantification of ESR1 , PGR , and ERBB2 mRNA expression in breast tumor samples [ 26 ]. The test results of the PAM50-based Prosigna breast cancer prognosis test, however, are affected by adjacent nontumor tissue.…”

Impact of age, intrinsic subtype and local treatment on long-term local-regional recurrence and breast cancer mortality among low-risk breast cancer patients

“…From the 532 FFPE samples with sufficient tumor tissue, RNA extraction were performed and expression of ESR1, PGR, ERBB2 and MKI67 were quantified with quantitative real time polymerase chain reaction (described in detail in Supplementary Material, available online at http://www.informahealthcare.com [13,[25][26][27]). If the quantification failed, data from medical records and immunohistochemistry (IHC) was used to determinate ER-, PR-and HER2-status.…”