Open Access Research article

Importance of pre-analytical steps for transcriptome and RT-qPCR analyses in the context of the phase II randomised multicentre trial REMAGUS02 of neoadjuvant chemotherapy in breast cancer patients

Patricia de Cremoux1*, Fabien Valet23, David Gentien4, Jacqueline Lehmann-Che5, Véronique Scott6, Carine Tran-Perennou1, Catherine Barbaroux1, Nicolas Servant3, Sophie Vacher7, Brigitte Sigal-Zafrani1, Marie-Christine Mathieu8, Philippe Bertheau9, Jean-Marc Guinebretière10, Bernard Asselain23, Michel Marty11 and Frédérique Spyratos7

Author Affiliations

1 Department of tumour Biology, Institut Curie, Paris 75005, France

2 Department of Biostatistics, Institut Curie, Paris 75005, France

3 Inserm U900, Institut Curie, Paris 75005, France

4 Translational Research Department, Institut Curie, Paris 75005, France

5 Department of Biochemistry, AP-HP, Saint-Louis Hospital, University Paris Diderot, Paris 75010, France

6 Translational Research Laboratory, Institut Gustave Roussy, Villejuif 94805, France

7 Department of Oncogenetic, Institut Curie, Hôpital René Huguenin, Saint Cloud 92210, France

8 Department of Pathology, Institut Gustave Roussy, Villejuif 94805, France

9 Department of Pathology, AP-HP, Saint-Louis Hospital, University Paris Diderot, Paris 75010, France

10 Department of Pathology, Institut Curie, René Huguenin Hospital, Saint Cloud 92210, France

11 Centre for Therapeutic Innovations in Oncology and Haematology, AP-HP, Saint-Louis Hospital, University Paris Diderot, Paris 75010, France

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BMC Cancer 2011, 11:215  doi:10.1186/1471-2407-11-215

Published: 1 June 2011

Abstract

Background

Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial.

Methods

This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA.

Results

Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy.

Conclusions

Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.

Keywords:
neoadjuvant setting; transcriptome; RT-qPCR; breast cancer; quality criteria