From the Bin to the Gene: Leftovers of Diagnostic Fine Needle Aspiration Cytology Can Be Successfully Used To Perform Gene Expression Profiling of Breast Carcinomas
L Annaratone, C Marchio, I Castellano, D Cantarella, E Allia, G Bussolati, E Medico, A Sapino. University of Turin, Turin, Italy; Institute for Cancer Research and Treatment (IRCC), Turin, Italy
Background: Molecular testing of breast cancer is emerging as an ancillary technique to conventional histopathological tools. Several predictive signatures based on differential expression of genes between distinct tumour groups have been described so far and one of these has recently gained FDA approval.The success of molecular tests is strictly related to an adequate sampling of the lesion and to the good preservation of material to be subjected to the analysis. Therefore, it is mandatory to use fresh samples that are representative of and enriched for the tumour cell population in order to obtain a sufficient yield of good quality RNA. Our aim was to investigate whether leftovers from diagnostic Fine Needle Aspiration (FNA) cytology can be successfully used to extract RNA of enough quantity and quality to be subjected to different types of molecular analysis.
Design: We collected 28 samples of FNA leftovers obtained from mammograpically detected opacities. FNAs were performed under ultrasound guidance using a multi-pass approach with a 22-gauge needle attached to a syringe in a pistol-grip holder. The aspirated material was immediately smeared and stained, then the syringe was washed in Trizol solution and the collected material was transferred in vials at 4°C. Following RNA extraction and quantification, RT-PCR for PGK and CK-19 genes were performed. Twelve samples with different RNA concentration were selected for microarray gene expression analysis, which was performed by using the HumanHT-12_V3 Expression BeadChip by Illumina.
Results: RNA was successfully extracted from all FNA samples with a mean RNA yield of 11.7 μg/FNA (range: 0.78-88.4 μg; median: 4.85 μg; mode 7.5 μg). RNA integrity and absence of DNA polymerase inhibitors were assessed by the percentage of samples positive for PGK housekeeping gene. Twenty-six out of 28 (92.8%) samples were RT-PCR positive for PGK. These 26 samples were tested for CK-19 gene expression and all of them were positive. Microarray gene expression analysis of selected cases demonstrated a good performance in all of the 12 samples.
Conclusions: FNA cytology leftovers can be successfully used to extract RNA of enough quantity and quality to reliably perform molecular analyses.
Monday, March 22, 2010 1:00 PM
Poster Session II # 53, Monday Afternoon