Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.
Author(s): Eikrem O, Beisland C, Hjelle K, Flatberg A, Scherer A, Landolt L, Skogstrand T, Leh S, Beisvag V, Marti HP
Publication: PLoS One, 2016, Vol. 11, Page e0149743
PubMed ID: 26901863 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare RNA and gene expression profiles from case-matched renal cell carcinoma (RCC) specimens that were formalin-fixed and paraffin-embedded (FFPE) or preserved in RNAlater. Both tumor and normal adjacent specimens were analyzed.
Conclusion of Paper
Compared to RNAlater-preserved specimens, case-matched FFPE specimens had a lower average RNA integrity number (RIN) and higher percentage of RNA fragments greater than 200 nt in length. A comparable number of genes were detected and mean expression was very strongly correlated between FFPE and RNAlater-preserved specimens (R=0.97). Clustering analysis segregated specimens based primarily on diagnosis (tumor versus normal adjacent), and no secondary clustering based on fixation method was observable. Further, there was considerable overlap between the twenty most differentially expressed genes and pathways between tumor and normal specimens among those preserved by FFPE and RNAlater. RNA levels of carbonic anhydrase 9 (CA9) correctly separated 30 of 32 normal adjacent and tumor specimens in this study and139 of 144 specimens based on published array data. Mean levels of CA9 were also higher in serum from metastatic patients (237 pg/ml) and from patients with non-metastatic tumors larger than 9 cm (112 pg/ml) than in patients with T1a stage tumors (54 pg/ml).
Studies
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Study Purpose
The purpose of this study was to compare RNA and gene expression profiles from case-matched RCC specimens that were preserved by FFPE or RNAlater. Two tumor and two normal adjacent 16-gauge core needle biopsies were obtained from each of sixteen patients, and preserved by FFPE or in RNAlater. RNA was extracted from FFPE specimens using the miRNeasy FFPE kit and from RNAlater-preserved specimens using the miRNeasy micro kit. RNA integrity was evaluated by bioanalyzer and NGS libraries were constructed using Illumina TruSeq1 Access. Levels of CA-9 were quantified by ELISA in the serum of 9 patients with metastatic tumors, 15 patients with non-metastatic tumors larger than 9 cm, and 14 patients with T1a stage tumors.
Summary of Findings:
Compared to RNAlater-preserved specimens, case-matched FFPE specimens had a lower mean RIN (2.3 versus 5.7) and a higher percentage of RNA fragments greater than 200 nt in length (DV200 of 75% versus 61%). For at least 8 specimens, a comparable number of genes were detected at greater than 15 copies per million in FFPE and RNAlater-preserved specimens (9164 genes in FFPE and 9205 genes in RNAlater). Further, there was 94% overlap in the identity of the genes detected and mean expression was very strongly correlated (R=0.97) between specimens preserved by FFPE and RNAlater. Based on cluster analysis, specimens segregated primarily by diagnosis (tumor versus normal adjacent) with no obvious clustering based on fixation method. Further, fourteen of the top 20 genes differentially expressed between normal adjacent and tumor specimens were common among FFPE and RNAlater-preserved specimens. Pathway analysis revealed 91 and 109 pathways affected by diagnosis in FFPE and RNAlater-preserved specimens, respectively. Of these pathways, 75% were common among both RNAlater and FFPE-preserved specimens; and, 12 of the 20 most affected pathways among FFPE specimens were also in the top 20 among RNAlater-preserved specimens. Affected pathways included the previously identified vascular endothelial growth factor (VEGF) and NOTCH pathways. Based on CA9 levels, 30 of 32 normal adjacent and tumor specimens were correctly separated, with exceptions including a normal adjacent specimen that contained tumor cells and a tumor specimen that contained normal adjacent tissue. Further, CA9 was able to correctly classify 139 of 144 specimens based on published array data. Immunohistochemistry confirmed elevated expression of neuronal pentraxin-2 (NPTX2) and CA9, as well as lower expression of uromodulin (UMOD) in renal carcinoma specimens compared to normal adjacent specimens. Mean levels of CA9 were higher in serum from metastatic patients (237 pg/ml) and from patients with non-metastatic tumors larger than 9 cm (112 pg/ml) than patients with T1a stage tumors (54 pg/ml) (P=0.0158 and P=0.0031, respectively).
Biospecimens
Preservative Types
- RNAlater
- Formalin
Diagnoses:
- Neoplastic - Normal Adjacent
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Automated electrophoresis/Bioanalyzer Protein ELISA Protein Immunohistochemistry RNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
RNAlater
ELISA Specific Targeted peptide/protein CA9
Immunohistochemistry Specific Targeted peptide/protein NPTX2
CA9
UMOD
Biospecimen Acquisition Biospecimen location Tumor
Normal adjacent
Metastatic tumor
Non-metastatic tumor >9 cm
T1a stage tumor