Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues.

Author(s): Tyekucheva S, Martin NE, Stack EC, Wei W, Vathipadiekal V, Waldron L, Fiorentino M, Lis RT, Stampfer MJ, Loda M, Parmigiani G, Mucci LA, Birrer M

Publication: J Mol Diagn, 2015, Vol. 17, Page 374-81

PubMed ID: 25937617 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effect of using prostate rather than ovarian tumor specimens, FFPE block storage duration, RNA input amount, necrosis content, and microarray technology platform on the correlation of gene expression between replicate FFPE specimens. The effect of microarray platform on the correlation between the log-fold changes in gene expression of high and low Gleason grade tumors in this study and a previous study was also examined.  Two to three 0.6 mm punches of tumor and normal adjacent tissue were obtained from three archival FFPE prostatectomy specimens stored for 11-21 years. Prostatectomy cores were deparaffinized in Citrisolv, digested overnight in proteinase K buffer at 55˚C, and RNA was extracted using RecoverAll Total Nucleic Acid Isolation kit. RNA was quantified using the NanoDrop and RiboGreen RNA Assay Kit. Expression was determined in 6 paired normal adjacent and tumor prostatectomy specimen using Nanostring and in 5 paired normal adjacent and tumor prostatectomy specimens after amplification with the NuGen WT-Ovation FFPE System V2 using Affymetrix Human Gene 1.0ST Array. Twenty-five mm scrolls of 10 µm sections of archived FFPE ovarian tumors (5 serous carcinoma and 6 clear cell carcinoma specimens, stored 4-7 years) were deparaffinized in xylene and RNA was extracted using the RNeasy FFPE extraction kit. Expression was determined in 2-3 technical replicates of all 11 ovarian tumors using Nanostring and after amplification with the NuGen WT-Ovation FFPE System V2 using the Affymetrix Human Gene 1.0ST. Array.

   Summary of Findings:

   Slightly more variability in the percent present calls was noted for prostate than ovarian specimens using the Affymetrix Human Gene 1.0ST Array (0.17-0.58 versus 0.40-0.68), but this was attributed to normalization effects due to differences in staining intensity. The percent present calls were not correlated with storage duration using either platform, nor were the correlation coefficients between technical replicates affected by block age.  Although increasing the RNA input from 50 ng to 100 ng did not affect the Affymetrix (amplified RNA) percent present rate, more probes were detected by NanoString when 200 ng was used rather than 100 ng (P=0.04) and this in turn resulted in more consistency in gene expression levels between replicates when 200 ng was used rather than 100 ng. The authors state the ovarian specimens with more extensive necrosis had lower expression correlations than those with less necrosis. As expected, correlation coefficients were lower when the intensity of the signal was lower. Only 2% of genes had expression intensities above an unspecified cut-off in one replicate and below in the other. Generally, expression of the 236 genes included on the NanoString was positively strongly to very strongly correlated with expression of corresponding genes on an Affymetrix array, but for 30% of genes in prostate and 13% of genes in ovary the correlation was less than 0.3. Fifteen genes (BCR, CASP10, CEBPA, CSF3, CYP1A1, FLT3, GATA1, HRAS, LMO2, MLH1, MLL, MPL, TFE3, WEE1, and WNT10B) were found to only be weakly correlated between platforms in both tissue types. The log-fold change between high and low Gleason grade tumors of the 150 genes in the Gleason signature was only modestly correlated between a previous study and this one using the Affymetrix array. However, the correlation between the log-fold changes in this study using NanoString and a previous study for the 10 genes in the Gleason signature also measured by NanoString was 0.89.

   Biospecimens

   • Tissue - Prostate
   • Tissue - Ovary

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Normal Adjacent
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	4-7 years 11-21 years
   Biospecimen Acquisition	Biospecimen location	Ovary Prostate
   DNA microarray Specific	Technology platform	Affymetrix Human Gene 1.0ST Array NanoString nCounter
   DNA microarray Specific	Template/input amount	50 ng 100 ng 200 ng
   DNA microarray Specific	Nucleic acid amplification	Unamplified (NanoString) Whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2 (Affymetrix)

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